Dissertations / Theses on the topic 'Lipid formulations'

Cancer is the second leading cause of death and is responsible for approximately 9.6 million deaths worldwide in 2018. Among all oncological diseases, lung cancer claims the highest mortality (male: 23.5%; female: 22%) and the second most new cases (male: 13%; female: 12%) in the US. Approximately 40% of newly diagnosed lung cancer patients are in the advanced stage IV, for which platinum-based chemotherapy is the first-line treatment, either by itself or in combination with surgery or radiotherapy. Cisplatin, the first-generation platinum-based anticancer chemotherapeutic agent, has the highest potency against lung cancer but carries many severe adverse effects. Cisplatin also induces drug resistance during long-term chemotherapy. Many more platinum complexes have been investigated as better alternatives, which led to the approval of carboplatin and oxaliplatin by Food and Drug Administration (FDA). In addition, miriplatin suspended in iodolipds (lipiodolization) was approved in Japan for the treatment of hepatocellular carcinoma (HCC) in 2009. Miriplatin has the same non-leaving group as oxaliplatin but different leaving groups of two myristate chains, which make it highly lipophilic. Several characteristics of solid tumors in lung cancer constitute a physiochemical barrier to the homogenous distribution and deep penetration of chemotherapy agents. Nanocarriers provide a promising platform to overcome the physiochemical barrier and to reduce the systemic toxicity of anticancer chemotherapy. In this study, miriplatin is formulated with various lipid-based nanocarriers including micelles and solid lipid nanoparticles (SLNs) thanks to its highly lipophilic structure. The goal of this thesis is to develop and evaluate miriplatin-loaded nano formulations against lung cancer. Miriplatin-loaded formulations were prepared by different methods, including thin film hydration and several scale-up methods including chloroform dripping, chloroform injection, chloroform evaporation, co-solvent evaporation, chloroform slow evaporation and co-solvent slow evaporation. Between the two types of nano formulations under this study, micelles were much smaller (~10 nm in diameter) and more homogeneous (PDI < 0.3), while SLNs were bigger (~ 100 nm in diameter) and more heterogeneous (PDI ~0.8). A quantification method of miriplatin was established using inductively coupled plasma-optical emission spectrometry (ICP-OES). The quantification of platinum recovery from different miriplatin-loaded nano formulations was facilitated by digestion with 70% nitric acid and heating. The co-solvent slow evaporation method to prepare miriplatin-loaded nano formulations improved the platinum recovery prominently from 10% to 70%. Thus, co-solvent slow evaporation has been established as a pharmaceutically viable scale-up method to prepare nano formulations of miriplatin. Miriplatin-loaded nano formulations of different compositions were negatively stained with uranyl acetate and then imaged by transmission electron microscopy (TEM), which showed the formulations’ size and morphology that were consistent with the size and PDI data from dynamic light scattering studies by the Malvern Zetasizer. In the TEM studies, micelles showed a morphology of spherical dots at around 10 nm in diameter while SLNs showed both spherical and rod structures with a size distribution from 50 to 150 nm. A three-dimensional multicellular spheroid (3D MCS) model of A549-iRFP cells was used for in vitro evaluation of the nano formulations’ activity against lung cancer. A549-iRFP cells were engineered from the common lung cancer cell line A549 to stably express the near-infrared fluorescent protein (iRFP). The viability of A549-iRFP 3D MCS after exposure to cisplatin or nano formulations was similar to A549 3D MCS. The anticancer activity of miriplatin-loaded nano formulations against 3D MCS was positively associated with the platinum recovery as quantified by ICP-OES. The miriplatin-loaded nano formulations that had been prepared by the co-solvent slow evaporation method showed substantial anticancer activities against A549 3D MCS and A549-iRFP 3D MCS, which were comparable to cisplatin. Taken together, miriplatin-loaded nano formulations were successfully prepared by co-solvent slow evaporation. The formulations were developed to carry favorable physiochemical properties to enhance the activities of platinum drugs against lung cancer.

La mucoviscidose est une maladie génétique grave et évolutive dont les atteintes pulmonaires sont, aujourd’hui, les causes principales de décès des patients. Du fait de l’absence ou du dysfonctionnement du canal chlore CFTR, les patients atteints de mucoviscidose présentent un mucus hypervisqueux notamment au niveau des voies respiratoires. Ce mucus est un environnement favorable à la colonisation par des bactéries opportunistes telles que le Staphylococcus aureus ou le Pseudomonas aeruginosa. La chronicité des infections couplées avec une inflammation importante conduisent à la dégradation progressive des fonctions respiratoires. Actuellement, hormis la greffe coeur/poumon, aucun traitement curatif n’est encore accessible pour l’ensemble des patients. L’approche par thérapie génique apparait être une bonne stratégie pour tenter de guérir tous les patients indépendamment du type de mutations dont ils sont porteurs. Il s’agit d’apporter une copie saine du gène CFTR à l’intérieur des cellules afin que celles-ci expriment une protéine fonctionnelle. Pour ce faire, de nombreuses barrières sont à franchir. Parmi elles, la présence de bactéries dans l’environnement cellulaire est un frein s’opposant au transfert de gènes notamment par vecteurs. Il semble pertinent de développer une formulation multifonctionnelle permettant d’une part d’éliminer les bactéries en surface et d’autre part, de transfecter les cellules cibles. Cette formulation doit rester efficace après qu’elle ait été aérosolisée. Au cours de ce travail, plusieurs formulations, incorporant des lipides cationiques et des composés argent, ont été mises au point
Cystic fibrosis is a genetic disease with lung damages as the current main causes of death. Due to the absence or dysfunction of the CFTR chloride channel, CF patients have hyper-viscous mucus, particularly in the respiratory tract. This mucus is an environment favorable to infection development by opportunistic bacteria such as Staphylococcus aureus or Pseudomonas aeruginosa. The chronicity of infections coupled with significant inflammation leads to the progressive degradation of respiratory functions. Currently, apart from the heart-lung transplantation, no cure is still available for all patients.The approach by gene therapy appears to be a good strategy to cure all patients regardless of the type of mutations they have. It is a matter of bringing a healthy copy of the CFTR gene into the cells so that they express a functional protein. To do this, many barriers must be overcome. Among them, the presence of bacteria in the cellular environment is a brake against the transfer of genes in particular by vectors. It seems pertinent to develop a multifunctional formulation that on the one hand eliminates surface bacteria and on the other hand transfect the target cells. This formulation must remain effective after it has been aerosolized. During this work, several formulations, incorporating cationic lipids and silver compounds, have been developed

The aim of this thesis is to investigate the physicochemical parameters which can influence drug loading within liposomes and to characterise the effect such formulations have on drug uptake and transport across in vitro epithelial barrier models. Liposomes composed of phosphatidylcholine (PC) or distearoyl phosphatidylcholine (DSPC) and cholesterol (0, 4, 8, 16 µM) were prepared and optimised in terms of drug loading using the hand-shaking method (Bangham et al., 1965). Subsequently, liposomes composed of 16 µM PC or DSPC and cholesterol (4 µM) were used to monitor hydroxybenzoate release and transport from Iiposomes. The MIT (3[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and crystal violet assays were employed to determine toxicity of the Iiposome. formulations towards the Caco-2 cell line, employed to model the epithelial barrier in vitro. Uptake and transport of mannitol, propranolol, glutamine and digoxin was measured in the presence and absence of Iiposome formulations to establish changes in absorption resulting from the presence of lipid formulations. Incorporation of the four hydroxybenzoates was shown to be influenced by a number of factors, including liposome composition and drug conformation. Methyl hydroxybenzo.ate (MP) was incorporated into the bilayer most effectively with percentage incorporation of 68% compared to 45% for butyl hydroxybenzoate (BP), despite its increased Iipophilicity. This was attributed to the decreased packing ability of BP within the hydrocarbon core of the lipid bilayer compared to MP. Release studies also suggested that the smaller MP was more strongly incorporated within the lipid bilayer with only 8% of the incorporated solute being released after 48-hours compared to 17% in the case of BP. Model transport studies were seen to reflect drug release profiles from the liposome bilayers with significantly (p < 0.01) higher amounts of BP partitioning from the liposome compared to MP, Caco-2 cell viability was maintained above 86% in the presence of all Iiposome formulations tested indicating the liposome formulations are non-toxic towards Caco-2 cells. Paracellular (apical-to-basolateral) transport of mannitol was significantly increased in the presence of DSPC, PC / DSPC:Cholesterol (16:4 µM; 1000 µg). Glutamine uptake and transport via the carrier-mediated route was Significantly (p < 0.01) increased in the presence of PC I DSPC:Cholesterol (16:0; 16:4 µM). Digoxin apical-to-basolateral transport was significantly increased (p < 0,01) in the presence of PC / DSPC:Cholesterol (16:0; 16:4 µM); thus reducing digoxin efflux via P-glycoprotein. In contrast, PC:ChoJesterol (16:0; 16:4 µM) significantly (p < 0.01) decreased propranolol uptake via the passive transcellular route. Bi-directional transport of propranolol was significantly (p < 0,01) decreased in the presence of PC/DSPC:Cholesterol (16:0; 16:4 µM). The structure of a solute is an important determinant for the incorporation and release of a solute from liposome formulations. PC, DSPC and cholesterol liposome formulations are nontoxic towards Caco-2 cell monolayers and improved uptake and transport of mannitol, glutamine. and digoxin across Caco-2 cell monolayers; thus providing a potential alternative delivery vehicle.

Ces travaux de thèse ont porté sur le développement de formulations innovantes et nanoparticulaire, destinées à améliorer la prise en charge des patients atteints de leucémie aigüe myéloïdes (LAM). Cette amélioration de la qualité de vie peut passer par le développement d’une formulation orale de décitabine.Trois stratégies de formulations différentes ont été développées : deux formulations de nanocapsules lipides (LNCs) avec encapsulation ou de décitabine, ou d’une prodrogue de décitabine (décitabine(C12)2) . La troisième stratégie a été le développement de particules de type liposomal, dans lesquelles la décitabine a été encapsulée. Après avoir été caractérisée sur des critères physicochimiques, chacune des stratégies basées sur les LNCs a été évaluée par des essais in vitro pour évaluer la perméabilité intestinale de la décitabine lorsqu’elle a été encapsulée. Une des stratégies a permis d’accroitre la perméabilité, in vitro, de la décitabine. L’activité sur la prolifération cellulaire a ensuite été évaluée sur des cellules humaines de LAM. Il a été démontré que l’encapsulation dans les LNCs améliore l’activité de la décitabine et de la décitabine (C12)2. Après l’ensemble de ces essais, en vue d’évaluer le potentiel avantages de ces formulations pour augmenter la demi-vie plasmatique de la décitabine, leurs stabilités dans du plasma humain a été évaluée. La décitabine (C12)2 libre et encapsulée permettent de limiter la dégradation rapide de la décitabine. Finalement, une étude de pharmacocinétique a été mise en place. L’encapsulation de la décitabine, en synthétisant au préalable une prodrogue permet d’augmenter les concentrations maximales atteintes
The aim of this phD work was to develop nanoparticle formulations to improve patients’quality of life in case of acute myeloid leukemia (AML). These formulations could, for example, allow an oral administration of decitabine. Three different formulations were developed: two were based on lipid nanocapsules (LNCs) with an encapsulation of decitabine or a decitabine prodrug (decitabine(C12)2). The third strategy was aliposomal formulation with a decitabine encapsulation. After being characterized on physico-chemical parameters, in vitro intestinal permeability studies were performed on LNCs strategies. One strategy was able to enhance decitabine permeability. Cell proliferation studies performed on human AMLcell lines showed that encapsulations into LNCs improve decitabine and decitabine(C12)2 activities. In order to evaluate the potential of these formulations to enhance decitabine plasma half-life, their stabilities in human plasma were then assayed. Free decitabine(C12)2 or encapsulated into LNCs has been shown to limit the rapid decitabine degradation. Finally, pharmacokinetic studies were performed. Decitabine encapsulation into LNCs with a previous decitabine prodrug synthesis was able to increase maximal plasma concentrations

Poorly water-soluble drugs (PWSDs), to date, require advanced formulation techniques to improve solubility and achieve the required plasma concentration to show a therapeutic effect when orally administered. Lipid-based formulations (LBFs) are an enabling strategy that is being used to improve the oral delivery of PWSDs. The aim of this study was to investigate the effect of lipid-based formulation, Type IIIA-LC, on the solubilization patterns of PWSDs, namely, carvedilol and felodipine. Solubility studies, for both drugs, were performed with LBF dispersed in -1) dog intestinal fluid (DIF), and 2) water, to identify and compare the extent of solubility in different matrices, and in silico to identify interesting patterns with any correlations in experimental and computational data. Solubility studies showed that carvedilol had better solubility in LBF when compared to felodipine. Computational studies showed that both drugs solubilized in the colloid in both digested and undigested states. Effect of drug loading had no significant difference on the solubilization patterns of both drugs. The maximum drug loading done was for 100 molecules though there is the possibility of the colloid having a higher capacity. Digestion did not seem to have a significant effect on the distribution of both drugs. In vitro and in silico data were in qualitative agreement and therefore, this computational model can be further used to study the specific processes causing solubilization, improvement, and development of new LBFs.

Fish meal (FM) and fish oil (FO) have traditionally been central in aquaculture feed formulation but the finite global supply situation limiting future use along with issues of contaminant levels in these feed ingredients have become critical issues. The objectives of the present study were to investigate alternative feed ingredients as substitutes for both FM and FO in feeds for Atlantic salmon (Salmo salar) to ensure optimal growth, feed efficiency and health of the fish as well as maintaining the nutritional quality of the fish product to the human consumer, especially the levels of n-3 highly unsaturated fatty acid (HUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in the flesh. The results of the present study revealed that there were no negative effects on growth performance, feed utilisation and apparent digestibility in Atlantic salmon when FO was substituted with vegetable oil (VO) but these parameters were affected when FM was replaced with alternative protein sources from plants and animals at high levels, despite dietary supplementation with crystalline amino acids and lecithin. Reduction in feed intake was a factor affecting growth retardation when FM inclusion decreased. However, replacing FM with alternative plant and animal proteins along with partial replacement of FO had no major effect on nutritional quality, particularly n-3 HUFA content of salmon tissues. Replacing Northern FO with decontaminated FO or blends of southern hemisphere FO and VOs strategies to reduce POP contaminants and retain high nutritional values in flesh were very successful. Dietary treatments and genetic origin of fish both had effects on tissue compositions and gene expression. All fish groups (strain/family), consist of CAL, LEAN and FAT strains, fed a diet containing VO showed significant differential expression of lipid metabolism-related genes compared to fish fed a FO diet with LEAN strain appearing to adapt to VO inclusion better than FAT strain. This thesis has demonstrated dual replacement of FM and FO with alternative raw materials in salmon feeds without a major negative impact on nutritional quality.

Compostos de origem vegetal podem apresentar inúmeros efeitos benéficos à saúde, havendo, entretanto, uma necessidade de desenvolver formulações que permitam viabilizar seu uso farmacêutico, alimentício, nutracêutico ou cosmecêutico. Syzygium aromaticum, conhecido popularmente como cravo da Índia, é uma espécie vegetal aromática com marcada atividade antioxidante, analgésica e antimicrobiana. A baixa solubilidade e estabilidade química, assim como a volatilidade dos principais compostos associados às atividades biológicas da planta justificam o desenvolvimento de formulações que melhorem suas propriedades físico-químicas e características de liberação. Formulações lipídicas têm sido cada vez mais usadas para o aumento da solubilidade de compostos no trato gastrointestinal e para o aumento da biodisponibilidade. O principal objetivo deste projeto foi investigar a produção de formulações lipídicas sólidas contendo compostos bioativos de S. aromaticum e avaliar o efeito da composição da formulação e das variáveis operacionais nas propriedades físico-químicas das partículas, estabilidade e permeação intestinal in vitro. O processo de extração dos compostos a partir da matéria-prima vegetal também foi estudado. A formulação lipídica foi otimizada com respeito ao tipo e a proporção dos lipídeos, do emulsificante e dos carreadores de secagem. Os processos de emulsificação e secagem também foram criteriosamente estudados. Os resultados mostraram que a composição da formulação teve efeitos significativos nas propriedades físico-químicas do produto e no desempenho da secagem. A formulação lipídica otimizada mostrou ser mais estável que a formulação não lipídica em condições de armazenamento de alta umidade. Com relação à permeação intestinal in vitro, utilizando eugenol como marcador, não foram observadas diferenças significativas entre estas duas formulações. Este projeto permitiu obter informações relevantes sobre a secagem por spray drying de formulações lipídicas contendo extratos vegetais. Esta rota tecnológica representa uma estratégia interessante na obtenção de formulações lipídicas estáveis que promovam o aumento da biodisponibilidade oral de compostos bioativos.
Plant-derived compounds can provide important benefits to human health. However, these compounds should be properly formulated in order to facilitate their pharmaceutical, nutraceutical, food or cosmetic applications. Syzygium aromaticum commonly known as Indian clove, is an aromatic tree with antioxidant, antimicrobial and analgesic properties. The poor water solubility and the volatility of the compounds associated to the biological activities, justify the development of formulations that improve its physicochemical and release properties. Lipid based formulations have gained special attention for oral delivery due to the improvement of solubility in the intestinal tract and increase of bioavailability. The main objective of this project was to investigate the production of solid lipidic formulations containing bioactive compounds of S. aromaticum and to test the effect of the formulation composition and the process variables on the physhicochemical properties of the particles, stability and in vitro intestinal permeation. The extraction methods of the compounds from the plant were also studied. The lipid formulation was optimized with regard to the type and proportion of the solid lipid, the surfactant and the drying carrier. The emulsification and the drying processes were carefully evaluated. Results showed that the formulation composition had significant effects on the physicochemical properties of the product and on the drying performance. The optimized lipid formulation was more stable than the formulation without lipids in high humidity stress storage conditions. With regard to the in vitro intestinal permeation using eugenol as marker compound, not significant differences were observed between the samples. This project allowed to obtain relevant information about the spray drying process of lipid formulations containing plant extracts. This technique could be an interesting strategy to obtain stable lipid formulations than enhance the oral bioavailability of bioactive compounds

Pseudomonas aeruginosa (PA) et Acinetobacter baumannii (AB) sont responsables d'infections pulmonaires chroniques souvent associées à la formation de biofilms, i.e. des agrégats bactériens piégés dans une matrice riche en polymères anioniques tels que l'alginate ou l’ADN. Au sein de ces biofilms, la diffusion des antibiotiques cationiques (ATB), tels que la tobramycine (TOB) et la colistine (COL), est en partie réduite due aux interactions avec les polymères de la matrice. Les principaux objectifs de cette thèse ont été dans un premier temps de développer un modèle de biofilm pulmonaire mimant ceux retrouvés dans les infections pulmonaires chroniques des patients atteints de mucoviscidose (CF), l'une des principales maladies pulmonaires dans laquelle ces biofilms se développent. Un nouveau système d’administration d'ATB, composé de nanoparticules lipidiques chargées de COL, a été par la suite développé et testé grâce à ce modèle de biofilms pulmonaires.Chez les patients atteints de CF, PA forme des biofilms constitués d'agrégats (50-100 μm) incorporés dans leur mucus pulmonaire. Un modèle de biofilm pulmonaire in vitro composé des polymères présents in vivo (alginate, mucines, ADN) et constitué de PA piégés dans des billes d'alginate, a été développé et évalué à l'aide de la COL et de la TOB. Ce modèle a été optimisé en évaluant l'effet de la taille des billes et du milieu de croissance dans lequel elles ont été dispersées. La modification de la taille des billes de 60 à 1200 µm et l'utilisation d'un milieu de dispersion simulant la composition du mucus pulmonaire ont permis de réduire davantage l'efficacité de la TOB dans ce modèle. Pour des concentrations élevées en TOB, des variants de PA formant de très petite colonies (SCV), également trouvés chez les patients atteints de CF, ont été observés dans le modèle, ce qui suggère une bonne imitation des conditions in vivo. Dans ce modèle, l'efficacité de la COL a été moins affectée que celle de la TOB, et aucune SCV a été observé. Des nanoparticules lipidiques PEGylées chargées de COL et contenant un adjuvant lipophile à la COL tel que le farnésol ont été développées pour augmenter son efficacité contre les biofilms. L'encapsulation de la COL a amélioré son efficacité par un facteur deux dans le modèle de biofilm pulmonaire à PA précédemment développée. Ces nanoparticules ont permis également de restaurer la sensibilité à la COL d'une souche résistant d’AB et ont amélioré son efficacité dans un modèle de biofilm d’AB formé sur une surface abiotique
Pseudomonas aeruginosa (PA) and Acinetobacter baumannii (AB) are responsible for chronic lung infections often associated with the development of biofilms, i.e. bacterial aggregates trapped in a self-produced matrix of anionic polymers such as alginate or DNA. Within these biofilms, bacteria are protected from cationic antibiotics (ATB) such as tobramycin (TOB) and colistin (COL), in part through their interaction with matrix polymers, which reduce their diffusion. The main objectives of this thesis were first to develop a lung biofilm model that mimics those found in chronic lung infections of patients with cystic fibrosis (CF), one of the main lung diseases for which biofilms develop. Then use this model to evaluate a new ATB delivery system made of lipid nanoparticles developed to treat lung biofilms.In CF patients, PA forms biofilms consisting of aggregates (50-100 μm) enclosed in their pulmonary mucus. An in vitro lung biofilm model composed of polymers present in vivo (alginate, mucins, DNA) and consisting of PA trapped in alginate beads has been developed and evaluated using COL and TOB. This model was refined by evaluating the effect of the size of the beads and the growth medium in which they were dispersed. Changing the size of the beads from 60 to 1200 µm and using a dispersion medium simulating the composition of mucus further decreased the effectiveness of TOB in this model. For high TOB concentrations, small-colony variants (SCV) of PA, also found in the CF patient, were observed in the model, suggesting a good mimicry of the conditions in vivo. In this model, COL efficacy was less impacted than that of TOB, and prevented the appearance of SCV. Pegylated lipid nanoparticles loaded with COL and containing a lipophilic adjuvant to COL such as farnesol have been developed to increase its effectiveness against biofilms. Encapsulation of COL improved its efficiency by two in the lung biofilm model PA developed, but also restored the sensitivity to COL of a resistant AB and improved its efficiency in an abiotic adherent biofilm model with AB

Le fromage fondu est un produit alimentaire de seconde transformation obtenu après mélange et cuisson de fromages, additionnés éventuellement d'autres ingrédients laitiers. Ce produit, dont la consommation croit dans de nombreuses régions du monde, présente une grande variété d'applications et peut être conservé plusieurs mois à température ambiante. Cependant, les mécanismes moléculaires sous-jacents à sa structuration biochimique demeurent mal connus et sa fabrication industrielle reste souvent empirique.L'objectif général de ce travail est d'améliorer la compréhension des étapes de structuration biochimique du fromage fondu de type « portion triangulaire tartinable », au cours des étapes de sa fabrication. Dans un premier temps, les propriétés physico-chimiques des sels de fonte, additifs ajoutés au fromage fondu, ont été étudiées dans des milieux modèles de complexités croissantes, de la solution aqueuse jusqu'au lait écrémé. La composition de ces sels de fonte, leur hydrolyse après traitement thermique ainsi que leur interaction avec certains constituants laitiers ont été évaluées par deux méthodes complémentaires : la chromatographie ionique et la RMN du phosphore. Par ailleurs, il a été montré que ces sels, via la chélation du calcium, induisaient la dissociation des caséines, modifiant les propriétés d'hydratation et d'émulsification de ces dernières.Dans un deuxième temps, le rôle des sels de fonte quant à la structuration des protéines, de l'eau, des minéraux et de la matière grasse a été analysé dans des matrices plus complexes : les portions triangulaires tartinables, aux étapes clés de leur fabrication. Un mécanisme d'interaction des constituants biochimiques majoritaires a été proposé, prenant en compte l'évolution des molécules sous l'effet des contraintes physiques et chimiques appliquées au cours du procédé
Processed cheese is manufactured by the secondary processing food industry, by mixing and heating cheese, along with other dairy ingredients. This product, whose consumption grows in many parts of the world, has a wide variety of applications and can be stored for several months at room temperature. However, the molecular mechanisms underlying its structure remain poorly understood and industrial production is often empirical. The general objective of this work is to improve the understanding of biochemical steps structuring spreadable processed cheese during its manufacture. At first, the physico-chemical properties of additives used in processed cheese, i.e. emulsifying salts, have been studied in simplified environments of increasing complexity, from aqueous solutions to skimmed milk. The composition of theses salts, their hydrolysis after heat treatment and their interaction with some dairy constituents were assessed by two complementary methods: ion chromatography and phosphorus NMR. Moreover, it has been shown that these salts, through the calcium chelation, induced casein dissociation and modified their hydration and emulsifying properties. Then, the role of these salts on the structure and interactions between proteins, water, minerals and fat were analyzed in spreadable processed cheese, at different steps of its manufacture. A mechanism of interaction between the major biochemical constituents has been proposed, taking into account the evolution of molecules under the influence of physical and chemical constraints applied during the process

There has been a lot of interest on solid lipid nanoparticles (SLNs) as these colloidal submicron drug dosage forms present a promising frontier in drug delivery. It is possible to incorporate susceptible drugs such as protein intended for oral delivery. Here, we aim to develop an oral delivery system based on SLNs to deliver the peptide hormone, insulin using the double emulsion (W/O/W) solvent evaporation technique for formulating the SLNs. The choice of lipids was carefully selected to incorporate acceptability to biological milieu. The main purpose of the work was to formulate SLNs to achieve different localisation of insulin within the SLNs, based on the three hypothetical models proposed by Muller et al. (2000). Following that, the effect of this localisation on the propensity of the SLNs to be taken up by absorptive cells was investigated. SLNs was successfully fabricated to achieve two insulin localisation models, namely the solid solution model and the core-shell model with drug-enriched shell. The zeta potential measurements was used to indirectly indicate the appropriate insulin localisation model. The zeta potential of the unloaded SLNs, insulin-loaded SLNs and surface-adsorbed insulin SLNs were recorded as -51.7 ± 1 mV, -45.8 ± 1 mV and -40.8 ± 1 mV respectively. In vitro cell studies showed a notable difference in the Caco-2 cell lines when the cells were exposed to SLNs of the two different insulin localisation models. Thus, different effects seen on the Caco-2 cells suggests that the localisation of insulin within SLNs can potentially influence its uptake, stressing the importance of characterising drug localisation in nanoparticles, as this eventually affects drug bioavailability.

This thesis was devoted to the development of innovative oral delivery systems for two different molecules. In the first part, microparticles (MPs) based on xylan and Eudragit® S-100 were produced and used to encapsulate 5-aminosalicylic acid for colon delivery. Xylan was extracted from corn cobs and characterized in terms of its physicochemical, rheological and toxicological properties. The polymeric MPs were prepared by interfacial cross-linking polymerization and spray-drying and characterized for their morphology, mean size and distribution, thermal stability, crystallinity, entrapment efficiency and in vitro drug release. MPs with suitable physical characteristics and satisfactory yields were prepared by both methods, although the spray-dried systems showed higher thermal stability. In general, spray-dried MPs would be preferable systems due to their thermal stability and absence of toxic agents used in their preparation. However, drug loading and release need to be optimized. In the second part of this thesis, oil-in-water microemulsions (O/W MEs) based on medium-chain triglycerides were formulated as drug carriers and solubility enhancers for amphotericin B (AmB). Phase diagrams were constructed using surfactant blends with hydrophilic-lipophilic balance values between 9.7 and 14.4. The drug-free and drug-loaded MEs presented spherical non-aggregated droplets around 80 and 120 nm, respectively, and a low polydispersity index. The incorporation of AmB was high and depended on the volume fraction of the disperse phase. These MEs did not reduce the viability of J774.A1 macrophage-like cells for concentrations up to 25 µg/mL of AmB. Therefore, O/W MEs based on propylene glycol esters of caprylic acid may be considered as suitable delivery systems for AmB.

L’objectif des travaux de cette thèse était de développer un système de délivrance stimulus-sensible innovant. Basé sur des vésicules lipidiques, il permet la libération d’une substance anti-cancéreuse hydrophile encapsulée dans leur cœur aqueux, sous l’effet de la lumière. Des porphyrines, incorporées dans leur bicouche, permettent, une fois illuminées, de générer de l’oxygène singulet qui oxyde les chaînes acyl insaturées des phospholipides. Cela induit une augmentation de la perméabilité des liposomes et permet la libération de leur cargo. Nous avons, dans un premier temps, effectué une sélection de phospholipides et de porphyrines permettant de construire le système. Les résultats expérimentaux ont pu être corrélés à une étude de simulation de dynamique moléculaire. L’ensemble a mis en exergue l’importance de la profondeur d’insertion de la porphyrine dans la bicouche lipidique et de sa proximité avec la double-liaison des phospholipides. Mais il a aussi montré les limites de ce système. Nous avons alors développé deux nouvelles molécules, dérivées de phospholipides naturels auxquels a été couplée la pheophorbide a. Malgré leur possible autoassemblage sous la forme de vésicules, ces derniers n’étaient pas stables et s’agrégeaient rapidement. Nous avons donc associé ces conjugués à des lipides classiques (DSPC, cholestérol) et analysé les propriétés des mélanges obtenus. Les propriétés photothermiques des systèmes conçus ont été confirmées, capables d’induire une élévation en température de 14°C. La chaleur générée, responsable d’une plus grande fluidité de la bicouche lipidique, a permis de favoriser la libération du cargo. Enfin, les deux conjugués synthétisés ont montré eux-mêmes une activité phototoxique (PDT), additionnée d’une sélectivité vis-à-vis de cellules du cancer de l’œsophage. Ces nouvelles molécules offrent donc de nombreuses opportunités pour le développement de systèmes multimodaux, bio-inspirés et biodégradables, pour la délivrance d’un médicament sous l’effet de la lumière
The aim of this work was to develop an innovative stimulus-responsive delivery system. Based on lipid vesicles, it allows the controlled release, by light, of a hydrophilic anti-cancer substance encapsulated in their aqueous core. Once illuminated, porphyrin molecules inserted into the lipidic bilayer, generate singlet oxygen which oxidizes the unsaturated acyl chains of the phospholipids. This induces an increase in the permeability of the liposomes and the release of their cargo. We first made a selection of phospholipids and porphyrins to build the system. Our experimental study could be correlated with results of molecular dynamics simulations. The whole work highlighted the importance of the depth of insertion of porphyrin into the lipid bilayer and its proximity to the double bond of phospholipids. But it also showed the limits of this system. We then developed two new molecules, derived from natural phospholipids, to which pheophorbide a was coupled. The conjugates were able to form self-assembled vesicles but were unstable and quickly aggregated. We therefore associated these conjugates with classical lipids (DSPC, cholesterol) and analyzed the properties of these mixtures. We highlighted photothermal properties of the designed systems, capable of inducing a temperature rise of 14 °C. The generation of heat, responsible for a greater fluidity of the lipid bilayer, subsequently promoted the encapsulated cargo release. Finally, the two synthesized conjugates showed a phototoxic activity (PDT), with selectivity towards esophageal cancer cells. These new molecules therefore offer many opportunities for the development of multimodal, bio-inspired and biodegradable systems, for the delivery of a drug under the effect of light

The research surrounding delivery carriers of hydrophobic drugs has evolved with the help of efficient tools brought together by nature’s most important process, self-assembly. This enabled scientists to create and tailor materials with multiple functionalities in order to suite specific needs. One such nanomaterial is the disc-shaped cargo carrier, named nanodiscs. Nanodiscs are noncovalent assemblies consisting of a phospholipid bilayer,whose hydrophobic edge is stabilized by two or more copies of membrane scaffold protein. Despite an abundance of research directed towards the development of an effective nanosystem for delivery into mammalian cells, superficial focus is given to characterizing the incorporation of hydrophobic drugs. This thesis describes the development of a simple approach to systematically study the relation between physical parameters related to loading and release of poorly water soluble drugs from nanodisc systems. For the nanodiscs reconstitution, two strategies have been studied, the first comprised of adding the protein to a mixture of lipid and detergent micelles, and second strategy involved adding protein stabilised in detergent micelles to the lipid. Two more strategies were employed for studying the loading of drugs into nanodiscs -the drugs were added either to a solution of nanodiscs, or loaded during the reconstitution of nanodiscs. The latter strategy showed an increase in drug incorporated per nanodiscs and a slower release rate. In the case of amiodarone and chlorambucil incorporation, a decrease in membrane thickness was seen to be correlated with the efficiency of drug loading within the POPC bilayer. In addition, the lipid composition within nanodiscs was varied in order to investigate its effect on nanodisc formation, drug loading and release kinetics. Finally, the protein component of empty and drug loaded nanodiscs was site-specifically modified via Sortase A mediated ligation,for traceable delivery in in vitro cell studies. Good internalisation of MSP by HeLa cells was observed when cells were treated with empty and chlorambucil loaded nanodiscs.

Les écrans solaires sont des formulations dont l’application permet de protéger la peau des effets néfastes du rayonnement ultraviolet (UV) et notamment de l’apparition des cancers cutanés. Ces formulations contiennent des filtres UV organiques et/ou inorganiques. Certains filtres UV organiques sont connus pour pénétrer la peau et engendrer des réactions allergiques et photo-allergiques. De plus, certains d’entre eux peuvent engendrer des effets toxiques sur les cellules cutanées vivantes et atteindre la circulation systémique. Les filtres UV inorganiques sont la plupart du temps incorporés dans les écrans solaires sous forme de nanoparticules afin d’améliorer les qualités esthétiques des écrans solaires minéraux. Les nanoparticules utilisées comme filtres UV inorganiques exerceraient des effets toxiques sur les cellules nucléées de la peau. Les nanoparticules et nanocapsules lipidiques sont des particules lipidiques submicroniques intéressantes pour formuler les actifs pharmaceutiques et cosmétiques et notamment les filtres UV. L’objectif de ce travail était de développer des suspensions de nanoparticules lipidiques pour formuler des filtres UV inorganiques et organiques en les maintenant à la surface de la peau tout en augmentant leur efficacité photo-protectrice. Ces travaux ont permis de développer des suspensions de nanoparticules lipidiques encapsulant des filtres UV inorganiques en augmentant leur pouvoir photo-protecteur. Cette étude a également mis en évidence l’intérêt de ces suspensions de nanoparticules lipidiques pour encapsuler un filtre UV organique et limiter sa perméation percutanée tout en augmentant son efficacité filtrante dans le domaine UV
Sunscreens are topical formulations that protect the skin against damages induced by ultraviolet (UV) radiations and notably skin cancers formation. Those formulations contain organic and/or inorganic UV filters. Some organic UV filters are known to penetrate the skin and trigger allergic and photo-allergic cutaneous reactions. Moreover, some of them are responsible for toxic effects on skin nucleated cells and could reach systemic circulation. Nanoparticles of inorganic UV filters are often incorporated into sunscreens to improve their aesthetic qualities. Nanoparticles used as inorganic UV filters could exercise toxic effects on skin nucleated cells. Lipid nanoparticles and nanocapsules are submicronic lipid particles interesting to formulate pharmaceutical and cosmetic active compounds and notably UV filters. The aim of this work was to develop lipid nanoparticles to entrap organic and inorganic UV filters maintaining them at skin surface while increasing their photo-protection efficiency. This study permitted to develop lipid nanoparticle suspensions entrapping inorganic UV filters enhancing their photo-protection efficiency. This work also highlighted the interest of these lipid nanoparticle suspensions to entrap an organic UV filter avoiding its percutaneous permeation while enhancing its photo-protection efficiency

Thesis (Ph. D.)--Ohio State University, 2005.
Title from first page of PDF file. Document formatted into pages; contains xvi, 110 p.; also includes graphics (some col.) Includes bibliographical references (p. 98-110). Available online via OhioLINK's ETD Center

Successful delivery of pharmaceuticals orally requires a firm understanding of how dosage forms behave during their passage through the gastrointestinal (GI) tract. In this study, the GI transit time and absorption of amphotericin B (AmB) solid lipid nanoparticles (SLN) were investigated in rats, using paracetamol (PAR) and sulphapyridine (SP) as indirect markers. A high encapsulation efficiency of 91.2% was obtained for the AmB SLNs. The SLNs were exhaustively characterised with regards to size, zeta potential (ZP), viscosity, density, migration propensity within agarose gel, in vitro drug release and morphology, to ensure similar disposition in the GI tract after simultaneous oral administration. Freeze-drying did not significantly alter the size or ZP of the AmB SLNs, and in vitro drug release from fresh and freeze-dried SLNs were identical. AmB, PAR and sulphasalazine (SSZ) (the latter being the prodrug of SP) were individually formulated into SLNs using beeswax and theobroma oil as the lipid matrix. The z-averages, polydispersity indices and ZPs of the SLNs ranged from 206.5-224.8 nm, 0.161-0.218 and |61.90|-|71.90| mV, respectively. Gel electrophoresis studies indicated a similar movement propensity among the three SLNs as their migration distances were identical (22.2-22.4 mm) within agarose gel. Scanning electron and atomic force microscopy studies revealed that all three SLNs were spherical in morphology and with similar surface characteristics. The SLNs were assessed for changes in size and surface charge on exposure to simulated GI fluids using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). On contact with the fluids, the particles had a slight increase in size due to ingress of the dissolution media. NTA results were found to be more beneficial than DLS as the latter was biased towards larger particles that were present possibly due to aggregation. After incubation in simulated gastric fluid followed by simulated intestinal fluid (mimicking gastric emptying), all the SLNs were found to be less than 350 nm in size and neutral in charge, which are optimal attributes for intestinal absorption. Time-of-flight secondary ion mass spectroscopic (ToF-SIMS) analyses revealed minimal drug amounts on the surfaces of the particles indicating that drug location was in the core of the SLNs. A developed and validated high-performance liquid chromatography (HPLC) method for simultaneous assay of the drugs in rat plasma using piroxicam as internal standard was found to be sensitive, accurate and precise, with drug recovery from plasma exceeding 92% in each case. A pilot GI transit study conducted in rats showed that the HPLC method was appropriate for the study. In the main study, the effects of food on the transit and absorption of the AmB SLNs were investigated. The presence of food slowed the transit of the SLNs in the GI tract. The gastric transit time of the AmB SLNs was estimated indirectly using PAR and was obtained as 1.71-2.25 hr. Caecal arrival time (CAT) of the AmB SLNs was estimated using SP detection in plasma as SSZ metabolism to produce SP occurs predominantly by the activity of colonic flora. In both fasted and fed states, CAT was 1.80-1.90 hr whereas transit time through the small intestine was 1.65-1.79 hr. A delayed rate of AmB absorption was observed in the fed state however, the extent of absorption was not affected by food. The percentage AmB absorption during the fasted state in the stomach, small intestine and colon were not significantly different from absorption within the respective regions in the fed state. In both states however, absorption was highest in the colon and appeared to be a summation of small intestinal absorption plus absorption proper within the colon. The study indicated that, AmB SLNs irrespective of food status were slowly but predominantly taken up via the lymphatic route and the small intestine was the most favourable site for their absorption. The data obtained indicate that it is possible to enhance the bioavailability of AmB through its incorporation into SLNs. Further enhancement of AmB bioavailability can be achieved through appropriate formulation interventions aimed at slowing transit of the SLNs in the small intestine. Finally, being a lipid-based system, the SLNs may have a potential to reduce the nephrotoxic effects of AmB.

Les vaccins représentent l’un des progrès majeurs de l’Histoire pour la santé publique, concrétisant notamment l’éradication de la variole en 1980. Les vaccins historiques, à base de pathogènes entiers atténués ou inactivés et donc très immunogènes ont été progressivement remplacés par des vaccins à sous-unités, beaucoup plus sûrs mais en contrepartie moins immunogènes. Des adjuvants tels que des vecteurs et des molécules immunostimulantes ont donc été incorporés dans les formulations vaccinales dans le but de générer des réponses immunitaires de grande amplitude. Cependant, les principaux adjuvants actuellement autorisés chez l’homme induisent exclusivement une réponse immunitaire humorale, à savoir la production d’anticorps permettant de neutraliser les pathogènes extracellulaires. Or, certains pathogènes comme le VIH requièrent une immunité cellulaire, indispensable à l’élimination du virus persistant dans les cellules infectées. Dans ce contexte, les adjuvants de vaccin sont en plein essor dans le but d’identifier de nouveaux candidats plus performants et sûrs. Nous décrivons ici la démarche suivie afin de proposer un vecteur lipidique nanoparticulaire (LNP), dont la stabilité, l’innocuité et la versatilité en font un outil idéal pour la délivrance d’antigènes. Nous avons dans un premier temps réalisé la preuve de concept sur la base de l’antigène modèle ovalbumine, dont la délivrance aux cellules immunitaires a permis d’augmenter significativement la réponse humorale in vivo chez la souris. D’autre part, l’induction d’une réponse cellulaire a été observée par la double délivrance de l’antigène et d’un immunostimulant. Plusieurs combinaisons et stratégies de vectorisations ont été évaluées, dans le but d’identifier la formulation la plus performante en vue d’une étude de protection anti-tumorale. Finalement, nous avons appliqué ces technologies au cas concret du VIH avec l’antigène de capside p24, ce qui s’est conclu par une étude d’immunogénicité chez le primate non-humain. L’ensemble de ces résultats met en lumière la versatilité des LNP et leur capacité à induire des réponses immunitaires de grande magnitude, à médiation humorale et cellulaire
The development of vaccines was one of the major health advances of the last century, with the success of smallpox eradication in 1980. Historical vaccines, based on attenuated or killed pathogens thus strongly immunogenic were finally replaced by subunit candidates, much safer but also poorly immunogenic. Therefore, adjuvants such as vectors and immunostimulants were incorporated in vaccine formulations in order to generate immune responses of high magnitude. However, actual adjuvants authorized in human vaccines only trigger humoral immune responses, with the production of antibodies which neutralize extracellular pathogens. Yet, some pathogens such as HIV require the induction of a cell-mediated immunity, necessary to eliminate viral reservoirs in infected cells. In this context, new adjuvant systems are being developed in order to identify the most efficient and safe candidates. Here we describe the approach followed to prepare a stable, safe and versatile vector consisting in lipid nanoparticles (LNP), for the delivery of antigens. We first report the proof of concept of antigen delivery based on the model ovalbumin, leading to the significant enhancement of humoral responses in vivo in mice. Thereafter, we focused on the induction of cell-mediated immune responses through the vectorization of both antigens and immunostimulants. Several combinations and vectorization strategies were assessed in the aim to identify the best prototype for a study of protection against tumor challenge. Finally, we applied these systems to HIV and its capsid antigen p24, which allowed us to conduct an immunogenicity study on a non-human primate model. Altogether, these results highlight the versatility of LNP and their ability to induce potent humoral and cell-mediated immune responses

Malaria affects millions of people annually especially in third world countries. Increase in resistance and limited research being conducted adds to the global burden of malaria. Mefloquine, known for unwanted adverse reactions and neurotoxicity, is highly lipophilic and is still used as treatment and prophylaxis. Lipid drug delivery systems are commonly used to increase solubility and efficacy and decrease toxicity. The most generally used lipid drug delivery system is liposomes. The lipid bilayer structure varying in size from 25 nm to 100 μm can entrap both hydrophilic and lipophilic compounds. Similar in structure and size to liposomes, Pheroid™ technology consist of natural fatty acids and is also able to entrap lipophilic and hydrophilic compounds. The aim of this study was to formulate liposomes and Pheroid™ vesicles loaded with mefloquine and evaluate the physiochemical characteristic of the formulations followed by efficacy and toxicity studies. Pheroid™ vesicles and liposomes with and without mefloquine were evaluated in size, morphology, pH and entrapment efficacy during three month accelerated stability testing. Optimization of size determination by flow cytometry lead to accurate determination of size for both Pheroid™ vesicles and liposomes. During the three months stability testing, Pheroid™ vesicles showed a small change in size from 3.07 ± 0.01 μm to approximately 3 μm for all three temperatures. Confocal laser scanning microscopic evaluation of the liposomes showed structures uniform in spherical shape and size. No difference in size or structure between the Pheroid™ vesicles with and without mefloquine were obtained. Significant increase (p=0.027) in size from 6.46 ± 0.01 μm to above 10 μm was observed for liposomes at all the temperatures. Clearly formed lipid bilayer structures were observed on micrographs. With the addition of mefloquine to the liposome formulation, a decrease in the amount of bilayer structures and an increase in oil droplets were found. Entrapment efficacy was determined by firstly separating the entrapped drug from the unentrapped drug utilizing a Sephadex®G50 mini column. This was followed by spectrophotometric evaluation by UV-spectrophotometry at 283 nm. Initial entrapment efficacy of both Pheroid™ vesicles and liposomes was above 60%. An increase in entrapment efficacy was observed for Pheroid™ vesicles. The addition of mefloquine to already formulated Pheroid™ vesicles illustrated entrapment efficacy of 60.14 ± 5.59% after 14 days. Formulations loaded with mefloquine resulted in lower pH values as well as a decrease in pH over time. Optimization of efficacy studies utilizing propidium iodide was necessary due to the similarity in size and shape of the drug delivery systems to erythrocytes. A gating strategy was successfully implemented for the determination of the percentage parasitemia. Efficacy testing of mefloquine loaded in Pheroid™ vesicles and liposomes showed a 186% and 207% decrease in parasitemia levels compared to the control of mefloquine. Toxicity studies conducted include haemolysis and ROS (reactive oxygen species) analysis on erythrocytes as well as cell viability on mouse neuroblastoma cells. Pheroid™ vesicles with and without mefloquine resulted in a dose dependent increase in ROS and haemolysis over time. A dose dependent increase in ROS and haemolysis in both liposome formulations were observed, but to a lesser extent. Mefloquine proved to be neurotoxic with similar results obtained when mefloquine was entrapped in liposomes. Pheroid™ vesicles seem to have neuroprotective properties resulting in higher cell viability. Mefloquine could be entrapped successfully in Pheroid™ vesicles and less in liposomes. Pheroid™ vesicles was more stable over a three months accelerated stability testing with more favourable characteristics. The increase in ROS levels of Pheroid™ vesicles could be responsible for the higher efficacy and haemolytic activity. DL-α-Tocopherol in Pheroid™ vesicles possibly acted as a pro-oxidant due to the presence of iron in the erythrocytes. DL-α-Tocopherol showed possible antioxidant properties in the neurotoxicity evaluation resulting in higher cell viability. Even though liposomes illustrated higher efficacy and little haemolysis and ROS production, no difference in neurotoxicity was observed together with unfavourable properties during stability testing makes this drug delivery system less favourable in comparison to Pheroid™ vesicles. Mefloquine was successfully incorporated into Pheroid™ vesicles resulted in high efficacy and showed possible neuroprotection and therefore makes it an ideal system for treatment of malaria.
Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2011

A number of therapeutic proteins are used for long in clinical practice. These include for example insulin, calcitonine, growth hormone, and parathyroid hormone for the treatment of various systemic disorders, as well as protein antigens in vaccine formulations. Due to the recent developments in biochemical engineering and in the comprehension of the physiopathology of many diseases, peptides and proteins are expected to become a drug class of increasing importance. Recently, novel biological drugs have for example been developed such as monoclonal antibodies, antibody fragments, soluble receptors, and receptor agonists or antagonists. These are mainly used for the treatment of auto-immune and inflammatory diseases (asthma, rheumatoid arthritis) and for the treatment of cancers. However, a major drawback of these biomolecules is the need to use parenteral administration. This is mainly due to the harsh pH conditions that proteins undergo by oral administration, leading to various physico-chemical degradations and loss of biological activity.

Pulmonary delivery of these proteins could constitute an alternative to parenteral delivery. Due to the very high surface area of the lungs, the low thickness of the alveolar epithelium and the high level of lung vascularisation, pulmonary administration can indeed provide fast systemic absorption of drugs, while avoiding hepatic first pass metabolism. On the other hand, drugs for local treatment can also be administered directly into the lung, which allows delivering high doses while limiting systemic side effects. Nevertheless, administration of drugs to the lungs requires some challenges to be taken up. It is indeed necessary to provide the drug as very small solid or liquid microparticles (1-5 µm) in order to reach the lungs. For solid microparticles, it is also needed to overcome the very high inter-particle interactions by using appropriate formulation strategies and by including deaggregation mechanisms in the inhalation device. Other issues are more specifically related to the pulmonary administration of proteins. These can indeed undergo physico-chemical degradations during processing, administration, and/or storage. Moreover, if systemic action is required, proteins will often need addition of an absorption enhancer to cross the alveolar epithelium because of their large molecular weight and hydrophilicity.

In this work, we developed formulations for pulmonary delivery of proteins using two model proteins. Insulin (5.8 kDa) was chosen as a model of small protein. It is also an application of systemic pulmonary delivery. On the other hand, an anti-IL13 monoclonal antibody fragment (54 kDa) was used as a model of larger protein. This molecule is currently in development for the treatment of asthma and provided an application for local pulmonary delivery. The formulation strategy was to produce dry powders using a combination of micronisation techniques (high speed and high pressure homogenisations), drying techniques (spray-drying, freeze-drying), and addition of lipid excipients. These lipid excipients were added as a coating around the protein particles and were expected to prevent protein degradations during processing and/or storage, essentially by avoiding contact with water. It could also improve the aerodynamic properties of the powders by modification of the surface properties of the particles and/or limitation of the capillary forces.

First, we evaluated insulin lipid-coated formulations and formulations without excipients, produced using high pressure homogenisation and spray-drying. In the case of lipid-coated formulations, a physiological lipid composition based on a mixture of cholesterol and phospholipids was used. We were able to obtain good aerodynamic features for the different formulations tested, with fine particle fractions between 46% and 63% versus 11% for raw insulin powder. These are high FPF values in comparison with those obtained for other protein formulations for inhalation currently under development, which often have an in vitro deposition of around 30%. Insulin presented a good stability in the dry state, even when no lipid coating was added.

The presence of a lipid coating of up to 30% (w/w) did not significantly improve the aerodynamic behaviour of the powders, but the coated formulations exhibited decreased residual moisture content after 3-month storage, which should be of interest for the long-term stability of the formulations.

In a second step, two of the developed insulin formulations were evaluated in a clinical study to determine whether the formulations give high deep lung deposition in vivo, and how insulin is absorbed into the systemic blood stream. This pharmaco-scintigraphic trial was performed on twelve type 1 diabetic patients using an uncoated formulation and a formulation coated with 20% (w/w) of lipids. The two formulations showed interesting features, with pharmacokinetic profiles that mimic the natural insulin secretion pattern. Bioavailability was within the ranges of two of the three dry powder insulins that have reached phase III clinical development. However, the formulation with a lipid coating exhibited a lower lung deposition in comparison with the uncoated formulation, which was not expected from the previous in vitro results. Additional in vitro experiments indicated that this lower performance was related to a decrease in the disaggregation efficiency of the powder at a sub-optimal inhalation flow-rate. An extensive training of the patients to the inhalation procedure could therefore improve the lung deposition of the coated formulation.

Finally, we developed and evaluated dry powder formulations of the anti-IL13 antibody fragment. These were produced using, successively, freeze-drying, high pressure homogenisation (HPH), and spray-drying. The influence of different types and concentrations of stabilising excipients was evaluated for each production step. Due to its more elaborated structure, the antibody fragment was found to be more sensitive than insulin to physico-chemical degradation, particularly during the HPH process, which led to different types of degradation products. These could partly be avoided by adding 50% sucrose during freeze-drying and 10% Na glycocholate or palmitic acid in the liquid phase during HPH (dispersing agents). However, the presence of a small fraction of insoluble aggregates could not be fully avoided. Further spray-drying of the suspensions in the presence of 10% Na glycocholate or palmitic acid led to the formation of a hydrophilic or hydrophobic coating around the particles, respectively. Na glycocholate was found to be particularly effective in protecting the antibody during spray-drying, which was found to be at least partly related to its ability to inhibit sucrose recrystallisation. However, the best formulation still presented a small fraction of insoluble aggregates (6%). The aerodynamic evaluation of the formulations showed FPFs that were compatible with lung deposition, with the formulation containing Na glycocholate presenting the highest FPF (42%). The formulation coated with palmitic acid presented a slightly lower FPF (35%). The aerodynamic properties of this formulation remained unchanged at a sub-optimal inspiratory flow rate, to the contrary of what was observed for the insulin formulation coated with 20% (w/w) cholesterol and phospholipids. Palmitic acid could therefore be of interest as a hydrophobic coating material, and provide long-term stability of protein drugs.

The work performed with the insulin and anti-IL13 molecules provided the proof-of-concept that it was possible to obtain dry powder protein formulations with appropriate aerodynamic properties and good overall physico-chemical stability, using simple production techniques and few selected excipients. The formulation strategy presented in this work could therefore be of interest for the future development of inhaled proteins for local or systemic applications.

Doctorat en sciences pharmaceutiques
info:eu-repo/semantics/nonPublished

Intravenous lipid emulsions are used ubiquitously through the medical field as a source of parenteral nutrition. Development of new formulations requires an understanding of the metabolism of the product. Current methods of rate determination and of metabolism analysis have several drawbacks. Radioactive labelled assays have lower biological relevance, are time consuming due to separation steps and require long substrate preparation. Existing fluorescence assays based around triglyceride hydrolysis are impractical in emulsion systems due to high signal-noise ratio as well as the use of non-specific fluorescent dyes. Colorimetric methods such as the non-esterified fatty acids (NEFA) assay is expensive, requires multiple steps and specialised machinery. Due to the limitations of these techniques we developed a novel fluorescent assay using a lipoprotein lipase specific substrate incorporated into lipid emulsions. The lipoprotein lipase substrate, EnzChek® Lipase Substrate, green fluorescent, 505/515, is based on a triglyceride structure with a fluorescent dye at the Sn1 position and a dark quencher at the Sn2 position. LPL cleaves preferentially at the Sn1 position of triglycerides, which separates the dye from the quencher creating a fluorescent signal. The signal can then be detected using a fluorescent plate reader. Both lipid emulsion particles and EnzChek are substrates for LPL, so the hydrolysis of EnzChek is analogous for native emulsions particles. Over time, in the presence of LPL, fluorescent signal increases as more EnzChek is hydrolysed in tandem with emulsion particles. We have designed a range of emulsions with varied oil and surfactant composition. Using the EnzChek emulsion assay detailed above we are able to follow the rate of metabolism in real-time. This assay has been tested and found to be robust and reproducible. It is able to investigate differences in metabolism between Soybean oil, medium-chain triglyceride and fish oil emulsions. As well as changes in surfactant type and concentration.

Hand hygiene is a major and fundamental topic in patient management to avoid health care-associated infections. According to the WHO alcohol-based hand rubs are the preferred means in clinical antisepsis. However, the frequent use of alcohol-based hand rubs can cause adverse events, which negatively impact user compliance, demonstrating a risk for patients. Adverse events are based on protein denaturation and stratum corneum lipid extraction. The aim of this work was to develop in vivo assays for lipid-processing enzymes in the stratum corneum as biomarkers to be routinely used in the evaluation of hand sanitiser formulations. Tape stripping is a well-established method to obtain stratum corneum samples. Fluorogenic substrates were used to develop selective enzymatic assays for phospholipase A1, A2, lipase, β-glucocerebrosidase, and sphingomyelinase activity. Consecutive tapes were removed to understand the activity – depth profiles of the enzymes. In addition, the established non-invasive analytical methods of transepidermal water loss and spectrophotometric intracutaneous analysis were combined with the enzymatic measurements in clinical studies. The characteristic activity – depth profiles of enzymes changed under induced skin barrier alterations by ethanol exposure, indicating compensatory and inflammatory responses. In human subjects, the formulation containing of 0.4% glycerol in addition to 70% ethanol resulted in a reduced transepidermal water loss and reduced enzyme activity. This was based on its hydrating properties of the skin. Niacinamide at 0.3% added an additional benefit on barrier integrity, increasing cohesion between corneocytes and reduced lipase activity. Glycine has hydrating properties and was able to reduce transepidermal water loss added to the 70% ethanol formulation. Glycine increased the activity of β-glucocerebrosidase in healthy skin and hence also offers an interesting new approach in hand sanitisers by potentially altering the Cl- flux in keratinocytes increasing lamellar body secretion and delivering higher amounts of intercellular lipids. The successfully developed assays demonstrate a minimally invasive method to identify the state of the skin barrier integrity in human subjects. This approach allowed the identification of the benefits of glycerol, niacinamide, and glycine in hand sanitisers, regarding the skin barrier integrity. Further excipients and compositions can be routinely studied with these methods in the future to optimise hand sanitiser formulations and consequently improve user compliance. However, the assays are not limited to the evaluation of hand sanitiser but the biomarkers can also be applied to study other formulations or active pharmaceutical ingredients as well as in the understanding, characterization, and monitoring of disease states of the skin.

Ce travail de thèse s’articule autour de la génération et de la caractérisation de capsules d’organo-hydrogel ou simplement bi-gels, obtenues par la technologie du prilling et destinées à la délivrance contrôlée d’un principe actif (P.A), après administration par voie orale. L’Efavirenz (EFV), un antirétroviral utilisé dans le traitement du VIH/Sida, a été le P.A utilisé comme modèle de molécule de faible solubilité dans l’eau. Il a été dissous dans l’organogel composé d’huile de tournesol et d’acide 12-hydroxystéarique (12-HSA). L’organogel a été caractérisé par sa température de transition de phase sol-gel-sol. Et la thermoréversibilité caractéristique de cet organogel n’a pas changé avec l’introduction de l’EFV. Pour la caractérisation des organogels comme véhicule de P.A, ils ont été produits à deux températures (5°C et 25 °C) et avec deux concentrations de 12-HSA (5% et 20%). Pour les essais de libération de l’EFV deux milieux de dissolution ont été utilisés (pH 1,2 et 6,8) avec et sans enzymes. Le profil de libération de l’EFV à partir de capsules bi-gels (2500 à3000 μm de diamètre) est essentiellement lié à la quantité d’organogélifiant dans le coeur, à la présence de la membrane d’alginate et à l’état physique de cette membrane (hydratée ou sèche). Une réduction de 50% a pu être observée à pH acide en présence de la membrane externe. A pH acide la libération est plus lente qu’à pH 6,8, quand la membrane perd sa fonction protectrice et le coeur organogel passe à régler la libération. Deux mécanismes de relargage sont observés : l’érosion et la diffusion, expliqués par le modèle de Korsmeyer-Peppas
This thesis focuses on the generation and characterization of organo-hydrogel capsules (bigels), manufactured by prilling technology for controlled drug delivery after oral administration. Efavirenz (EFV), an antiretroviral medications used to treat HIV/AIDS, is the active pharmaceutical ingredient (API) used as a model molecule of low solubility in water. It was dissolved in the organogel, which is compound of sunflower oil and 12-hydroxystearic acid (12-HSA). The organogel was characterized by its phase transition temperature sol-gel-sol. The typical thermoreversibility of this organogel has not changed with introduction of EFV. The organogels were produced at two temperatures (5 °C and 25 °C) and with two concentrations of 12-HSA (5% and 20%) for being characterized as an API vehicle. Two dissolution media were used with and without enzymes (pH 1.2 and 6.8), for EFV release quantification. The EFV release profile from bi-gels capsules (diameter from 2500 to 3000 μm) is essentially related to the amount of organogelator in their core, to the presence of the alginate membrane and to the state physics of this membrane (hydrated or dry). The release of EFV has reduced 50% at acid pH in the presence of the external membrane. In simulated gastric fluid, the release is slower than at pH 6.8 (simulated intestinal fluid). In the intestine, the membrane loses its protective function and the organogel’s core begins to control the release of EFV. Two release mechanisms are observed: erosion and diffusion, which can be explained by the Korsmeyer-Peppas model

Ces travaux de thèse ont porté sur la formulation de gels muco-adhésifs piégeant des nanocapsules lipidiques (LNCs) de curcumine. L’objectif est d’améliorer la biodisponibilité de la curcumine en la solubilisant au sein des LNCs piégées dans un gel, utilisées pour augmenter l’absorption intestinale. La première étape du travail a permis d’identifier le Transcutol®HP et le Kolliphor®HS15 comme excipients compatibles avec la préparation des LNCs tout en assurant une solubilisation suffisante de la curcumine. Deux formulations de LNCs de curcumine ont été proposées : l’une avec le Transcutol®HP/Labrafac®WL1349/Labrafil® M1944CS et l’autre avec le Captex®8000 en tant que constituants du cœur des particules. La formulation avec le Transcutol®HP a été optimisée pour obtenir des particules de 63nm avec une distribution étroite (PdI=0.17) et un rendement d’encapsulation de 92%. Avec le Captex®8000 des particules de 57nm avec un PdI<0.1 ont été obtenues. En revanche, pour les deux formulations, l’étape de purification constitue une étape critique non résolue. Un gel d’alginate de sodium, de géométrie cylindrique compatible avec une insertion dans des gélules, a également été développé. La capacité des gels à absorber l’eau dans différents milieux biomimétiques a été suivie dans le temps in vitro. Une différence de comportement entre les gels lyophilisés et les gels non lyophilisés a été mise en évidence. Enfin, l’encapsulation de deux fluorochromes dans les LNCs a permis de suivre l’intégrité des LNCs lors de la formulation des gels par la technique de transfert d'énergie par résonance de type Förster (FRET). Les LNCs restent intactes pendant l’étape de gélification et la capacité du gel à piéger les LNCs a été démontrée
The aim of this phD work was to develop a muco-adhesive gel entrapping curcumin loaded lipid nanocapsules (LNCs). The objective was to improve the bioavailability of curcumin by its solubilization into LNCs entrapped into a gel to increase intestinal absorption. Firstly, Transcutol@HP and Kolliphor@ HS15 were identified as suitable excipients to formulate LNCs With a sufficient solubility of curcumin. Two formulations of curcumin LNCs were M1944CS or Captex@8000, which form the core of particles. The TranscutoPHP formulation was optimized to obtain 63nm particles With a narrow distribution (Pdl=0.17) and 920/0 of encapsulation efficiency. Particles With a mean diameter of 57nm and Pdl

Platinum-refractory ovarian cancer is considered an incurable disease as current treatments are only palliative. Improvements in treatment will be realized as our understanding of the unique signaling pathways driving disease development increases and new therapeutics targeting these pathways are developed. It’s important to recognize, however, that at this time new molecularly targeted agents are not replacing the drugs being used to treat cancer; they are being used in combination with existing standards of care. In light of this, it is important to explore novel approaches using existing agents that are designed to achieve maximum therapeutic benefits in dosage forms that are well tolerated. Like most cancers, ovarian cancer is treated with a combination of drugs selected on the basis of complementary mechanisms of action and non-overlapping toxicities. Synergistic drug combinations can achieve therapeutic effects equal to that achieved with single agents, but at significantly lower and better tolerated doses. The factors that govern synergistic drug:drug interaction are, however, poorly understood and it is argued in this thesis that drug interactions favoring synergy will be influenced by drug exposure time. An effective method to enhance drug exposure time involves the use of drug carriers and a goal of this thesis was to develop an effective combination regimen against recurrent ovarian cancer using a novel lipid nanoparticle (LNP) formulation of topotecan and Doxil®; a LNP formulation of doxorubicin that has already been approved for the treatment of relapsed ovarian cancer. The LNP formulation of topotecan developed through this thesis research, referred to as TopophoreC™, was 2-to-3-fold more toxic than free topotecan, however this product candidate showed significantly better anti-tumor activity when compared to free topotecan administered at equivalent doses. Combinations of this LNP topotecan formulation with Doxil® were therapeutically superior to combinations of free topotecan and Doxil® as judged in two models of ovarian cancer. On the basis of these studies, it can be concluded that interaction between TopophoreC™ and Doxil® affect the pharmacokinetic behavior of Doxil® however the results provide proof of concept data to support the use of TopophoreC™ and Doxil® combination for treatment of recurrent ovarian cancer.

Ces travaux visent à déterminer l‘intérêt de deux types de nanosystèmes (NS) coeur-couronne dans des applications dermatologiques ou cosmétiques. Les nanocapsules lipidiques (LNC) sont obtenues par une méthode déjà décrite dans la littérature. Leur formule est modifiée pour incorporer un actif cosmétique d‘intérêt. Les nanocapsules d‘alginate (ANC) sont développées au moyen de plans d‘expériences. Elles sont composées d‘un coeur huileux et d‘une coque d‘alginate de calcium gélifiée obtenue par gélification ionique de surface d‘une nanoémulsion. Des méthodes basées sur le phénomène de fluorescence nous permettent de mettre en évidence l‘endocytose des ANC par les kératinocytes. Leur contenu est rapidement libéré dans le cytoplasme. Une étude sur différents modèles ex vivo montre que les deux nanosystèmes permettent aux molécules encapsulées d‘atteindre les couches vivantes de l‘épiderme. ANC et LNC sont stables plusieurs mois dispersées dans des formes galéniques semi-solides. Ces deux NS sont donc adaptés à la délivrance de molécules actives dans la peau
Two types of core-shell nanosystems have been evaluated for dermatological and cosmetic applications. Lipid nanocapsules (LNC) are obtained by a method that has already been described in the literature. Their composition is adapted for incorporation of a specific cosmetic ingredient. Alginate nanocapsules (ANC) are developed with the aid of experimental design. They consist of a triglyceride core with a rigid calcium alginate shell obtained by ionic gelation of the surface of a nanoemulsion. By incorporating fluorophores into these nanosystems, they can be studied by advanced spectral fluorescence imaging methods. We were thus able to show that ANC are first internalized into keratinocytes by endocytosis, and once inside the cells, their contents are rapidly released into the cytoplasm. A study of different ex vivo skin model systems has shown that both nanosystems enable active substances to reach the living epidermis. When incorporated into gels similar to those used as galenic forms for topical administration, LNC and ANC remain stable for months. They can thus be used as vectors for delivering active substances to the skin

Local delivery of medication to the lung is highly desirable as the principal advantages include reduced systemic side effects and higher dose levels of the applicable medication at the site of drug action. This administration could be particularly useful for patients with specifically chronic pulmonary infections or pulmonary diseases, such as cystic fibrosis, asthma or lung cancer.

In order to deliver a high dose range of medication for highly-dosed drugs such as antibiotics, “carrier-free” DPI formulations of tobramycin were developed with the aim of minimizing the use of excipients. Briefly, dry powders were prepared by spray drying various suspensions of tobramycin in isopropanol.

First, as particle size is a key parameter in defining drug deposition in the lungs, the new Spraytec® laser diffraction method specifically modified for measuring the PSD of aerosolized drug was evaluated. The dispersion properties of various dry powder formulations were investigated using different laser diffraction and impaction apparatuses at different flow rates and using different inhalator devices. Different correlations between geometric and aerodynamic size data were demonstrated in this study. As a potential application, for the flow rate, the different inhalation devices and the drug formulations examined, the tobramycin fine particle fraction could be predicted from measurements obtained from the Spraytec® using linear relationships. Correlations (R² > 0.9) between the MMAD and the percentage of particles with a diameter below 5 µm could be demonstrated between the results obtained from the laser diffraction technique and the impaction method. Consequently, the Spraytec® laser diffraction technique was proved to be an important tool for initial formulation and process screening during formulation development of DPIs.

In order to modify the surface properties of the raw tobramycin powder, different powder compositions were formulated with the aim of studying the influence of the concentration of tobramycin in drug suspensions used for spray-drying, the lipid film composition (cholesterol:Phospholipon ratio) and the coating level (in percentage) on the physicochemical and aerodynamic characteristics of the antibiotic.

The results indicated that the application of a lipid coating around the active particles allowed an improvement in particle dispersion from the inhalator, decreasing raw powder agglomeration and thus enhancing drug deposition deep in the lungs. Moreover, these results seemed to be influenced by the amount and composition of the lipids in the formulations. The evaluation of the influence of the coating level showed that the deposition of only 5% w/w lipids (on a dry basis) was sufficient to improve particle dispersion properties during inhalation. The FPF, which is around 36% for the uncoated micronized tobramycin, was increased to up to about 68% for the most effective lipid-coated formulation. Of particular importance, these results revealed the need to add sufficient amounts of covering material in order to significantly modify the particle surface properties and reduce their tendency to agglomeration, while limiting the lipid level in the formulations in order to avoid any undesirable sticking and to allow the delivery of more of the active drug to the deep lung.

Another approach used to modify the surface properties of raw tobramycin was to coat the micronized particles with nanoparticles of the drug, produced by high pressure homogenization. The evaluation of the influence of the level of nanoparticle coating of the micronized particles showed that the presence of nanoparticles in the formulations improved the particle dispersion properties during inhalation. One microparticle was completely covered with a single layer or several layers of nanoparticles, in function of the percentage of nanoparticles in the mixture. Coating the fine drug particles with particles in the nanometer range was believed to reduce Van Der Waals forces and powder agglomeration. These various layers of nanoparticles also allowed a decrease in the cohesion of the powder by improving the slip between the particles.

On the other hand, suspensions containing solely nanoparticles were spray dried with various concentrations of surfactant in order to produce easily dispersible and reproducible micron-size agglomerates of nanoparticles during inhalation. The evaluation of the influence of the concentration of surfactant showed that deposition of only 2% w/w (on a dry basis) of Na glycocholate is sufficient to improve particle dispersion properties during inhalation. Consequently, the use of nanoparticles in dry powder formulations increased the FPF from 36% for the uncoated micronized tobramycin to about 61% for this latter formulation.

To modify the balance between the different forces of interactions without the need for any excipient, the influence of formulation components on the aerosolization characteristics of spray-dried tobramycin through the use of various proportions of water in the solvent used to prepare initial suspensions was investigated. These results showed that it is possible to modify the surface properties of the particles by coating the particles of drug with a homogeneously distributed film of the active compound dissolved in a solvent system containing a mixture of different solvents such as isopropanol and water. During nebulization of the suspension, droplets are composed of one or more particles in solid state surrounded with solvent containing the dissolved drug. It is hypothesized that during the drying step, dissolved tobramycin forms a coating of the amorphous drug around particles in suspension. The coating of drug particles can thus be used as an alternative approach that permits the modification of the surface properties of the particles, increasing the flowability, the desagglomeration tendency and the fine particle fraction deposited in the deep lung. So, the evaluation of the influence of the water content of the suspensions and the effect of the inlet temperature during spray-drying showed that the addition of 2% water v/v is sufficient to improve particle dispersion during inhalation. Of particular interest, as tobramycin is a very hygroscopic drug, the addition of water turned out to be a critical step. It was thus important to add a small amount of water to the solvent system and to process the drying step at a high temperature to produce formulations containing solely the active drug and showing a FPF of up to 50%.

Moreover, stability studies demonstrated that these optimized formulations (lipid-coated formulation, nanoparticle formulation and amorphous drug-coated formulation) were stable over a long time period at various ICH temperature and relative humidity storage conditions (25°C/60% RH, 30°C/65% RH and 40°C/75% RH). The formulations were shown to keep their crystalline state, initial PSD, redispersion characteristics and deposition results for more than twelve months.

In order to confirm these encouraging results, two optimized formulations (one with a lipid coating and another with amorphous drug coating) were selected and compared to the only commercially available tobramycin formulation for inhalation, Tobi® (nebulizer solution), by performing a combined in vivo scintigraphic and pharmacokinetic evaluation of tobramycin DPIs in nine CF patients.

In comparison with Tobi®, it was estimated that lung deposition, expressed as a percentage of the nominal dose, was 7.0 and 4.5 times higher for the lipid-coated and amorphous tobramycin-coated formulations, respectively. Moreover, the pharmacokinetic data, adjusted to the same drug dose as that of the Tobi® deposited in the lungs, showed that the AUC values were found to be 1.6 times higher for Tobi® than for DPI formulations. So this evaluation confirmed the superiority of dry powder formulations in terms of drug deposition and reduced systemic exposure in comparison with the conventional comparator product, Tobi®.

Thus, these new and orginal tobramycin DPI formulations based on the use of very low excipient levels and presenting very high lung deposition properties, were shown to offer very good prospects for improving the delivery of drugs to the pulmonary tract and to the widest possible patient population.

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

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

Les peptides thérapeutiques peuvent traiter de nombreuses pathologies de manière efficace et sélective. Leur biodisponibilité orale est limitée par une forte dégradation liée à l’action de protéases dans le lumen intestinal et par une faible absorption par la barrière intestinale. Nous avons évalué la capacité des nanoparticules lipidiques solides (SLN) et des vecteurs lipidiques nanostructurés (NLC) à augmenter la biodisponibilité orale d’un peptide modèle : le Leuprolide (LEU). L’augmentation de sa lipophilie par formation d’une paire d’ions hydrophobe (HIP) avec le docusate permet d’augmenter significativement le taux d’encapsulation du LEU dans les nanovecteurs. Ceux-ci sont obtenus par homogénéisation haute pression avec une taille de 120 nm et une structure en plaquettes. Malgré leur stabilité dans les milieux gastro-intestinaux simulés, une libération importante de l’actif est observée dans le milieu intestinal à jeun. Vis-à-vis de la dégradation protéolytique, les NLC montrent une protection significative du LEU en présence de trypsine. L’évaluation du passage intestinal sur des monocouches de Caco-2 (modèle entérocyte) et de Caco-2/HT29-MTX (modèle sécrétant des mucines) révèle une internalisation des nanovecteurs mais aucune amélioration de l’absorption. En effet, la morphologie en plaquettes des SLN et NLC associée à la faible stabilité de l’HIP dans le milieu provoquent une libération importante du LEU, annulant la capacité de transport des nanovecteurs à travers la barrière intestinale. Il convient d’améliorer la stabilité de la hydrophobisation pour augmenter la biodisponibilité orale des peptides via l’encapsulation dans des nanovecteurs lipidiques solides
Therapeutic peptides are able to treat a wide variety of diseases with selective and potent action. Their oral bioavailability is strongly limited by an important proteolytic activity in the intestinal lumen and poor permeation across the intestinal border. We have evaluated the capacity of Solid Lipid Nanoparticles (SLN) and Nanostructured Lipid carriers (NLC) to overcome both oral bioavailability limiting aspects, using Leuprolide (LEU) as model peptide. Lipidization of LEU by formation of a Hydrophobic Ion Pair (HIP) with docusate enables a significant increase of peptide encapsulation efficiency in both SLN and NLC. The nanocarriers, obtained by high pressure homogenization, measured 120 nm and were stable in simulated gastro-intestinal fluids. However, due to particles platelet-shape, an important quantity of LEU is released in simulated fasted state intestinal fluid. Regarding the protective effect towards proteolytic degradation, only NLC maintain LEU integrity in presence of trypsin. Intestinal transport, evaluated on Caco-2 (enterocyte-like model) and Caco-2/HT29-MTX (mucin-secreting model) monolayers, show nanocarriers internalization by enterocytes but no improvement of LEU permeability. Indeed, the combination of nanoparticles platelet-shape with the poor stability of the HIP in the transport medium induces a high burst release of the peptide, limiting nanoparticles capacity to transport LEU across the intestinal border. Stability of peptide lipidization needs to be improved to withstand biorelevant medium to benefit from the advantages of encapsulation in solid lipid nanocarriers and consequently improve their oral bioavailability

Les Lactones Macrocycliques (LMs) sont des antiparasitaires largement utilisé en médecine vétérinaire contre les endo et les ectoparasites. Nos travaux révèlent pour la première fois l'implication des lipoprotéines dans le transport plasmatique des 5 différentes lactones (Ivermectine, Doramectine, Moxidectine, Abamectine, prinomectine) chez les différentes espèces animales cibles y compris l'homme. De plus nous avons pu mettre en évidence l'impact des perturbations du profil plasmatique en lipides et lipoprotéines sur la pharmacocinétique et la distribution plasmatique des LMs. Au même titre nous avons démontré le rôle crucial de la circulation lymphatique dans l'absorption intestinale de la moxidectine ce que nous a permis d'optimiser leur biodisponibilité suite à une coadministration des lipides par voie orale. De même la mise en oeuvre d'une formulation liposomale de l'ivermectine a produit une biodisponibilité convenable toutefois une évaluation de la distribution tissulaire et de l'efficacité de ces formulations prometteuses s'avérera nécessaire. En conclusion nos travaux suggèrent que les lipides et les lipoprotéines plasmatiques jouent un rôle prépondérant dans le transport et le devenir des LMs dans l'organisme hôte
Macrocyclic Lactones (MLs) are potent anthelmintic drugs widely used for control of both internal and external parasites in domestic animals and livestock. In a first step our work shows that the five MLs tested (Ivermectin, Doramectin, Moxidectin, Abamectin, Eprinomectin) were extensively distributed into plasma lipoproteins with a preferential association to HDL in both animal species including humans. In the second step we have reported the effect of dyslipidemias on the plasma distribution and pharmacokinetic of moxidectin in human and rabbit model. On the other hand our investigation clearly indicated the major contribution of lymphatic circulation in the process of intestinal absorption of moxidectin and subsequently to its systemic bioavailability. Our findings could allow the enhancement of oral MLs bioavailability by using lipid-based ormulation. Finally our data demonstrated that the use of liposomal formulation represents promising tool by improving the bioavailability and the efficacy of ivermectin and related drugs. Furthermore, in vivo and in vitro investigations are needed to demonstrate the ability of liposome to improve the ivermectin efficacy. In conclusion our works suggest that the lipids and lipoproteins play a primordial role in the transport and disposition of MLs in the host's organism

Le purinorécepteur P2X7 joue un rôle majeur dans les phénomènes de dégénérescences (Alzheimer, DMLA) et mort cellulaires. Récemment des études ont montré que l’activation basale de ce récepteur est indispensable dans le processus de cicatrisation et de prolifération cellulaire. Nous avons essayé de mieux comprendre le paradoxe de cette activation du récepteur P2X7 qui oriente vers la prolifération et même les métastases tumorales, mais aussi vers les mécanismes de dégénérescence cellulaire. L'influence du microenvironnement (Lipides, matrice extracellulaire, oxygène) apparait essentielle pour comprendre ces différents effets. Notre premier objectif a été d’étudier l’impact de la modulation du microenvironnement du récepteur P2X7 par des composants de la matrice extracellulaire. Ainsi, sur un modèle de cicatrisation cutanée, nous avons mis en évidence l’impact de la taille des fragments du hyaluronate de sodium (composant prédominant dans la matrice extracellulaire). Nos résultats ont montré que l’activation du récepteur P2X7 dépend du poids moléculaire du hyaluronate de sodium. Notre deuxième objectif a été de moduler le microenvironnement lipidique du récepteur P2X7. Nous avons sélectionné une huile riche en acides gras insaturés et avons ainsi étudié son effet sur l’activation du récepteur P2X7. Sur nos modèles de cicatrisation, nous avons mis en évidence qu’une modulation du microenvironnement lipidique du récepteur P2X7 influence son activation. D’autre part la modulation du microenvironnement lipidique sur des cellules tumorale de mélanome active les voies de dégénérescence ouvrant la perspective de nouvelles stratégies thérapeutiques
The purinoceptor P2X7 plays a role in cytotoxic degenerative processus (Alzheimer, AMD) and cell death. Recent studies have shown that basal activation of this receptor is essential in the healing process and cell proliferation. We tried to understand the paradox that activation of P2X7 receptor that directs to the same proliferation and tumor metastasis, but also to the mechanisms of cell degeneration. The influence of the microenvironment (lipids, extracellular matrix, oxygen) appears essential to understand these effects. Our first objective was to study the impact of the modulation of P2X7 receptor microenvironment by extracellular matrix components. On cell monolayer model of wound healing we have highlighted the impact hyaluronan molecular weight (predominant component in the extracellular matrix). Our results showed that activation of P2X7 receptor is dependent on hyaluronan molecular weight. Our second objective was to modulate lipid microenvironment P2X7 receptor. We selected an oil rich in unsaturated fatty acids and thus have studied its effect on the activation of P2X7 receptor. In our models of healing and melanoma cell, we have demonstrated that modulation of lipid microenvironment affects P2X7 activation

Le déficit en transporteur de la créatine est une maladie rare neurologique dans laquelle la perte de fonctionnalité du transporteur de la créatine (SLC6A8) conduit à une absence de créatine au niveau cérébral et à des retards de développement majeurs chez les enfants. A l'heure actuelle, aucune thérapie efficace n'est disponible.Une approche thérapeutique potentielle est le développement de molécules prodrogues de la créatine plus lipophiles qui franchiront les membranes cellulaires de façon passive et la recherche d'une formulation galénique susceptible d'emmener la prodrogue vers les cellules cibles d'intérêt, les neurones. Ainsi, dans cette thèse, nous proposons une nouvelle voie de synthèse originale d'esters de la créatine à longue chaîne aliphatique. Ces composés présentent des propriétés pharmacologiques intéressantes : nous montrons qu'il existe une relation de structure-activité entre la taille de la chaîne aliphatique (et donc la lipophilie) et la capacité de la molécule à être internalisée dans les cellules endothéliales cérébrales, astrocytaires et neuronales, constituant l'unité neurovasculaire. Il ressort de nos observations expérimentales que l'ester dodécylique de créatine est le meilleur candidat médicament. De plus, après avoir été internalisé dans les fibroblastes des patients présentant un déficit fonctionnel du transporteur de la créatine, l'ester dodécylique subit une conversion par les estérases cellulaires, libérant ainsi la créatine dans le compartiment intracellulaire.La formulation galénique permettant de protéger ces esters de créatine jusqu'au cerveau repose, elle, sur la nanovectorisation, par encapsulation de l'ester dodécylique de créatine dans des NanoCapsules Lipidiques. L'avantage de cette formulation est de permettre également un ciblage actif vers la Barrière Hémato-Encéphalique, obstacle majeur dans le développement de thérapies ciblant le Système Nerveux Central. Nos observations expérimentales mettent en exergue cette double stratégie thérapeutique pour le traitement du déficit en transporteur de la créatine.Ce travail a été soutenu financièrement par la Fondation Lejeune.

La rectocolite hémorragique (RCH) fait partie des Maladies Inflammatoires Chroniques de l’Intestin (MICI) et reste à ce jour incurable. Au-delà de l’administration d’anti-inflammatoires, dont la curcumine (CC) en présente les effets, une des voies à explorer est la réparation de la muqueuse intestinale par un apport exogène de phosphatidylcholine (PC) et de lysophophatidylcholine, déficitaires à plus de 70% chez les patients atteints de RCH. Pour répondre à cette problématique, nous nous sommes intéressés à la conception de systèmes lipidiques pour la voie orale permettant le transport simultané de CC et de PC. Nous avons exploré dans un premier temps la nature des interactions mises en jeu entre les deux principes actifs au sein de deux systèmes membranaires modèles, les bicouches liposomales et les monocouches (ou films de Langmuir). Il en ressort que l’équilibre céto/énolique de la curcumine et donc l’interaction avec la PC soit influencée par la concentration et le confinement. D’autre part, l’ajout d’un céride, le palmitate de cétyle, complexant la curcumine favoriserait, pour certaines compositions, la miscibilité avec la PC tout en rigidifiant le mélange ainsi obtenu. Sur la base de ces résultats, nous avons réalisé des suspensions de particules solides lipidiques, tout en variant la méthodologie de formulation, diffusion-évaporation de solvant vs double émulsion et la nature du stabilisant, émulsifiants vs nanoparticules. La suspension obtenue par double émulsion s’avère être la plus efficace en termes d’encapsulation, de protection en milieu gastrique simulé et de libération prolongée des principes actifs dans un milieu intestinal simulé. Enfin, la capacité des nanoparticules solides à stabiliser l’interface solide/liquide des SLN, semblable à l’effet de Pickering, a aussi été démontrée. L’ensemble de cette étude est une preuve de concept quant au potentiel des suspensions de nanoparticules lipidiques solides pour l’administration orale de curcumine et de phosphatidylcholine exogène
Ulcerative colitis (UC) is an Inflammatory Bowel Disease (IBD) and it is still nowadays incurable. Beyond the administration of anti-inflammatory drugs, including curcumin (CC), one of the pathways to be explored is the repair of the intestinal mucosa by an exogenous supply of phosphatidylcholine (PC) and lysophophatidylcholine, which are over 70% deficient in patients with RH. To address this issue, we were interested in the design of lipid systems for the oral delivery and allowing the simultaneous carry of CC and PC. We first explored the nature of the interactions involved between the two active ingredients within two model membrane systems, liposomal bilayers and monolayers (or Langmuir films). It turns out that the keto/enol equilibrium of curcumin and therefore the interaction with PC is influenced by both concentration and confinement. On the other hand, the addition of a ceride, cetyl palmitate, complexing curcumin allows, for some compositions, the miscibility with PC while stiffening the as obtained mixture. Based on these results, we formulated various dispersions of solid lipid particles, by varying the methodology of colloidal engineering, diffusion-solvent evaporation vs double emulsion and the nature of the stabilizer, emulsifiers vs nanoparticles. The suspension obtained by the double emulsion method appears as the most efficient in terms of encapsulation, protection in simulated gastric medium and sustained release of the active ingredients in a simulated intestinal medium. Finally, the ability of solid nanoparticles to stabilize the solid / liquid interface of SLNs, similar to the Pickering effect, has also been demonstrated. The entire study is a proof of concept regarding the potential of solid lipid nanoparticle suspensions for the oral administration of curcumin and exogenous phosphatidylcholine

Tactile perception is considered an important contributor to the overall consumer experience of a product. However, what physical properties that create the specifics of tactile perception, are still not completely understood. This thesis has researched how many dimensions that are required to differentiate the surfaces perceptually, and then tried to explain these dimensions in terms of physical properties, by interconnecting human perception measurements with various physical measurements. The tactile perception was assessed by multidimensional scaling or magnitude estimation, in which methods human participants assign numbers to how similar pairs of surfaces are perceived or to the relative quantity of a specified perceptual attribute, such as softness, smoothness, coarseness and coolness. The role of friction and surface texture in tactile perception was investigated in particular detail, because typically tactile exploration involves moving (at least) one finger over a textured surface. A tactile approach for measuring friction was developed by means of moving a finger over the surfaces, mounted on a force sensor. The contribution of finger friction to tactile perception was investigated for surfaces of printing papers and tissue papers, as well as for model surfaces with controlled topography. The overarching research goal of this thesis was to study, systematically, the role of texture in tactile perception of surfaces. The model surfaces displayed a sinusoidal texture with a characteristic wavelength and amplitude, fabricated by surface wrinkling and replica molding techniques. A library of surfaces was manufactured, ranging in wavelengths from 270 nm up to 100 µm and in amplitudes from 7 nm up to 6 µm. These surfaces were rigid and cleanable and could therefore be reused among the participants. To my knowledge, this is the first time in a psychophysical experiment, that the surface texture has been controlled over several orders of magnitude in length scale, without simultaneously changing other material properties of the stimuli. The finger friction coefficient was found to decrease with increasing aspect ratio (amplitude/wavelength) of the model surfaces and also with increasing average surface roughness of the printing papers. Analytical modeling of the finger’s interaction with the model surfaces shows how the friction coefficient increases with the real contact area, and that the friction mechanism is the same on both the nanoscale and microscale. The same interaction mechanism also explains the friction characteristics of tissue paper. Furthermore, it was found that the perceptions of smoothness, coarseness, coolness and dryness are satisfactorily related to the real contact area at the finger-surface interface.  It is shown that it is possible to discern perceptually among both printing papers and tissue papers, and this differentiation is based on either two or three underlying dimensions. Rough/smooth and thin/thick were the two main dimensions of surface feel found for the printing papers, whereas friction and wavelength were strongly related to the perceptual cues employed in scaling the model surfaces. These experimental results support the duplex theory of texture perception, which holds that both a “spatial sense”; used to discriminate the roughest textures from the others, and a “vibration sense”; used to discriminate among the smoother textures, are involved. The perception of what is considered rough and smooth depends on the experimental stimulus context. It is concluded that friction is important for human differentiation of surface textures below about 10 µm in surface roughness, and for larger surface textures, friction is less important or can even be neglected. The finger friction experiments also allowed the following conclusions to be drawn: (i) The interindividual variation in friction coefficients is too large to allow direct comparison; however, the trends in relative friction coefficients for a group of participants are the same. (ii) Lipids are transferred to the test surface of study, and this lowers the friction. (iii) Many of the studies point to a characteristic frequency during sliding of about 30 Hz, which is both characteristic of the resonance frequency of skin and the expected frequency associated with the fingerprints. (iv) The applied load in surface interrogation is in fact regulated in response to the friction force. The limits in tactile perception were indirectly researched by similarity scaling experiments on the model surfaces. Wrinkle wavelengths of 760 nm and 870 nm could be discriminated from untextured reference surfaces, whereas 270 nm could not. The amplitude of the wrinkles so discriminated was approximately 10 nm, suggesting that nanotechnology may well have a role to play in haptics and tactile perception.
Taktil perception bidrar starkt till den sammantagna upplevelsen av en produkt, men hur materials olika ytegenskaper påverkar och styr perceptionen är ännu inte helt klart. Den här avhandlingen undersöker hur många och vilka egenskaper som är viktiga när känslan mellan två ytor jämförs. Tillvägagångssättet är tvärvetenskapligt där fysikaliska mätningar kopplas ihop med perceptions mätningar där människor används som instrument. Två typer av perceptionsförsök har utförts, multidimensionell skalning där försökspersoner sätter siffror på hur lika två ytor känns, samt magnitud estimation där i stället intensiteten på specifika perceptuella storheter som t.ex. upplevt lenhet, upplevd mjukhet och upplevd strävhet bedömdes. Eftersom taktil perception innebär kontakt samt relativ rörelse mellan hud och ytor, har fokus i avhandlingen varit att undersöka hur friktion och ytans struktur (ytråhet) påverkar och bidrar till den taktila perceptionen. Förutom fysikaliska mätningar på friktion och ytstruktur har värmekonduktivitet, mjukhet samt olika standard mätningar inom pappersindustrin mätts. En metod för att mäta friktion mellan ett finger och olika ytor har utvecklats för att i möjligaste mån återspegla friktionskomponenten i upplevt taktil perception. Friktionskoefficienter beräknades och jämfördes mellan alla ytor. De stimuli som har studerats är tryckpapper och mjukpapper samt modellytor, gjorda för att systematiskt undersöka hur ytstruktur påverkar perceptionen. Tillverkningsmetoden för modellytorna valdes så att ytorna var tåliga och kunde tvättas och därmed återanvändas. Strukturen på ytorna bestod av ett vågformat mönster där våglängden varierade mellan 270 nm och 100 µm och amplituden mellan 7 nm och 6 µm. Enligt vår vetskap är det första gången som strukturer i de här skalorna har gjorts utan att samtidigt ändra andra material egenskaper. Friktionskoefficienten minskade med ökad kvot mellan amplituden och våglängden på modellytorna samt med ytråheten på tryckpappren. En analytisk modell tillämpades på kontakten mellan ett finger och ytorna som visade att friktionskoefficienten beror av den verkliga kontaktarean. För de mycket grövre mjukpappren uppmättes inga stora skillnader i friktion förmodligen för att kontakarean mellan de olika mjukpapprena var lika. Den faktiska kontakarean visade sig också vara viktig för perceptionen av lenhet, strävhet, torrhet och svalhet. Det visade sig vara en stor perceptuell skillnad mellan olika typer av tryckpapper och mjukpapper utifrån hur stimuli placerade sig på en taktil karta. För de tre materialen användes enbart två alternativt tre egenskaper hos materialet för att särskilja mellan alla olika par. För tryckpapper verkade en viktig dimension kunna beskrivas av alla de perceptuella och fysikaliska egenskaper som har med kontaktarean att göra, d.v.s. lenhet, svalhet, torrhet, ytråhet, värmekonduktivitet samt friktion. För att taktilt särskilja mellan olika ytor där bara strukturen är varierade, kunde friktion och våglängden relateras till spridningen i kartan. Båda studierna stödjer duplex theory of texture perception, där ett spatialt sinne används för att särskilja en av de grövre ytorna från en slät, och ett vibrationssinne för att särskilja mellan olika släta strukturer. Friktionen visade sig alltså vara en viktig fysikalisk egenskap för strukturer under åtminstone 10 µm i ytråhet. Från fingerfriktions mätningar kunde även följande slutsatser dras: (i) Stora skillnader i friktionskoefficient mellan olika personer uppmättes, men trenderna mellan olika individer var samma, vilket gör att relativa skillnader i friktion från en individ är representativa. (ii) Lipider (fingerfett) som överförs från fingret till ytan vid kontakt sänker friktionen. (iii) Frekvensinnehållet i friktionskraften varierar mellan olika ytor och den frekvenstopp som ses vid 30 Hz kan möjligtvis bero på fingrets struktur eller resonansfrekvensen på huden. (iv) Den pålagda kraften under en friktionsmätning visar sig omedvetet regleras av den friktionskraft som fingret möter under rörelse.  Hur små strukturer som kan diskrimineras har indirekt undersökts genom likhetsförsöket på modellytorna där försökspersoner skulle bedöma hur lika alla par av ytor kändes. Resultaten visade att ytorna med våglängder på 760 nm och 870 nm upplevdes olika jämfört med referens ytor utan något systematiskt mönster, medan ytan med 270 nm i våglängd inte kunde särskiljas. Amplituden på ytan som kunde diskrimineras var endast ca 10 nm, vilket indikerar att nanoteknologi mycket väl kan bidra inom haptiken och för att i framtiden kontrollera den taktila perceptionen.

QC 20121026

碩士
弘光科技大學
化妝品科技研究所
100
Following the solid lipid nanostructured, nanostructured lipid carriers（NLC） was developed as new carrier system type these years. In this study, combined hot homogenization and sonication technique was employed to produce Nanostructured lipid carriers（NLC）.The influences of mixed lipid composition, phase ratio of emulsion, homogenization speed and time on both particle size and other physicochemical properties of the NLC were characterized by using Laser Particle Size Analyzer and thermogravimetric analysis. As a result, this combined technique Sonication Technigue provides 34.60 % particle size reduction with narrow particle size distributions behave under temperature 80 ℃. On the other hands, the faster stirring speed and longer homogenization time makes smaller particle size. The size of nanostructured lipid carriers（NLC）was significantly influenced by homogenization speed and time. As 3000 rpm/20 min. homogenization condition increased up to 9000 rpm/40 min., the obtained particles showed pronounced 59.15 % particle size reduction. Stabled thermal property of NLC was verified by thermogravimetric analysis（TGA）under temperature 130 ℃condition. These results indicated that the NLC in this study produced by combined technique under optimal conditions could potentially be exploited as a cosmetic active carrier with narrow particle size distribution and thermally stable properties. Cosmetic activity of alcoholic crude extract of stem and leave of Eleutherococcus Senticosus, and its water, ethyl acetate and hexane partial fractions（E-W, E-EA and E-Hex）were evaluated in this study. E-W extract possess highest DPPH radical scavenging effect（IC50:6.57 mg/ml）.The DPPH radical scavenging effect of E-Hex and E-EA were 7.99 mg/ml and 10.00 mg/ml. The inhibit rate of E-W was 40 % which showed stronger values than Scutellaria baicalensis in 500 ppm concentration. The total amount of phenolic compounds of E-W extract (480.90 mg of GAE /g) is much higher than E-EA（411.60 mg of GAE /g） and E-Hex（299.90 mg of GAE /g）. The performance of E-W was better than and Scutellaria baicalensis which was well-known anti-oxidation property. E-W partition fraction presented inhibition of tyrosinase activity at the IC50 value of 2.42 mg/ml, showed higher whitening potential than the reference of Scutellaria baicalensis under concentration of 2.70 mg/g condition. The SPF-boosting properties of extracts were evaluated by in vitro SPF assay. Results shows formulation contains E-W fraction, boosted the SPF value from 21.32 ± 5.44 (without E-W) to 36.23 ± 6.69 (with 3% E-W). The SPF value increased 41.2% compared with the without E-W one. The larger additive of E-W concentration caused higher SPF value. Summarily, our study showed that the SPF of sun block with NLC, NLC-5 was 34.82 higher than without NLC one, Base-5（23.99）.The SPF value of NLC-5 increased 30.10 % compared with the Base-5. The Abs（358nm） value of sun block with NLC, NLC-5 was 1.358 higher than without NLC one, Base-5 (1.156). The SPF of sun block with extract of Eleutherococcus Senticosus was 49.03 ± 4.91 increasing 30.10 % compared with the Base-5. The sun block adding extract of Eleutherococcus Senticosus had well anti-oxidation property and UV protective capability. Our study verified that the physical property of sun block with NLC had well performance. After Transdermal delivery analysis, the new component not only reduced the damage of chemical sun block agent, but also couldn’t cause allergic reaction because of nothing of carrier permeating the skin. The new effective component could be applied in cosmetics application with anti-oxidation property, white and SPF enhancement.

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2016
Hydrochlorothiazide (HCT), an antihypertensive drug from the diuretic class, is being widely used for paediatric treatment of hypertension. This drug, belonging to the Class IV of the Biopharmaceutical Classification System (BCS), is known for its low aqueous solubility and low membrane permeability, thus representing a challenge for the formulators. In addition, such drugs are frequently associated to oral bioavailability issues. In the common practice, HCT is formulated as a suspension, which carries serious stability and dose accuracy concerns. Consequently, alternative formulations for this drug are currently being sought, wherein lipid-based systems, in particular lipid nanoparticles, showed the best features for controlled drug delivery. In this context, lipid nanoparticles were developed with the aim of improving both oral bioavailability and therapeutic effects of HCT in the paediatric treatment of hypertension. Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) with different components were prepared by hot high-shear homogenisation, followed by ultrasonication. NLC6 composed of a 9:1 Precirol® ATO 5 – ricin oil blend and Tween® 80 as surfactant gave the best results. In fact, this formulation presented particles with submicron size, uniformly distributed, with the highest EE% and LC%, and enhanced stability after 1 month of storage at 4 °C as well as under simulated gastrointestinal conditions. In addition, its drug release profile was superior to that of HCT suspension. These promising results confirmed the potential of the proposed NLC delivery technology for the oral administration of HCT and other Class IV drugs to paediatric patients.
A hidroclorotiazida (HCT), diurético anti-hipertensor, tem sido muito utilizada no tratamento pediátrico da hipertensão. A baixa solubilidade em água e baixa permeabilidade definem a inclusão deste fármaco na Classe IV do Sistema de Classificação Biofarmacêutica (SCB) e tornam-no um desafio para os formuladores. Estes fármacos são frequentemente associados a problemas de biodisponibilidade oral. Geralmente, a HCT é formulada em suspensão, apresentando inerentes limitações de estabilidade e precisão das doses. Deste modo, várias formulações alternativas têm sido estudadas, de entre as quais os sistemas lipídicos, em particular as nanopartículas lipídicas, revelaram as melhores características para o controlo da libertação deste fármaco. Neste estudo, foram desenvolvidas nanopartículas lipídicas com o objetivo de aumentar a biodisponibilidade oral e os efeitos terapêuticos da HCT no tratamento pediátrico da hipertensão. Nanopartículas lipídicas sólidas (NLS) e vetores lipídicos nanoestruturados (NLC) com diferentes composições foram produzidos pelo método de homogeneização a alta pressão a quente, seguido de ultrasonicação. A formulação NLC6 com Precirol® ATO 5 – óleo de rícino, numa relação de 9:1, e com o tensioativo Tween® 80 conduziu aos melhores resultados. Com efeito, através desta formulação obtiveram-se partículas com tamanho submicrométrico, distribuição uniforme, caracterizadas por elevadas EE% e LC%, e estáveis após 1 mês de armazenamento a 4 °C e em condições de simulação gastrointestinal. Além disso, o perfil de libertação de fármaco deste sistema foi superior, comparativamente à suspensão de HCT. Estes resultados promissores confirmaram o potencial da tecnologia NLC proposta na administração oral de HCT e outros fármacos de Classe IV, em doentes pediátricos.