Yes / Three-dimensional printing (3DP) has the potential to cause a paradigm shift in the manufacture of pharmaceuticals, enabling personalised medicines to be produced on-demand. To facilitate integration into healthcare, non-destructive characterisation techniques are required to ensure final product quality. Here, the use of process analytical technologies (PAT), including near infrared spectroscopy (NIR) and Raman confocal microscopy, were evaluated on paracetamol-loaded 3D printed cylindrical tablets composed of an acrylic polymer (Eudragit L100-55). Using a portable NIR spectrometer, a calibration model was developed, which predicted successfully drug concentration across the range of 4–40% w/w. The model demonstrated excellent linearity (R2 = 0.996) and accuracy (RMSEP = 0.63%) and results were confirmed with conventional HPLC analysis. The model maintained high accuracy for tablets of a different geometry (torus shapes), a different formulation type (oral films) and when the polymer was changed from acrylic to cellulosic (hypromellose, HPMC). Raman confocal microscopy showed a homogenous drug distribution, with paracetamol predominantly present in the amorphous form as a solid dispersion. Overall, this article is the first to report the use of a rapid ‘point-and-shoot’ approach as a non-destructive quality control method, supporting the integration of 3DP for medicine production into clinical practice. / Open Access funded by Engineering and Physical Sciences Research Council United Kingdom (EPSRC), UK for their financial support (EP/L01646X).

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: The primary aim of this study was to investigate the feasibility of the amphiphilic block copolymer poly((vinylpyrrolidone)-b-poly(vinyl acetate)) (PVP-b-PVAc) as a vehicle for hydrophobic anti-cancer drugs. PVP-b-PVAc block copolymers of constant hydrophilic PVP block length and varying hydrophobic PVAc block lengths were synthesized via xanthate-mediated controlled radical polymerization (CRP). The methodology consisted of growing the PVAc chain from a xanthate end-functional PVP. In an aqueous environment the amphiphilic block copolymers selfassembled into spherical vesicle-like structures consisting of a hydrophobic PVAc bilayer membrane, a hydrophilic PVP corona and an aqueous core. The self-assembly behaviour and the physicochemical properties of the self-assembled structures were investigated by 1H NMR spectroscopy, fluorescence spectroscopy, transmission electron microscopy (TEM) and dynamic and static light scattering. Drug loading studies were performed using a model hydrophobic drug, clofazimine, and a common anti-cancer drug paclitaxel (PTX) to evaluate the potential of the PVP-b-PVAc block copolymers for drug delivery,. Clofazimine and PTX were physically entrapped into the hydrophobic domain of the self-assembled PVP-b-PVAc block copolymers via the dialysis method. The drug-loaded PVP-b-PVAc block copolymers were characterized regarding particle size, morphology, stability and drug loading capacity in order to assess their feasibility as a drug vehicle. The polymer vesicles had a relatively high drug loading capacity of 20 wt %. The effect of the hydrophobic PVAc block length on the drug loading capacity and encapsulation efficiency were also studied. Drug loading increased with increasing the hydrophobic PVAc block length. The effect of the drug feed ratio of clofazimine and PTX on the drug loading capacity and encapsulation efficiency were also investigated. The optimal formulation for the drug-loaded PVP-b-PVAc was determined and further investigated in vitro. The size stability of the drugloaded PVP-b-PVAc block copolymers was also assessed under physiological conditions (PBS, pH 7.4, 37 °C) and were stable in the absence and presence of serum. PVP-b-PVAc block copolymers were tested in vitro on MDA-MB-231 multi-drug-resistant human breast epithelial cancer cells and normal MCF12A breast epithelial cells to provide evidence of their antitumor efficacy. In vitro cell culture studies revealed that the PVP-b-PVAc drug carrier exhibited no cytotoxicity towards MDA-MB-231 and MCF12A cells, confirming the biocompatibility of the PVP-b-PVAc carrier. In vitro cytotoxicity assays using clofazimine-PVPb- PVAc formulations showed that when MDA-MB-231 cells were exposed to the formulations, an enhanced therapeutic effect was observed compared to the free drug. Cellular internalization of the PVP-b-PVAc drug carrier was demonstrated by fluorescent labeling of the PVP-b-PVAc carrier. Fluorescence microscopy results showed that the carrier was internalized by the MDAMB- 231 cells after 3 hours and localized in the cytoplasm and the perinuclear region. Overall, it was demonstrated that PVP-b-PVAc block copolymers appear to be promising candidates for the delivery of hydrophobic anti-cancer drugs. / AFRIKAANSE OPSOMMING: Die studie is gebaseer op die gebruik van amfifieliese blokkopolimere van poli((Nvinielpirolidoon)- b-poli(vinielasetaat)) (PVP-b-PVAc) as potensiële geneesmiddeldraers. PVP-b-PVAc blokkopolimere van konstante hydrofiliese bloklengte en verskillende hydrofobiese bloklengte is voorberei via die RAFT/MADIX-proses. Blokkopolimere met vinielasetaat is vanaf poli(N-vinielpirolidoon) met ‘n xantaatendfunksie voorberei. In ‘n wateromgewing vorm die PVP-b-PVAc blokkopolimere vesikel strukture met ‘n hydrofobiese membraan en ‘n hydrofiliese mantel. Die fisies-chemiese eienskappe van die PVP-b-PVAc blokkopolimere is gekarakteriseerd met gebruik van KMR spektroskopie, fluoresent spektroskopie, transmissie elektronmikroskopie (TEM) en dinamiese en statiese lig verstrooiing. Die potensiaal van PVP-b-PVAc as ‘n geneesmiddeldraer is ondersoek deur gebruik te maak van die hydrofobiese geneesmiddel, clofazimine, en ‘n anti-kanker geneesmiddel, paclitaxel. Clofazimine en paclitaxel is ge-inkapsuleer in die hydrofobiese gedeelte van die blokkopolimere via die dialise-metode. Clofazimine-PVP-b-PVAc en paclitaxel-PVPb- PVAc blokkopolimere is gekarakteriseerd met betrekking tot die partikel grootte, morfologie, stabiliteit en laai kapasitiet om die PVP-b-PVAc blokkopolimere as geneesmiddeldraers te evalueer. Die PVP-b-PVAc geneesmiddeldraer het ‘n relatiewe hoë laai kapsiteit van 20 gew % aangetoon. Die invloed van die bloklengte op die laai kapasitiet is ook ondersoek en beskryf. ‘n Toename in die laai kapasitiet is gesien met ‘n toename in die hydrofobiese bloklengte. Die invloed van die hoeveelheid geneesmiddel op die laai kapasitiet en die inkapsuleer doeltreffendheid is ook ondersoek. Die optimale formulasie is gevind en verder gebruik vir in vitro studies. Die stabiliteit van die geneesmiddeldraer in fisiologiese omstandighede (pH 7.4, 37 °C) is ook beskryf. Resultate toon aan dat die sisteem stabiel is onder hierdie omstandighede in die afwesigheid en aanwesigheid van serum. In vitro eksperimente is op MCF12A epiteel-borsselle en MDA-MB-231 epiteelborskankerselle getoets om die anti-tumoraktiwiteit te ondersoek. Resultate toon aan dat die PVP-b-PVAc geen sitotoxiese effek op die selle het nie, wat aandui dat die polimere bioverenigbaar is. Verder is dit bewys dat die PVP-b-PVAc geneesmiddel formualsie ’n hoër sitotoxisiteit besit as die vry-geneesmiddel. Fluoresent studies het aangetoon dat die geneesmiddeldraer na 3 uur opgeneen word deur MDA-MB231 selle en gelokaliseerd is in die sitoplasma en in die omgewing van die kern van die selle. In die algemeen is dit aangetoon dat PVP-b-PVAc blokkopolimere potensiële kandidate vir die lewering van hydrofobiese geneesmiddels is.

High throughput screening techniques that are routinely used in modern drug discovery processes result in a higher prevalence of poorly water-soluble drugs. Such drugs often have poor bioavailability issues due to their poor dissolution and/or permeability to achieve sufficient and consistent systemic exposure, resulting in sub-optimal therapeutic efficacies, particularly via oral administration. Alternative formulations and delivery routes are demanded to improve their bioavailability. Nanoparticulate formulations of poorly water-soluble drugs offer improved dissolution profiles. The physiology of the lung makes it an ideal target for non-invasive local and systemic drug delivery for poorly water-soluble drugs. In Chapter 2, a particle engineering process ultra-rapid freezing (URF) was utilized to produce nanostructured aggregates of itraconazole (ITZ), a BCS class II drug, for pulmonary delivery with approved biocompatible excipients. The obtained formulation, ITZ:mannitol:lecithin (1:0.5:0.2, w/w), i.e. URF-ITZ, was a solid solution with high surface area and ability to achieve high magnitude of supersaturation. An aqueous colloidal dispersion of URF-ITZ was suitable for nebulization, which demonstrated optimal aerodynamic properties for deep lung delivery and high lung and systemic ITZ levels when inhaled by mice. The significantly improved systemic bioavailability of inhaled URF-ITZ was mainly ascribed to the amorphous morphology that raised the drug solubility. The effect of supersaturation of amorphous URF-ITZ relative to nanocrystalline ITZ on bioavailability following inhalation was evaluated in Chapter 3. The nanoparticulate amorphous ITZ composition resulted in a significantly higher systemic bioavailability than for the nanocrystalline ITZ composition, as a result of the higher supersaturation that increased the permeation. In Chapter 4, pharmacokinetics of inhaled nebulized aerosols of solubilized ITZ in solution versus nanoparticulate URF-ITZ colloidal dispersion were investigated, under the hypothesis that solubilized ITZ can be absorbed faster through mucosal membrane than the nanoparticulate ITZ. Despite similar ITZ lung deposition, the inhaled solubilized ITZ demonstrated significantly faster systemic absorption across lung epithelium relative to nanoparticulate ITZ in mice, due in part to the elimination of the phase-to-phase transition of nanoparticulate ITZ. / text

Computational methods such as linear free energy relationships (LFERs) offer a useful high-throughput solution to quickly evaluate drug developability, e.g. membrane permeability, organic solvent/water partition coefficients, and solubility. LFERs typically assume the contribution of structural components/functional groups to the overall properties of a given molecule to be constant and independent. This dissertation describes a series of studies in which linear free energy relationships were developed to predict solvation of small organic molecules in lipid formulations, specifically, triglyceride containing solvents and phospholipid-based liposomes. The formation of intermolecular HBs in triglyceride solvents (homogenous with H-bond accepting ability) and intramolecular HBs within the bilayer barrier domain (hydrocarbon-like) proved to be the major factors to consider in developing LFERs to account for the increased oil/water partition coefficients and enhanced bilayer permeability of small organic molecules. The triglyceride solvent/water partition coefficients of a series of model compounds varying in polarity and H-bond donating/accepting capability were used to establish a correlation between the solvent descriptors and the ester concentration in these solvents using the Abraham LFER approach. The LFER analyses showed that the descriptors representing the polarizability and H-bond basicity of the solvents vary systematically with the ester concentration. A fragment-based LFER to predict membrane permeability or 1,9- decadiene/water partition coefficients of small organic molecules including small peptides was systematically constructed using a total of 47 compounds. Significant nonadditivity was observed in peptides in that the contribution of the peptide backbone amide to the apparent transfer free energy from water into the bilayer barrier domain is considerably smaller than that of a “well-isolated” amide and greatly affected by adjacent polar substituents on the C-termini. In order to explain the phenomenon of nonadditivity, the formation of intramolecular HBs and inductive effects of neighboring polar groups on backbone amide, were investigated using FTIR and MD simulations. Both spectroscopic and computational results provided supportive evidence for the hypothesis that the formation of intramolecular HBs in peptides is the main reason for the observed nonadditivity of Δ(ΔG°)-CONH-. The MD simulation results showed that the inductive effect of neighboring groups is not as important as the effect of intramolecular HBs.

Miltefosine (hexadecylphosphocholine, HePC), a synthetic antitumor designed from natural phospholipids, is clinically approved for cutaneous metastases of breast cancer and cutaneous lymphoma. This drug acts mainly at cellular membrane level, where it accumulates and interferes with lipid metabolism and lipid-dependent signaling pathways leading the cells to apoptosis. However, HePC systemic and peroral administration induces hemolysis and mucosal toxicity, respectively. To overcome these limitations, we investigated the protective properties of colloidal polymeric micelles (PM) composed by Pluronics, triblock copolymers of poly(ethylene oxide) and poly(propylene oxide). We found that both Pluronic composition and concentration modulate the hemolytic profile of incorporated drug (HePC-PM) by increasing the drug amount to cause in vitro hemolysis. Moreover, small-angle X-ray scattering (SAXS) was used to assess structural information of interactions between HePC and PM. Additionally, we showed that HePC-PM prevented mucosal irritation, decreasing bleeding and lysis of blood vessels in a chicken chorioallantoic membrane model. Interestingly, HePC-PM increased the in vitro selective cytotoxicity against cervix tumor cells rather healthy fibroblasts, suggesting a differential uptake of these nanostructures by tumor cells. Furthermore, we also found that HePC induces cytotoxicity and decrease cell survival, migration and proliferation in osteosarcoma cells in vitro. We showed that cytotoxicity by HePC is associated with caspase-3 activation, DNA fragmentation, apoptotic-like bodys formation and inhibition of both constitutive and cytokine-stimulated Akt/PKB phosphorylation. HePC-PM clearly reduces the drug cytotoxic effects. Finally, we demonstrated that Pluronic F127 polymeric micelles are efficient for cargo delivering the encapsulated drug preferentially into tumor cells rather than healthy cells. These findings together suggest that Pluronic F127 PM reduce drug side effects and provide a potential alternative for systemic delivery of HePC, as well as other amphiphilic drugs. / Miltefosina (hexadecilfosfocolina, HePC), um fármaco antitumoral sintético desenvolvido a partir de fosfolipídios naturais, é clinicamente aprovada para o tratamento tópico de metástases de câncer de mama e linfomas cutâneos. Atua principalmente nas membranas celulares, onde se acumula e interfere no metabolismo lipídico e nas vias de sinalização dependentes de lipídios levando as células à apoptose. No entanto, quando administrada sistemicamente ou oralmente a HePC induz hemólise e toxicidade de mucosas, respectivamente. Para superar estas reações adversas investigamos os efeitos protetores conferidos por micelas poliméricas coloidais (PM) compostas por Pluronics, copolímeros tribloco de poli(óxido de etileno) e poli(óxido de propileno). Inicialmente, encontramos que a composição e concentração do Pluronic modulam o perfil hemolítico do fármaco encapsulado (HePC-PM), aumentando a quantidade necessária de HePC para causar hemólise in vitro. Além disso, utilizamos o espalhamento de raios-X a baixo ângulo (SAXS) para obter informações estruturais das interações entre HePC e PM. Em seguida, mostramos que HePC-PM preveniu a irritação da mucosa, diminuindo a hemorragia e a vasoconstricção em membrana corioalantóica de ovos embrionados. Estudos in vitro demonstraram que a HePC-PM aumentou seletivamente a citotoxicidade contra células de carcinoma HeLa em relação a fibroblastos saudáveis, sugerindo captação diferencial dessas nanoestruturas pelas células tumorais. Além disso, relatamos que, in vitro, a HePC induz citotoxicidade, diminui a sobrevivência, migração e proliferação osteossarcomas. Esta citotoxicidade está associada à ativação da caspase-3, fragmentação do DNA, formação de corpos apoptóticos e inibição da fosforilação de Akt/PKB. Adicionalmente, HePC-PM reduz os efeitos citotóxicos nestas linhagens. Finalmente, demonstramos que as micelas poliméricas de Pluronic F127 são eficientes para a entrega intracelular fármacos preferencialmente em células tumorais, e em menor grau em células saudáveis. Em conjunto, os dados sugerem que este sistema nanoestruturado reduz a toxicidade da HePC e representa uma alternativa potencial para a administração sistêmica deste e de outros fármacos anfifílicos.

De acordo com o conceito de sistemas de liberação controlada, o presente estudo foi baseado na utilização de polímeros hidrofílicos biocompatíveis, formadores de hidrogéis, para o desenvolvimento de matrizes na forma de filmes finos. Os polímeros utilizados para a formação das matrizes foram a quitosana proveniente das cascas de camarão, o amido de milho modificado e a poli(N-vinil-2-pirrolidona) - PVP. As matrizes foram reticuladas utilizando glutaraldeído. O fármaco escolhido para testar a capacidade de liberação dos dispositivos foi o anti-inflamatório não esteroidal (AINE) diclofenaco sódico. Para obtenção das matrizes com propriedades adequadas para essa finalidade, foram testadas misturas de quitosana-amido e quitosana-PVP. Após a triagem qualitativa, os dispositivos foram avaliados quanto à citotoxidade, intumescimento máximo, fração gel, parâmetros cinéticos associados à absorção de vapor de água e à capacidade de liberação de diclofenaco sódico in vitro. As formulações de quitosana-PVP foram as que apresentaram melhores propriedades para a aplicação proposta nesse estudo, se destacando a formulação A3, com alto percentual de liberação, boas propriedades de manuseio, poucos componentes na formulação diminuindo o potencial alergênico e aprovação no teste de citotoxicidade em células de camundongo (NCTC) pelo método de incorporação do vermelho neutro. / According to the concept of drug delivery systems, this study has based on the use of biocompatible hydrophilic polymers hydrogels-forming for the development of matrices in the form of thin films. The polymers used for forming the matrices were chitosan from shrimp shells, modified maize starch and poly(N-vinyl-2-pyrrolidone) PVP. The matrices were cross-linked using glutaraldehyde. The drug chosen to test the ability of the devices release was the non-steroidal anti-inflammatory drug (NSAID) sodium diclofenac. Mixtures between chitosan-starch and chitosan-PVP tested to obtain the matrices with suitable properties for this purpose. The devices after qualitative screening had evaluated for cytotoxicity, maximum swelling, gel fraction, kinetic parameters associated with absorbing water vapor and the release of diclofenac sodium able to in vitro. The formulations based on chitosan-PVP were the presents the best properties, in evidence formulation A3, with high percentage of delivery, good handing properties, few compounds/components reducing the allergenic potential and successful in vitro cell viability red uptake cytotoxicity assay, using cell culture mouse cells (NCTC).

Os benefícios gerados pela luz do sol foram observados desde a origem da humanidade. A terapia fotodinâmica é a mais nova modalidade de tratamento com luz, que envolve a administração do fármaco fotossensível, o qual se deposita preferencialmente nas células que se deseja a ação, seguido por seletiva iluminação da lesão com luz na região do visível. A ftalocianina de cloro-alumínio (AlClPc) e a indocianina verde são apontados como potenciais fármacos nesta terapia, sendo bastante utilizados atualmente. Foram preparados complexos de ciclodextrinas de ftalocianina de cloro-alumínio e hidroxipropil-beta ciclodextrina e beta-ciclodextrina, incorporados em lipossomas com a indocianina verde, com o propósito de avaliar o sinergismo da ação, proporcionado pela associação de sistemas de liberação e de fármacos fotossensíveis, na terapia fotodinâmica. Quando avaliado isoladamente os complexos de ftalocianina e ciclodextrinas demonstraram valores diferentes da constante de associação demonstrando maior complexação com hidroxipropil-beta ciclodextrina. No entanto, os derivados de ciclodextrinas estudados melhoraram as propriedades fotofísicas do fármaco. Na avaliação das propriedades fotobiológicas, nenhuma toxicidade foi observada para a ftalocianina complexada com ciclodextrinas nem com lipossomas. Na avaliação do complexo de ftalocianina de cloro-alumínio/ciclodextrinas na presença de luz, houve um alto índice de morte celular. No caso da associação dos complexos de AlClPc incorporados nos lipossomas juntamente com a ICV as preparações também demonstraram que as modificações geradas pela incorporação dos fármacos fotossensíveis favoreceram as propriedades espectroscópicas necessárias para a ação dos fármacos na PDT, com conseqüente redução da agregação e alterações significativas nas propriedades fotofísicas e fotobiológicas dos fármacos. Os complexos de ciclodextrinas incorporados nos lipossomas com indocianina verde se apresentaram estáveis como sistemas de liberação, após liofilização, garantindo a característica de vesícula unilamelar pequena. Neste trabalho observa-se claramente o sinergísmo garantido por associação dos dois fármacos fotossensíveis e de sistemas de liberação sugerindo uma nova área de pesquisa na terapia fotodinâmica. / The benefits generated by sunlight were observed from the origin of humanity. Photodynamic therapy is the new form treatment of light, which involves the administration of the photosensitizer drug which is targeting preferentially in diseased cells, followed by selective illumination of the lesion with visible light. The aluminum chloride phthalocyanine and indocyanine green are studied as potential drugs this therapy and often used. Were development complex of hydroxypropyl-beta cyclodextrin and beta-cyclodextrin with aluminum chloride phthalocyanine entrapment in liposomes with indocyanine green in order to studed the synergism of action, provided by association of both release and photosensitizer drugs, in photodynamic therapy. Complexes of phthalocyanine with different cyclodextrins derivatives showed different values of the association constant showing greater complexation with hydroxypropyl-beta cyclodextrin.However the derivatives of cyclodextrins studied improved the drug photophysical properties. In evaluating the photobiological properties, no toxicity was observed for phthalocyanines complexed with cyclodextrins or liposomes.Althought the evaluation of phthalocyanine complex excited by light, showed a higher rate of cell death. When the complexes where incorporated in liposomes plus indocyanine green photosensitizer drugs improved the spectroscopic properties necessary for the action of drugs in PDT reduced aggregation and significant improved in the photophysical and photobiological properties of drugs. Complexes of cyclodextrins incorporated in liposomes with indocyanine green showed stable drug delivery systems, after freeze-drying and keeping a small unilamellar vesicle.This work showed that there is clearly synergism by the association two photosensitizers and drug delivery systems and represents a great contribution for a new area of research in photodynamic therapy.

Carbohydrate recognition is one of the most sophisticated recognition processes in biological systems, mediating many important aspects of cell-cell recognition, such as inflammation, cell differentiation, and metastasis. Consequently, lectin-glycan interactions have been intensively studied in order to mimic their actions for potential bioanalytical and biomedical applications. Galectins, a class of ß-galactoside-specific animal lectins, have been strongly implicated in inflammation and cancer. Galectin-3 is involved in carbohydrate-mediated metastatic cell heterotypic and homotypic adhesion via interaction with Thomsen-Friedenreich (TF) antigen on cancer-associated MUC1. However, the precise mechanism by which galectin-3 recognizes TF antigen is poorly understood. Our thermodynamic studies have shown that the presentation of the carbohydrate ligand by MUC1-based peptide scaffolds can have a major impact on recognition, and may facilitate the design of more potent and specific galectin-3 inhibitors that can be used as novel chemical tools in dissecting the precise role of galectin-3 in cancer and inflammatory diseases. Another lectin, odorranalectin (OL), has been recently identified from Odorrana grahami skin secretions as the smallest cyclic peptide lectin, has a particular selectivity for L-fucose and very low toxicity and immunogenicity, rendering OL an excellent candidate for drug delivery to targeted sites, such as: (1) tumor-associated fucosylated antigens implicated in the pathogenesis of several cancers, for overcoming the nonspecificity of most anticancer agents; (2) the olfactory epithelium of nasal mucosa for enhanced delivery of peptide-based drugs to the brain. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2015.

A incidência de casos de melanoma tem aumentado em todo o mundo sendo que, apesar do diagnóstico precoce e do advento das terapias moleculares, o número de pacientes que morrem com a doença em estágio avançado continua em elevação. Deste modo as pesquisas atuais têm focado no desenvolvimento de diferentes estratégias para a disponibilização de terapias eficazes e acessíveis. Nesse contexto, a via de administração transdérmica constitui uma alternativa promissora para aumentar a eficácia local e sistêmica de fármacos, incluindo agentes antitumorais. Diversos métodos têm sido desenvolvidos para maximizar a permeação cutânea de fármacos, destacando-se, entre esses, a ablação térmica por radiofrequência (RF). Esse processo resulta na criação de vários microcanais entre a epiderme e a derme, pelos quais diversas moléculas podem passar em direção às camadas mais profundas da pele. Nesse estudo, a eficácia da infusão transdérmica de etoposídeo por dispositivo de radiofrequência fracionada foi avaliada em modelo de melanoma murino. Camundongos da linhagem C56BL/6 foram divididos nos seguintes grupos experimentais: 1) controle; 2) tratados com radiofrequência; 3) tratados com a aplicação tópica de etoposídeo; e 4) tratados com radiofrequência e posterior aplicação tópica de etoposídeo. Os tratamentos foram realizados durante o período de 28 dias. O peso corpóreo, o volume tumoral e o perfil hematológico foram avaliados semanalmente. Ao término do tratamento os animais foram eutanasiados e procedeu-se a coleta da massa tumoral e dos órgãos (pulmão, baço, rins, linfonodos e fígado) para análise histopatológica. As células tumorais obtidas das massas tumorais foram analisadas quanto às alterações do ciclo celular e do potencial transmembrânico mitocondrial. Os resultados demonstraram que o tratamento com etoposídeo isolado reduziu a sobrevida dos animais e ocasionou alterações histológicas indicativas de toxicidade. Em contrapartida, a infusão transdérmica do etoposídeo por dispositivo de radiofrequência promoveu redução significativa do volume tumoral, em comparação com todos os grupos experimentais, sem ocasionar mortalidade. Esse tratamento também diminuiu a plaquetocitose e elevou o número de eritrócitos em comparação com os outros grupos. A análise histopatológica dos órgãos dos animais tratados com RF + etoposídeo evidenciou que não houveram alterações significativas na arquitetura tecidual. Ainda, o grupo tratado com RF + etoposídeo foi o que apresentou o maior percentual de células estacionadas na fase S/G2M e com mitocôndrias inativas, evidenciando o aumento da eficácia demonstrada no estudo in vivo. O conjunto de resultados sugere que o tratamento com a radiofrequência seguida do etoposídeo resulta em melhor resposta antitumoral do quimioterápico, com baixos índices de toxicidade sistêmica / The incidence of melanoma cases has increased worldwide and, despite early diagnosis and targeted molecular therapy, the number of patients dying from metastatic disease continues to rise. Thus, current research has focused on the development of different treatment strategies to provide efficient and accessible solutions. In this sense, transdermal delivery is a promising alternative enhancing the local and systemic efficacy of drugs, including antitumor agents. Several methods have been developed to improve the skin permeation of drugs, highlighting, among those, the radiofrequency thermal ablation (RFA). This process results in the creation of many microchannels between the epidermis and the dermis through which several molecules can pass towards the deeper layers of the skin. In this study, the efficacy of the transdermal delivery of etoposide by a fractional radiofrequency device was evaluated in a murine melanoma model. C56BL/6 lineage mice were divided into the following experimental groups: 1) control; 2) treated with radiofrequency; 3) treated with topical applications of etoposide; and 4) treated with radiofrequency followed by topical applications of etoposide. The animals were treated for 28 days and the body weight, tumor volume and hematological profile were analyzed weekly. At the end of the treatments, the animals were euthanized and the tumor mass and organs (lung, spleen, kidneys, lymph nodes and liver) were collected for histopathological analysis. Tumor cells obtained from the tumor masses were analyzed for changes in the cell cycle and mitochondrial transmembrane potential. The results showed that the treatment with etoposide alone reduced the survival of the animals and caused histological changes indicating toxicity. On the other hand, the transdermal delivery of etoposide by a radiofrequency device resulted in a significant reduction of the tumor volume, in comparison with all the experimental groups, not causing mortality. This treatment also decreased thrombocytosis and increased the number of red blood cells compared to the other groups. The histopathological analysis of the organs from animals treated with RFA + etoposide demonstrated that there was no significant change in tissue architecture. Furthermore, the group treated with RFA + etoposide presented the highest percentage of cells with inactive mitochondria and interruption at the S/G2M stage, corroborating the increased efficacy of the in vivo study. The set of results indicates that the treatment with radiofrequency followed by etoposide results in better antitumor responses of chemotherapy, with low toxicity rates

A doença de Chagas é causada pelo Trypanosoma cruzi e acomete milhões de pessoas, principalmente as de baixa renda em países subdesenvolvidos. É considerada uma doença negligenciada, não existindo uma terapia eficaz contra os parasitas na fase crônica da doença. Estudos preliminares demonstraram que o ácido ursólico apresenta atividade tripanocida, entretanto, este fármaco possui baixa solubilidade em água, o que prejudica a sua biodisponibilidade. Com o intuito de viabilizar a terapia com ácido ursólico, as nanopartículas poliméricas são sistemas de liberação promissores, devido a sua capacidade de liberação modificada. Além disso, os sistemas nanoencapsulados destacam-se pela alta eficiência de encapsulação do fármaco, proteção contra degradação, e menor possibilidade de causar toxicidade. O objetivo deste trabalho foi o desenvolvimento e a caracterização de nanopartículas de policaprolactona para a veiculação de ácido ursólico, visando à otimização da terapia da Doença de Chagas. O estudo teve início com o desenvolvimento das nanopartículas poliméricas contendo ácido ursólico. A formulação obtida neste estudo pela técnica da nanoprecipitação apresentou menor valor de tamanho de partícula (173,17±4,20) e índice de polidispersividade (0,09±24,77), com perfil monomodal, potencial zeta de -36 mV e eficiência de encapsulação de 94,1±1,31%. O tamanho das partículas observado na microscopia eletrônica de varredura demonstrou ser compatível com os valores observados nas análises de espalhamento dinâmico de luz, embora tenha apresentado uma característica agregada e ligeiramente esférica. Através da determinação do coeficiente de partição do ácido ursólico foi possível avaliar a alta lipofilicidade do ácido ursólico. Na determinação do coeficiente de solubilidade do ácido ursólico, o lauril sulfato de sódio foi o tensoativo de escolha para o estudo in vitro do perfil de liberação, solubilizando aproximadamente 300 ?g.mL-1 de fármaco, mas não foi possível a realização do estudo in vitro do perfil de liberação, devido a sua característica altamente lipofílica. O estudo da citotoxicidade por ensaio de resazurina mostrou que a formulação escolhida não alterou a viabilidade celular de células LLCMK2, portanto sem toxicidade para o meio biológico, bem como evidenciou a capacidade das nanopartículas poliméricas contendo ácido ursólico de reduzir a viabilidade dos parasitas em aproximadamente 50%. A avaliação da atividade biológica do ácido ursólico em camundongo C57BL/6 infectados com a cepa Y do Trypanosoma cruzi apresentou uma redução acentuada (p<0,001) dos tripomastigotas quando comparados ao grupo I, sugerindo uma liberação sustentada do ácido ursólico nesse modelo de nanopartículas poliméricas. Como conclusão, as nanopartículas poliméricas contendo ácido ursólico podem ser propostas como uma abordagem quimioprofilática da doença de Chagas, considerando a necessidade dessa medida de segurança para pacientes que recebem transfusão sanguínea no Sistema Único de Saúde no Brasil. / Chagas disease is caused by parasite Trypanosoma cruzi and affects millions of lowincome in developing countries and because of that it is neglected by the pharmaceutical industry and there is no effective therapy against parasites in the chronic phase of the disease. Preliminary studies showed that ursolic acid presents tripanocidal activity, however, it has low water solubility, which reduces its bioavailability. Among the existing drug delivery systems, polymeric nanoparticles play a central role, due to their ability to sustain or control the release of drugs. Moreover, nanocoated systems are distinguished by high drug encapsulation efficiency, protection from degradation, and less likely to cause irritation. The aim of this work is development and characterization of the polymeric nanoparticles containing ursolic acid, aiming to optimize the treatment of Chagas disease. The study began with the development of polymeric nanoparticles containing ursolic acid. The formulation obtained in this study by the nanoprecipitation technique showed the lowest particle size (173.17 ± 4.20) and polydispersity index (0.09 ± 24.77), with monomodal profile, zeta potential of -36 mV and encapsulation efficiency was 94.1% ± 1.31. The size of the particles observed by scanning electron microscopy showed to be compatible with the values observed in the analysis of dynamic light scattering, although it had an aggregate and slightly spherical characteristic. By determining the coefficient of ursolic acid partition was possible to evaluate the high lipophilicity of ursolic acid. In determining the solubility coefficient of ursolic acid, sodium lauryl sulfate was the surfactant of choice for studying in vitro release profile, solubilizing approximately 300 ?g.mL-1 of the drug, but it has not been possible to conduct the study in vitro release profile, due to its highly lipophilic character. The study of resazurin cytotoxicity assay showed that the formulation did not alter the cell viability of LLCMK2 cells, and therefore, without toxicity to the biological environment and demonstrated the ability of the polymeric nanoparticles containing ursolic acid to reduce the viability of parasites in approximately 50 %. The evaluation of biological activity of ursolic acid in mice C57BL/6 mice infected with the Y strain of Trypanosoma cruzi showed a marked reduction (p<0.001) of trypomastigotes when compared to group I, suggesting a sustained release of ursolic acid in polymeric nanoparticles model. In conclusion, polymeric nanoparticles containing ursolic acid may be proposed as a chemoprophylactic approach of Chagas disease, considering the need for this safety measure for patients receiving blood transfusion Health System in Brazil.