Les nanotechnologies appliquées à la médecine, et plus particulièrement à l’oncologie, ont permis le développement d’une nouvelle classe d’entités, appelées communément nanomédicaments ou médicaments vectorisés. Ce projet de recherche a pour objectif d’encapsuler du docétaxel dans un vecteur lipidique unilamellaire furtif, puis de greffer en surface le trastuzumab afin d’en améliorer le profil pharmacocinétique, notamment en optimisant la spécificité de la phase de distribution. Les résultats obtenus montrent qu’il est possible de développer un immunoliposome furtif de 140 nm encapsulant 90 % de docétaxel avec un taux de greffage de trastuzumab de 30 %. L’approche en cytométrie de flux que nous avons développée et appliquée a permis une quantification absolue du nombre d’anticorps présents en surface. In vitro, un double screening en culture 2D et en sphéroïde 3D a démontré la supériorité antiproliférative de l’immunoliposome comparativement à tous les autres traitements, indépendamment du statut Her2 des lignées étudiées. Les études in vivo ont confirmé cette supériorité, y compris comparativement au T-DM1, l’antibody-drug conjugate de référence dans la pathologie. Les études de biodistribution ont montré que l’accumulation de notre forme vectorielle dépendait de la taille et du degré de vascularisation des tumeurs, plus que statut Her2 tumoral. En conclusion, nous avons démontré l’intérêt thérapeutique de développer des formes vectorielles dans la prise en charge du cancer du sein, comparativement aux traitements standard. Une optimisation de la phase de distribution explique la supériorité antiproliférative obtenue avec l’immunoliposome. / The application of nanotechnology in medicine, especially oncology, has allowed for the development of a new class of entities, commonly called nanomedicine or vectorized medicine.This research project aims to encapsulate docetaxel in a stealthy, unilamellar, lipidic vector, then graft trastuzumab onto its surface to improve its pharmacokinetic profile, specifically by optimizing the specificity of the distribution phase.The results show that it is possible to develop a stealthy immunoliposome of 140 nm encapsulating 90% docetaxel and a trastuzumab grafting rate of 30 %. The flow cytometry approach that we developed and applied allowed for an absolute quantification of the number of antibodies present on the surface. In vitro, a double screening in 2D culture and in 3D spheroid showed the antiproliferative superiority of the immunoliposome compared to all the other treatments, regardless of the Her2 status in the cells studied. In vivo studies have confirmed said superiority compared to T-DM1; the benchmark antibody-drug conjugate for this pathology. Biodistribution studies have shown that the accumulation of our vector depends moreover on the size and degree of tumor vascularization than its Her2 status. In conclusion, we have demonstrated the therapeutic value of developing vector forms in the management of breast cancer therapy compared to standard treatments. The optimization of the distribution phase explains the antiproliferative superiority obtained by using the immunoliposome.

O câncer de mama representa um grave problema de saúde pública. Dentre os fármacos empregados, destaca-se o paclitaxel, um agente citotóxico eficaz, porém associado a severos efeitos colaterais. A metformina hidrocloreto tem obtido resultados promissores para o tratamento de neoplasias, porém é bastante hidrofílica, fator limitante da biodisponibilidade. A rapamicina tem demonstrado sinergismo com paclitaxel e potente atividade antitumoral. Todavia, é um fármaco lipofílico e possui desvantagens. Sistemas nanoestruturados de fármacos como lipossomas PEGlados são largamente empregados para a melhora da farmacocinética e potencialização da ação terapêutica. Ademais, a funcionalização de lipossomas com anticorpos monoclonais pode permitir a entrega seletiva do fármaco encapsulado à célula alvo. No presente trabalho objetivou-se desenvolver e caracterizar lipossomas e imunolipossomas funcionalizados com trastuzumabe, contendo paclitaxel, metformina hidrocloreto e/ou rapamicina, bem como avaliar as formulações através de estudos in vitro e in vivo. Os resultados mostraram que a metformina hidrocloreto foi encapsulada com baixa eficiência, menor que 20%, ao passo que paclitaxel e rapamicina puderam ser co-encapsulados com adequados valores de eficiência de encapsulação, equivalente a 56,32% para paclitaxel e 73,31% para rapamicina, e tamanho de partícula nanométrico, de 136,95 nm em composição biocompatível baseada em SPC:Col:DSPE-PEG(2000). Os dois fármacos apresentaram liberação lenta, e foram convertidos às formas molecular e amorfa, respectivamente para paclitaxel e rapamicina quando encapsulados. Os imunolipossomas foram funcionalizados com elevada eficiência com trastuzumabe e mantiveram o tamanho nanométrico, com adequados valores de encapsulação dos fármacos. Ainda, mostrou-se o sinergismo entre paclitaxel e rapamicina coencapsulados em lipossomas em células triplo negativas (4T1) e houve sinergismo entre os dois fármacos, mediado pelo anticorpo em imunolipossomas frente à linhagem celular HER2 positiva (SKBR3), em virtude do aumento do uptake celular mediado pelo trastuzumabe. Finalmente, os resultados obtidos in vitro foram confirmados in vivo, sendo que os lipossomas com paclitaxel e rapamicina coencapsulados foram capazes de controlar o crescimento tumoral em modelo de câncer de mama triplo negativo, ao passo que o imunolipossoma com os dois fármacos permitiu o controle do crescimento de tumores xenográficos HER2 positivos, cuja média de volume tumoral correspondeu a 25,27%, 44,38% e 47,78% das médias dos volumes tumorais de controle negativo, positivo e lipossoma, respectivamente. Portanto, a formulação desenvolvida nesse trabalho tem potencial para ser avaliada em estudos clínicos. / Breast cancer represents a severe public health problem. Among the drugs used in the treatment, paclitaxel is an effective cytotoxic drug, but associated with side effects. Hydrocloride metformin has shown promising results for cancer treatment, however it is very hydrophilic, a limiting factor for bioavailability. Rapamycin has demonstrated synergism with paclitaxel and potent anticancer activity, though it is a lipophilic drug with drawbacks that compromise its bioavailability. Nanostructured drug delivery systems, such as PEGylated liposomes are largely employed for pharmacokinetics improvement and enhancement of therapeutic effect. Furthermore, the functionalization of liposomes with monoclonal antibodies enables the selective delivery of the loaded drug to the target cell. In the present work, we aimed to develop and characterize liposomes and immunoliposomes functionalized with trastuzumab, containing paclitaxel, hydrocloride metformin and/or rapamycin, as well as to evaluate the formulations through in vitro and in vivo studies. The results showed that hydrocloride metformin was encapsulated with low efficiency, less than 20%, on the other hand paclitaxel and rapamycin could be co-loaded with suitable values of encapsulation efficiency, 56.32% for paclitaxel and 73.31% for rapamycin and nanometric particle size, 136.95 nm, based on a SPC:Chol:DSPE-PEG(2000) composition. The two drugs displayed slow release, and were converted to molecular and amorphous form, respectively for paclitaxel and rapamycin when encapsulated. The immunoliposomes were developed with high efficiency with trastuzumab and kept the nanometric size, with adequate encapsulation of drugs. Moreover, herein it was shown the synergism between paclitaxel and rapamycin co-loaded in liposomes in triple negative cells (4T1) and there was synergism between the two drugs mediated by the antibody in immunoliposomes in the HER2-positive cell line (SKBR3), due to the improved cell uptake mediated by trastuzumab. Finally, the results obtained in vitro were confirmed in vivo. Co-loaded paclitaxel and rapamycin were able to control tumor growth in a triple negative breast cancer animal model, while the immunoliposome containing the two drugs allowed for better control of tumor growth in a HER2-positive breast xenograft model, whose average tumor volume corresponded to 25.27%, 44.38% and 47.78% of the tumor volumes of positive control, negative control and liposome, respectively. Therefore, the formulation developed herein has potential to be evaluated in clinical trials.

O câncer de mama representa um grave problema de saúde pública. Dentre os fármacos empregados, destaca-se o paclitaxel, um agente citotóxico eficaz, porém associado a severos efeitos colaterais. A metformina hidrocloreto tem obtido resultados promissores para o tratamento de neoplasias, porém é bastante hidrofílica, fator limitante da biodisponibilidade. A rapamicina tem demonstrado sinergismo com paclitaxel e potente atividade antitumoral. Todavia, é um fármaco lipofílico e possui desvantagens. Sistemas nanoestruturados de fármacos como lipossomas PEGlados são largamente empregados para a melhora da farmacocinética e potencialização da ação terapêutica. Ademais, a funcionalização de lipossomas com anticorpos monoclonais pode permitir a entrega seletiva do fármaco encapsulado à célula alvo. No presente trabalho objetivou-se desenvolver e caracterizar lipossomas e imunolipossomas funcionalizados com trastuzumabe, contendo paclitaxel, metformina hidrocloreto e/ou rapamicina, bem como avaliar as formulações através de estudos in vitro e in vivo. Os resultados mostraram que a metformina hidrocloreto foi encapsulada com baixa eficiência, menor que 20%, ao passo que paclitaxel e rapamicina puderam ser co-encapsulados com adequados valores de eficiência de encapsulação, equivalente a 56,32% para paclitaxel e 73,31% para rapamicina, e tamanho de partícula nanométrico, de 136,95 nm em composição biocompatível baseada em SPC:Col:DSPE-PEG(2000). Os dois fármacos apresentaram liberação lenta, e foram convertidos às formas molecular e amorfa, respectivamente para paclitaxel e rapamicina quando encapsulados. Os imunolipossomas foram funcionalizados com elevada eficiência com trastuzumabe e mantiveram o tamanho nanométrico, com adequados valores de encapsulação dos fármacos. Ainda, mostrou-se o sinergismo entre paclitaxel e rapamicina coencapsulados em lipossomas em células triplo negativas (4T1) e houve sinergismo entre os dois fármacos, mediado pelo anticorpo em imunolipossomas frente à linhagem celular HER2 positiva (SKBR3), em virtude do aumento do uptake celular mediado pelo trastuzumabe. Finalmente, os resultados obtidos in vitro foram confirmados in vivo, sendo que os lipossomas com paclitaxel e rapamicina coencapsulados foram capazes de controlar o crescimento tumoral em modelo de câncer de mama triplo negativo, ao passo que o imunolipossoma com os dois fármacos permitiu o controle do crescimento de tumores xenográficos HER2 positivos, cuja média de volume tumoral correspondeu a 25,27%, 44,38% e 47,78% das médias dos volumes tumorais de controle negativo, positivo e lipossoma, respectivamente. Portanto, a formulação desenvolvida nesse trabalho tem potencial para ser avaliada em estudos clínicos. / Breast cancer represents a severe public health problem. Among the drugs used in the treatment, paclitaxel is an effective cytotoxic drug, but associated with side effects. Hydrocloride metformin has shown promising results for cancer treatment, however it is very hydrophilic, a limiting factor for bioavailability. Rapamycin has demonstrated synergism with paclitaxel and potent anticancer activity, though it is a lipophilic drug with drawbacks that compromise its bioavailability. Nanostructured drug delivery systems, such as PEGylated liposomes are largely employed for pharmacokinetics improvement and enhancement of therapeutic effect. Furthermore, the functionalization of liposomes with monoclonal antibodies enables the selective delivery of the loaded drug to the target cell. In the present work, we aimed to develop and characterize liposomes and immunoliposomes functionalized with trastuzumab, containing paclitaxel, hydrocloride metformin and/or rapamycin, as well as to evaluate the formulations through in vitro and in vivo studies. The results showed that hydrocloride metformin was encapsulated with low efficiency, less than 20%, on the other hand paclitaxel and rapamycin could be co-loaded with suitable values of encapsulation efficiency, 56.32% for paclitaxel and 73.31% for rapamycin and nanometric particle size, 136.95 nm, based on a SPC:Chol:DSPE-PEG(2000) composition. The two drugs displayed slow release, and were converted to molecular and amorphous form, respectively for paclitaxel and rapamycin when encapsulated. The immunoliposomes were developed with high efficiency with trastuzumab and kept the nanometric size, with adequate encapsulation of drugs. Moreover, herein it was shown the synergism between paclitaxel and rapamycin co-loaded in liposomes in triple negative cells (4T1) and there was synergism between the two drugs mediated by the antibody in immunoliposomes in the HER2-positive cell line (SKBR3), due to the improved cell uptake mediated by trastuzumab. Finally, the results obtained in vitro were confirmed in vivo. Co-loaded paclitaxel and rapamycin were able to control tumor growth in a triple negative breast cancer animal model, while the immunoliposome containing the two drugs allowed for better control of tumor growth in a HER2-positive breast xenograft model, whose average tumor volume corresponded to 25.27%, 44.38% and 47.78% of the tumor volumes of positive control, negative control and liposome, respectively. Therefore, the formulation developed herein has potential to be evaluated in clinical trials.

Cancer is one of the leading causes of death in the world. For women in the U.S. and the European countries, breast cancer is the most common type and it continuously threatens the lives of the patients and causes huge economic losses. Chemotherapy and endocrine therapy are the common treatments for recurrence prevention and metastatic cancer symptom palliation. However, the uses of these therapies are meanwhile largely limited because their toxic side effects and non-specificity usually lead to low quality lives of the patients. Low aqueous solubility, multi-drug resistance, degradation of drug, limited intra-tumor diffusion and etc. are other limitations of conventional chemotherapies and endocrine therapies. Nanoparticle based drug carriers were extensively studied for therapeutic drug delivery. Many carriers could be loaded with high dose of hydrophobic and hydrophilic drugs, protect the drug from the surrounding in vivo environment during the transportation, specifically target and enter the tumor cells and slowly release the drug thereafter. Advanced nanoparticle drug carriers are studied driven by the need of a more efficient drug delivery. The 3rd generation of nanoparticle based drug carriers are recently developed. They usually consist of more than one type of nanoparticles. Different part of the particle has more specialized functions. Therefore, by carefully selecting from the conventional nanoparticle carriers, a 3rd generation particle could have the properties such as high loading capacity of multiple drugs, prolonged half-life in circulation, higher tendency of accumulating at the tumor site, improved specificity to the tumor cells, higher cell uptake rate and accurately triggered controlled release, and combination of the above-mentioned properties. In our study, a paclitaxel loaded nanoparticle supported immunoliposome was assembled for metastatic breast cancer drug delivery. Functionalized single walled carbon nanohorn or poly(lactic-co-glycolic acid) was encapsulated in the polyethylene glycol (PEG) coated liposome for high drug loading and controlled release. Anti-Her2 antibody or Herceptin® was grafted onto the surface of the liposome for a higher affinity to the Her2 overexpressing breast cancer cells. Firstly, the conjugation of protein to the surface of liposome and PEGylated liposomes were investigated. Proteins with or without membrane binding domain were conjugated to liposome and PEGylated liposomes through covalent and non-covalent binding for comparison. A modified enzyme-linked immune sorbent assay was developed for surface grafted protein quantification. Secondly, the encapsulation of solid nanoparticle into PEGylated immunoliposome was investigated. Results showed a new structure of solid nanoparticle in PEGylated immunoliposome at a 1:1 ratio was formed during the repeated freeze-thawing process. Supported immunoliposomes with high homogeneity in size and structure were purified by sucrose density gradient centrifugation. Thirdly, the drug loading, triggered release, cell binding, cell uptake and cell toxicities of the supported immunoliposome were studied. Release results showed a minimum drug leakage in serum at body temperature from the particle. The release was initiated with a minor burst trigged by low pH inside the tumor cell and followed with a long term linear pattern. Cell assay results showed the highest binding affinity of the antibody or Herceptin® grafted nanoparticles to Her2 overexpressing cell lines and a lysosomal intracellular distribution of the endocytosised particles. In the final study, a fabrication process for polymeric material nanoparticles was established. The process was capable of providing accurate control of the particle size with significant high output rates, thus largely extends the scope of materials for supporting the immunoliposome. / Ph. D.

O carcinoma de células escamosas (SCC) é um tumor maligno de origem epitelial no qual o receptor de fator de crescimento epidermal (EGFR) está superexpresso e associado a malignização. O cetuximabe é um anticorpo monoclonal, capaz de se ligar seletivamente ao EGFR. A combinação de cetuximabe com o quimioterápico hidrofílico 5-fluorouracil (5-FU) é utilizada na clínica pela via intravenosa e é associada a efeitos colaterais. A administração tópica de 5-FU também é realizada a partir de cremes convencionais, porém sua eficácia é limitada a doenças pré-cancerosas e o carcinoma basocelular. Estima-se que a conjugação do cetuximabe à superfície de lipossomas, os quais podem ser administrados na pele em associação a um método físico, como a iontoforese, seja capaz de direcionar a liberação do 5-FU para as camadas da pele onde os tumores estão presentes, além de aumentar o uptake celular em linhagem EGFR positiva, viabilizando o tratamento tópico de SCC. Assim, o objetivo do presente trabalho foi avaliar a influência do cetuximabe conjugado a um lipossoma (imunolipossoma) na penetração cutânea passiva e iontoforética do 5-FU e na regressão do SCC. Para tanto, um método analítico e um método bioanalítico foram desenvolvidos e validados para quantificação de 5-FU. Lipossomas convencionais foram preparados a partir de diferentes composições lipídicas, métodos de preparo e razão fármaco/lipídeo para selecionar aqueles que encapsulassem maior porcentagem de 5-FU. Lipossomas compostos por 1,2-distearoil-sn-glicero-3-fosfocolina e colesterol (DSPC:Chol, 55:45), preparados por hidratação do filme lipídico, na razão fármaco/ lipídeo 0,1, de aproximadamente 140 nm, foram escolhidos por encapsularem cerca de 50% de 5-FU. Para obtenção dos imunolipossomas, o cetuximabe foi primeiramente conjugado ao lipídeo DSPE acoplado ao ligante maleimida(polietilenoglicol)-2000 (DSPE-PEG-Mal), resultando em 94% de eficiência de conjugação. Os imunolipossomas foram então obtidos da mesma forma que os lipossomas e apresentaram tamanho e porcentagem de encapsulação do 5-FU semelhante a apresentada pelos lipossomas. Estudo in vitro em linhagem EGFR positiva (A431) mostrou sinergismo entre 5-FU e imunolipossoma, resultando em valores de IC50 cerca de 3 vezes menores do que o apresentado pelo 5-FU em solução. O uptake celular do imunolipossoma foi 3,5 vezes maior do que o do lipossoma. Nos estudos de penetração cutânea in vitro observou-se que, em relação a uma solução de 5-FU, lipossomas e imunolipossomas diminuíram a quantidade de 5- FU que atravessou a pele. A iontoforese aumentou a penetração do 5-FU a partir de todas as formulações. Neste caso, o acúmulo de 5-FU na epiderme viável, onde os tumores estão presentes, foi duas vezes maior quando este estava encapsulado no imunolipossoma em relação ao lipossoma. In vivo, as formulações foram administradas por via subcutânea ou tópica usando iontoforese em modelo xenográfico de SCC. O tratamento com os imunolipossomas diminuiu o crescimento tumoral em mais de 60% em relação ao controle e em torno de 50% em relação aos tratamentos com solução e lipossoma de 5-FU. A administração tópica por iontoforese apresentou maior redução tumoral do que a subcutânea quando os tumores foram tratados com solução de 5-FU e lipossomas, mas foi igualmente eficaz para os imunolipossomas. Análise imunohistológica revelou que o potencial de proliferação celular foi reduzido para os grupos tratados. Desta forma, a administração por iontoforese de imunolipossomas contendo 5-FU é uma estratégia promissora para o tratamento tópico de SCC / Squamous cell carcinoma (SCC) is a malignant tumor of epithelial origin in which the epidermal growth factor receptor (EGFR) is overexpressed and associated with malignization. Cetuximab is a monoclonal antibody, able to selectively bind EGFR. The combination of the chemotherapy with the hydrophilic drug 5-fluorouracil (5- FU) and cetuximab is applied in the clinic by intravenous injection and is associated with side effects. Cetuximab conjugation onto liposomal surface, which can be administered topically onto the skin using physical methods, such as iontophoresis, is able to direct 5-FU release to the skin layers where tumors are localized and to increase cellular uptake in EGFR positive cells, making possible the topical treatment of SCC. Thus, the objective of this work was to investigate the influence of cetuximab conjugation to liposomes (immunoliposomes) in the passive and iontophoresis skin penetration of 5-FU and SCC regression. For this purpose, an analytical and a bioanalytical method were developed and validated for 5-FU quantification. Then, conventional liposomes were prepared using different lipid compositions, preparation methods and drug to lipid ratios in order to select those able to load higher percentages of 5-FU. Liposomes composed by 1,2-Distearoylsn- glycero-3-phosphocholine and cholesterol (DSPC:Chol, 55:45) prepared by the thin lipid film hydration with drug/lipid ratio 0.1 with approximately 140 nm, were chosen because they encapsulated about 50% of 5-FU. For the obtainment of immunoliposomes, cetuximab was first coupled to the lipid DSPE linked to maleimide (polyethylene glycol)-2000 (DSPE-PEG-Mal) as an anchor for the antibody conjugation, resulting in 94% coupling efficiency. The immunoliposomes were obtained similarly to liposomes, with similar particle size and loading efficiency of 5-FU. In vitro studies using EGFR positive cells (A431) showed synergism for 5-FU and cetuximab, resulting in IC50 values about 3 times lower than 5-FU solution. Cellular uptake of immunoliposomes increased 3.5-fold compared to the liposomes. In vitro skin penetration studies revealed that, compared to the 5-FU solution, liposomes and immunoliposomes reduced the amount of 5-FU that passed through the skin. Iontophoresis increased the amount of 5-FU retained in viable epidermis for all formulations. In this case, the amount of 5-FU in viable epidermis, where tumors are localized, was 2 times higher when it was encapsulated in immunoliposomes compared to liposomes. In vivo, the formulations were administered subcutaneously or topically with iontophoresis in xenograft animal model of SCC. Treatment with immunoliposomes reduced tumor growth more than 60% compared to the negative control and about 50% compared to the treatments with 5-FU solution and liposomes. The topical administration using iontophoresis resulted in improved tumor reduction compared to the subcutaneous administration when tumors were treated with 5-FU solution and liposomes, but was equally effective for the immunoliposomes. The histological analysis showed the reduction of cellular proliferation for the treated groups. In conclusion, the administration of immunoliposomes containing 5-FU using iontophoresis is a promising strategy for the topical treatment of SCC