Mucins in ovarian cancer diagnosis and therapy

Ovarian cancer is the most lethal gynecologic malignancy and the five-year survival rate is only 35% after diagnosis. Epithelial ovarian cancer is a highly metastatic disease characterized by widespread peritoneal dissemination and ascites. The death incidences from ovarian cancer could be significantly lowered by developing new methods for the early diagnosis and treatment of this fatal disease. Several potential markers have been identified recently. However, mucins are the most promising markers for ovarian cancer diagnosis. Mucins are large extracellular, heavily glycosylated proteins and their aberrant expression has been implicated in the pathogenesis of a variety of cancers, including ovarian cancer. This review will summarize known facts about the pathological and molecular characteristics of ovarian cancer, the current status of ovarian cancer markers, as well as general information about mucins, the putative role of mucins in the progression of ovarian cancer and their potential use for the early diagnosis and treatment of this disease

Exploring and harnessing PEG-immune system interactions to engineer targeted stealth nanoparticles

Effective nanoparticle drug delivery to tumor cells typically relies on prolonged systemic circulation of the nanoparticles to allow for extravasation and accumulation in tumor tissue, as well as targeting ligands on the nanoparticles that can mediate receptor-specific uptake by target tumor cells. Due to the ability of polyethylene glycol (PEG) to effectively reduce nonspecific protein binding and cell clearance, PEGylation has become a commonplace strategy for formulating long-circulating nanoparticle systems. However, the precise characteristics (e.g., PEG molecular weight and density) that influence the interactions between PEG-coated nanoparticles and phagocytic immune cells remain poorly understood for many nanoparticle systems, and findings from human studies suggest that the body is further able to mount PEG-specific humoral responses to PEG-coated agents. Additionally, the presence of targeting ligands on the nanoparticle surface may also compromise the extended circulation profile of PEG-coated nanoparticles. To address these challenges and gaps in our understanding, in this dissertation quantitative approaches and systematic analyses were utilized to 1) evaluate the interactions between phagocytic cells and various PEG coatings on polymeric nanoparticles, 2) determine the prevalence and concentrations of different anti-PEG antibody isotypes amongst the general human population, and 3) apply an alternative approach for PEGylated nanoparticle delivery to tumors. The results indicated that extremely dense PEG coatings (RF/D >> 2.8) are required to effectively minimize nonspecific clearance by immune cells. Using competitive ELISAs and engineered antibody standards, the anti-PEG IgG1-4 and IgM levels in a large number of healthy human samples were quantified, with the majority of samples possessing detectable anti-PEG IgG and/or IgM. Finally, a multistep targeting (i.e., pretargeting) approach was tested for the delivery of biotin PEG-modified nanoparticles to disparate tumor cells in vitro and in vivo. The analytical methodologies and overall findings described here can inform future studies of PEGylated nanoparticle-immune system interactions and nanoparticle targeting strategies.Doctor of Philosoph

Technology market transfer plan launch of DELIVES to E.U. Market©

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em BiotecnologiaThe pharmaceutical industry faces one of the biggest waves of patent expirations. The magnitude of the potential loss of sales is such that Datamonitor, predicts that between 2011 and 2012 the revenue from the drug will decrease for the first time in four decades. Alternatively, the technologies for Drug Delivery (DD), which consist of the science of delivering an active ingredient in place of the body where this is necessary, in the right quantities at the right time in the most effective and convenient, are boosting the growth of pharmaceutical companies, increasing its revenue by extending the product life cycle and through new formulations and combinations. According to this perception, this marketing plan was developed for a future company, LIFE DELIVERY, which holds the patent of a new biomaterial, and intends to launch a new product - Deliver | Improving life quality - for the market. DELIVES is a New Targeted Drug Delivery (TDD), which consists of a DD directed to release the drug locally in the area of interest, extremely versatile and can be precisely adjusted to achieve the specific objectives of each client, providing a controlled release of drugs and elaborated through a process environmentally friendly. Using three types of segmentation (geographic, industry type and application) and applying different evaluation criteria to selected segments, the final conclusions point to the following target customers: large pharmaceutical companies, the European Union, acting in the markets of the following therapeutic areas: inflammation and Musculosketal, metabolic disorders and cardiovascular diseases, the latter being the ideal customers for market entry strategy. According to the perceived value, of target customers - translated in cost-saving -, LIFE DELIEVERY will adopt a position based on three main pillars: quality, innovation and organizational image. The business model adopted aims to license the technology, using different communication strategies, and € 322.078.48, is the cost to implement this marketing plan in the first year of life of the company

A nanoliposome-based drug delivery system for cancer

The current project shows that the sulfatide-containing nanoliposome (SCL) drug delivery system could be an effective and safe nanocarrier for the anticancer agent doxorubicin to target tumours with high expression of tenascin-C. Moreover, SCL encapsulation could be a new strategy for the treatment of diseases in the central nervous system

Nanoceria for ROS-Mediated Cancer Therapy: A Receptor-Targeted Theranostic Approach

Combining the multi-step processes of diagnosis and treatment of cancers is critical to the timely provision of treatments in order to improve survival rates. Theranostics exploits both diagnostic and therapeutic agents in a single system, thus initiating the treatment of cancer as soon as it is diagnosed. It also helps to guide the treatment and predict the therapeutic response of individual patients to drugs, thereby offering personalised and tailored treatment strategies. Nanoceria, which possesses intrinsic anti-cancer properties, is employed as the therapeutic component. Cerium can exist in two oxidation states, Ce3+ and Ce4+, and it can switch between these two states. The redox switching and associated anti-cancer activity of nanoceria is pH-dependent. Thus, it acts selectively as a pro-oxidant in the acidic pH of cancer cells, thereby elevating ROS production and killing the cancer cells, while it acts as an antioxidant in the basic pH of normal cells, thereby scavenging ROS and protecting the normal cells. A fluorophore-tagged ligand (FITC-EGF), which can bind specifically to the receptors that are overexpressed in cancers and so detecting these receptors/cells, was used as the diagnostic component. Since the epidermal growth factor receptor (EGFR) is a biomarker in various cancers, then it was selected as the diagnostic target. This receptor-targeted strategy also enhances the bioavailability of nanoparticles at the tumour site through receptor-mediated endocytosis. Rod-shaped nanoceria (~50 nm L x ~12 nm Ø), which was fabricated by hydrothermal synthesis, characterised (TEM, XRD, Raman, XPS, FTIR, DLS), and functionalised with FITC-tagged EGF to form the final theranostic formulation. The theranostic performance of this system was evaluated using EGFR-positive cancer cell lines. The diagnostic and therapeutic efficiency was examined for several cancers, including fibrosarcoma (HT-1080), melanoma (A375-P, A375-MA1), breast cancer (MDA-MB-231), pancreatic cancer (PANC-1), and cervical cancer (HeLa). The performances of the system were studied using 2D (all types) and 3D models (melanoma only). The ROS-mediated cytotoxicity of nanoceria towards cancer cells was confirmed by cell viability, cytotoxicity, ROS measurements (DCFDA, MitoSOX), and live-dead imaging (calcein AM and EthD-1) studies. The specificity of the interaction between EGF-nanoceria and EGFR receptor was confirmed using western blotting and the intracellular uptake levels were determined both qualitatively (bioTEM) and quantitatively (ICP-MS). The diagnostic activity was confirmed by laser scanning confocal imaging. Nanoceria was observed to induce significant ROS-mediated cytotoxicity toward all the cancer models studied, which is attributed to EGFR overexpression in these cancers. Fluorescence imaging confirmed the diagnostic potential of the system. A significant increase in the oxidative stress levels was detected in the 3D models in comparison to the 2D models, suggesting the suitability of 3D models for research applications. Finally, the hemocompatibility studies (hemolysis assay) demonstrated the suitability of nanoceria-based theranostic formulations for in vivo use

Development of polypeptide-based therapeutics for the treatment of castration resistant prostate cancer

El cáncer de próstata (CaP) es el segundo cáncer más frecuente en los hombres. Los estudios han establecido el gen de fusión (T2E) compuesto por TMPRSS2 (serina proteasa dependiente de andrógenos) y ERG (factor de transcripción de la familia ETS) como un biomarcador potencial de CaP. La progresión en el CaP incluye tanto el receptor de andrógenos (AR) como el receptor del factor de crecimiento de insulina 1 (IGF-1R), y el tratamiento con un anticuerpo anti-IGF-1R (AVE1642) ha demostrado un gran potencial en el tratamiento de los pacientes con CaP T2E-positivos. Por ello, el desarrollo de terapias personalizadas basadas en polímeros terapéuticos puede favorecer el tratamiento de CaP para un subtipo específicos de pacientes. La inhibición de IGF-1R empleando AVE1642 generó un efecto antitumoral en las células de CaP que expresan T2E. Investigamos la actividad antitumoral del AVE1642 después de su conjugación con un polipéptido biodegradable y biocompatible (ácido poli-L-glutámico (PGA)). La línea celular VCaP T2E-positiva tratada con AVE1642 y PGA-AVE1642 respondió a ambos tratamientos, en cambio las líneas celulares de CaP T2E-negativas no mostraron actividad, demostrando que la actividad del tratamiento AVE1642 requiere la expresión de T2E. También descubrimos que la conjugación del PGA al AVE1642 alteró el tráfico celular. La microscopía confocal/STORM en la línea celular VCaP determinó que el AVE1642 colocalizaba principalmente con endosomas y clatrina, y el PGA-AVE1642 colocalizaba mayormente con caveolina-1 pero no con endosomas ni clatrina. Además, nosotros evaluamos el mecanismo de acción descubriendo un patrón de inhibición diferente. Mientras que el AVE1642 inhibió la vía PI3K, el PGA-AVE1642 inhibió tanto la vía PI3K como la vía MAPK. Los análisis posteriores in vivo en un modelo ortotópico de ratón de CaP recientemente desarrollado utilizando la línea celular VCaP con expresión de luciferasa revelaron una mayor actividad antitumoral para el PGA-AVE1642 en comparación con AVE1642. Estudiamos el mecanismo de acción en las muestras tumorales, las cuales una vez más, encontramos que el tratamiento con PGA-AVE1642 inhibió las dos vías de señalización PI3K y MAPK. Además, estudios posteriores basados en el microambiente tumoral mostraron que el tratamiento con PGA-AVE1642 presentaba una alta inhibición de la funcionalidad y madurez de los vasos sanguíneos tumorales, de la proliferación celular y de la angiogénesis en comparación con el tratamiento con el AVE1642. La terapia de combinación utilizando AVE1642/PGA-AVE1642 con abiraterona resultó en un efecto antitumoral sinérgico en las células T2E-positivas. Curiosamente, los estudios in vivo también demostraron esta sinergia a través de la terapia de combinación empleando AVE1642 y abiraterona, por el contrario, la terapia de combinación empleando PGA-AVE1642 y abiraterona reveló la misma eficacia antitumoral en comparación con la terapia PGA-AVE1642 libre. Para explorar este resultado, investigamos las vías de señalización en las muestras tumorales, las cuales mostraron la inhibición de las vías de señalización PI3K y MAPK después de ambas terapias de combinación. Nuestros estudios demostraron que la conjugación de los polipéptidos mejora la terapia del AVE1642 en el modelo avanzado de CaP, la cual podría representar una terapia prometedora para los tumores de CaP T2E-positivos.Prostate Cancer (PCa) is the second most prevalent cancer in men. Studies have demonstrated the presence of a TMPRSS2 (androgendependent serine protease) and ERG (ETS family transcription factor) fusion gene (T2E) as a potential PCa biomarker. PCa progression involves both the Androgen Receptor (AR) and Insulin-like growth factor 1 receptor (IGF-1R), and treatment of an anti-IGF-1R inhibitor (AVE1642) results in potential therapeutic effects in T2E positive cells. Therefore, the development of personalized therapies based on polymer therapeutics may support the treatment of the specific PCa subtype. IGF-1R inhibition by AVE1642 therapy provides anti-tumoral effects in T2E expressing PCa cells. We investigated AVE1642 anti-tumoral activity after conjugation to a biodegradable and biocompatible polypeptide (poly-L-glutamic acid (PGA)). T2E-expressing VCaP PCa cells responded to both PGA-AVE1642 and AVE1642, while T2E negative PCa cell lines did not, demonstrating that AVE1642 activity requires T2E expression. While AVE1642 treatment led to IGF-1R internalization, the PGA-AVE1642 displayed enhanced stability and affinity binding to IGF-1R preventing the IGF-1R internalization. We discovered that PGA-conjugation to AVE1642 altered the cellular trafficking. Confocal/STORM microscopy demonstrated that the AVE1642 mainly colocalized with endosomes and clathrin, while PGA-AVE1642 mostly colocalized with caveolin-1 but not with endosomes or clathrin. We evaluated the mechanisms of action, discovering a different pathway inhibition. While the free AVE1642 inhibited the PI3K pathway through IGF-1R inhibition, PGA-AVE1642 inhibited both the PI3K and MAPK pathways. We established an orthotopic PCa mice model employing luciferase expressing VCaP cells. Our in vivo results revealed higher anti-tumoral activity for PGA-AVE1642 compared with AVE1642. We also studied the mechanism of action in tumor samples, which again suggested PI3K and MAPK pathway inhibition after PGA-AVE1642 treatment. Furthermore, tumor microenvironment studies in ex vivo samples suggested stronger PGA-AVE1642 effect on vessel functionality and maturity, and an improvement of cellular proliferation and angiogenesis inhibition in comparison with AVE1642. The combination therapy AVE1642/PGA-AVE1642 with abiraterone results in synergistic anti-tumoral effect in T2E positive cells. Interestingly, in vivo studies also demonstrated this synergy through the combination therapy with AVE1642 and abiraterone, in contrast, combination therapy using PGA-AVE1642 and abiraterone revealed same anti-tumoral effectiveness in comparison with free PGA-AVE1642 therapy. In order to explore this outcome, we investigated the signaling pathways in tumor samples, which showed PI3K and MAPK pathway inhibition after both combination therapies. Our studies demonstrated that the polypeptide-conjugation improves AVE1642 therapy in advanced PCa model which could represent a promising therapy for T2E positive PCa tumors