Antitumor activity of liposomal formulations oof bis-naphthalimide derivatives and erlotinib, in vitro study in pancreatic cancer cell lines

Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines Dissertation of the Master Degree in Pharmaceutical Technology Rita Isabel Henriques Sério Vidal This work was performed under the supervision of Prof. Doutora M. Helena Vasconcelos and co-supervision of Doutora Sofia Costa Lima - Outubro, 2013 - Dissertation of the Master Degree in Pharmaceutical Technology ii Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines DECLARATION ACCORDING TO THE PRESENT LEGISLATION, IT IS NOT AUTHORIZED THE REPRODUCTION OF ANY PART OF THIS DISSERTATION; The author Dissertation of the Master Degree in Pharmaceutical Technology iii Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines AGRADECIMENTOS Chegado o fim desta jornada, resta-me distribuir agradecimentos por todos que me acompanharam. Em primeiro lugar quero agradecer à minha orientadora, Professora Doutora Helena M. Vasconcelos, pela confiança depositada em mim, dando-me a oportunidade de desenvolver a minha tese nesta área. Agradeço-lhe por todos os ensinamentos e apoio na área de cultura e análise celular. Agradeço à minha co-orientadora, Doutora Sofia Costa Lima, pelo grande apoio e disponibilidade na partilha dos seus conhecimentos, tanto a nível de vetores lipossómicos bem como a nível de cultura celular. Não posso deixar de agradecer pela sua paciência, e por tentar sempre incutir-me espírito crítico e o entendimento do real porquê das coisas. Gostaria de agradecer à Professora Doutora Anabela Cordeiro da Silva, por todo o apoio, pela disponibilização de equipamento e do seu laboratório, mas com maior importância devo agradecer pela confiança depositada e pelas palavras de incentivo e encorajamento. Agradeço à Doutora Raquel Lima, que me iniciou no universo da cultura celular e que sempre esteve disponível para todas e quaisquer questões levantadas. Agradeço por todos os ensinamentos, pela disponibilidade nas análises de citometria (nem sempre foram fáceis), e por todas as conversas de encorajamento e palavras de apoio quando as coisas corriam menos bem. Não posso deixar de agradecer a Diana Sousa que foi um apoio incondicional, sempre com um sorriso e grande otimismo, fazendo-me sempre acreditar que tudo estaria a correr bem. À minha colega e amiga Vanessa Duarte agradeço-lhe por ser a melhor companheira que poderia pedir para fazer esta travessia comigo. Durante estes 2 anos de Mestrado foi o meu maior apoio e nunca me deixou pensar sequer em desistir. Agradeço-lhe pela sua presença em todos os momentos, nos risos, nos choros e pela sua amizade. Não teria sido a mesma coisa sem ti. Agradeço ao Nuno, que desde o momento que pensei em ingressar no Mestrado se manteve a meu lado, e acreditou sempre em mim (mais do que eu, por vezes). Agradeçolhe por toda a paciência nas minhas crises existenciais e ausências em prol do Mestrado, e por toda a força que me dá todos os dias. Por último, mas não menos importante gostaria de agradecer à minha família, em particular à minha mãe que desde pequena me incentiva a ultrapassar e redefenir os meus limites, acreditando sempre que serei mais uma vez capaz de me superar. Dissertation of the Master Degree in Pharmaceutical Technology iv Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines ABSTRACT Pancreatic cancer (PC) remains one of the most aggressive types of cancer, resulting in a 5 years survival rate of less than 5%, lacking in new therapies that present higher rates of success. New therapeutic alternatives using nanotechnology can be an interesting approach to novel or already commercialized drugs used for PC therapy. Liposomes are nanocarriers able to enhance stability and half-life of the loaded compound, increase solubility of poorly water soluble compounds, and improve bio-distribution and active targeting to the required pathological area of interest. The purpose of this work was to evaluate the in vitro pancreatic antitumor activity (in the BxPC-3 cell line) of bis-naphthalimide derivatives BNIPDaCHM and NPA, and of a commercial tyrosine kinase inhibitor, erlotinib (Tarceva®). In addition, the most potent compounds were further evaluated in vitro, following their incorporation in liposomal nontargeted formulations and within anti-EGFR immunoliposomes. Cytotoxicity assays were carried out with the sulforhodamine B assay, in human pancreatic cancer BxPC-3 cells. To understand the mechanisms of action of the compounds/drug, the cell cycle profile of BxPC-3 cells treated with BNIPDaCHM and erlotinib was analyzed by flow cytometry. In addition, the levels of apoptotic programmed cell death in cells treated with the compounds was analyzed by flow cytometry, and confirmed by immunodetection. Moreover, compound-loaded liposomes formulations were obtained by lipid film hydration, and extruded. Anti-human EGFR antibody coupling to liposomes was performed by a carbodiimide reaction, in order to obtain immunoliposomes. Finally, the liposomes as well as immunoliposomes’ sizes, polydispersity index and zeta potential measurements were determined by dynamic light scattering. Encapsulation efficiency of the compounds in the liposomal formulations was determined by fluorimetry and the liposomal formulations were observed by transmission electron microscopy. From the three compounds tested, the most potent were BNIPDaCHM and erlotinib (IC50 concentrations of 1.3±0.05 µM and 1.7±0.06 µM, respectively). The cell cycle profile evaluation indicated that treatment of cells with the IC50 concentration of BNIPDaCHM caused a 13% decreased in the G1 phase of the cell cycle and treatment with 2xIC50 concentration of erlotinib caused a 15% decrease in the G1 phase of the cell cycle. Drug and compound treatments also caused a slight increase in the % of programmed cell death by apoptosis (erlotinib caused 18.9±1.7% and BNIPDaCHM caused 16.7±1.8 % apoptosis, whereas the control levels were 11.7±1.8 % and 10.8±1.7%, respectively). Liposome formulations presented a mean size > 200 nm, and encapsulation efficiencies of 40.6% for erlotinib-loaded liposomes and 28.5% for BNIPDaCHM-loaded liposomes. Dissertation of the Master Degree in Pharmaceutical Technology v Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines The in vitro cytotoxic evaluation of compound loaded liposomes resulted in a minor alteration of the IC50 values (erlotinib-loaded liposomes presented IC50 concentrations of 1.8±0.1 µM and BNIPDaCHM-loaded liposomes had IC50 concentrations of 1.5±0.1 µM). Immunoliposomes coupling yields were ≤ 50% for all formulations. In vitro activity was analyzed for the erlotinib-loaded immunoliposomes and BNIPDaCHM-loaded immunoliposomes, resulting in a decrease of the IC50 concentration values (IC50 1.2 µM and IC50 0.9 µM, respectively). In conclusion, the preliminary results obtained in this work indicated that the proposed compounds presented some potential for the treatment of pancreatic cancer, and results may also suggested an enhancement in the anti-tumoral activity of the drug/compound when combined with anti-EGFR immunoliposomes. However, further studies need to be undertaken to confirm the enhanced anti-tumor activity of these immunoliposomes. In addition, further work will need to be carried out in order to continue these studies, using other cell lines and animal models, aiming to verify the mechanism of action of these compounds. Keywords: Lipossomes; immunoliposomes; bis-naphthalimide derivatives; erlotinib; pancreatic cancer; BxPC-3 cells. Dissertation of the Master Degree in Pharmaceutical Technology vi Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines RESUMO O cancro pancreático continua a ser um dos tipos de cancro mais agressivos, resultando numa taxa de sobrevivência menor a 5%, devido à falta de novas terapias que apresentem maiores taxas de sucesso. Novas alternativas terapêuticas que utilizem a nanotecnologia poderão ser uma abordagem interessante, quer para novos fármacos como para fármacos que já se encontrem a ser comercializados para a terapia do cancro pancreático. Os lipossomas são nanovetores capazes de melhorar a estabilidade do composto encapsulado, aumentar a solubilidade de compostos pouco solúveis em sistemas aquosos, e aumentar a biodistribuição e direcionamento ativo para a área patológica de interesse. O objetivo deste trabalho foi avaliar a atividade anti-tumoral in vitro (na linha celular pancreática BxPC-3) de dois compostos derivados de bisnaftalimidas, BNIPDaCHM e NPA, e de um fármaco inibidor da tirosina quinase já comercializado, erlotinib (Tarceva®). Para além disso, os compostos mais potentes foram avaliados in vitro após terem sido incorporados em lipossomas e em imunolipossomas anti-EGFR. Os ensaios de citotoxicidade foram efetuados através do ensaio de sulforodamina B em células de cancro pancreático, BxPC-3. De forma a compreender os mecanismos de ação dos compostos/fármaco, o ciclo celular das células BxPC-3 após tratamento com o compostos BNIPDaCHM e o fármaco erlotinib foi analisado através de citometria de fluxo. Os níveis de morte apoptótica nas células tratadas com BNIPDaCHM e erlotinib também foram analisados através de citometria de fluxo, e confirmados por imunodeteção. Por outro lado, formulações lipossómicas com o composto/fármaco encapsulado foram obtidas através da técnica de hidratação do filme lipídico e extrusão. O anticorpo antihumano EGFR foi ligado à superficíe lipossómica através de uma reação de carbodiimida, de forma a se obterem imunolipossomas. Finalmente, as formulações de lipossomas bem como de imunolipossomas foram caraterizadas em termos de tamanho, índice de polidispersão e potencial zeta através de dispersão de luz dinâmica (DLS). A eficácia de encapsulação do composto e fármaco nas formulações lipossómicas foi determinada por fluorimetria e os lipossomas foram observados por microscopia de transmissão eletrónica (TEM). Dos três compostos testados, os mais potentes foram o BNIPDaCHM e o erlotinib (concentrações de IC50 1.3±0.05 µM e 1.7±0.06 µM, respetivamente). A avaliação do perfil do ciclo celular indicou que o tratamento das células com a concentração correspondente ao IC50 do BNIPDaCHM causou uma diminuiçao de 13% na fase G1 do ciclo celular, e que o tratamento com a concentração correspondente a 2xIC50 do erlotinib causou uma diminuição de 15% na fase G1 do ciclo celular da linha BxPC-3. Dissertation of the Master Degree in Pharmaceutical Technology vii Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines Os tratamento com o fármaco e o composto também causaram um ligeiro aumento na % da morte celular por apoptose (erlotinib causou 18.9±1.7% e o BNIPDaCHM causou 16.7±1.8% de apoptose, considerando as percentagens de controlo 11.7±1.8% and 10.8±1.7%, respetivamente). As formulações de lipossomas não funcionalizados apresentaram um tamanho médio >200nm, e eficiências de encapsulação de 40.6% para os lipossomas com erlotinib encapsulado e de 28.5% para os lipossomas com BNIPDaCHM encapsulado. A avaliação da citotoxicidade in vitro dos lipossomas com composto/fármaco incorporado resultou numa ligeira alteração dos valores de IC50 (lipossomas com erlotinib apresentaram concentrações de IC50 de 1.8±0.1 µM e lipossomas com BNIPDaCHM obtiveram concentrações de IC50 de 1.5±0.1 µM). O rendimento da reação de ligação dos anticorpos anti-EGFR para produção dos imunolipossomas foi ≤50% para todas as formulações. A atividade anti-tumoral foi analisada in vitro para os imunolipossomas com erlotinib e BNIPDaCHM incorporados, resultando num decréscimo das concentrações de IC50 (IC50 1.2 µM e IC50 0.9 µM, respetivamente). Em conclusão, os resultados preliminares obtidos neste trabalho indicaram que os compostos propostos apresentam algum potencial para o tratamento do cancro pancreático, e os resultados podem também sugerir que há um melhoramento na atividade anti-tumoral do fármaco/composto quando combinados com imunolipossomas anti-EGFR. No entanto, estudos adicionais precisam de ser realizados, de modo a confirmar a atividade anti-tumoral melhorada com os imunolipossomas. Além disso, estudos adicionais precisam de ser realizados utilizando outras linhas celulares e modelos animais, visando a verificação do mecanismo ação destes compostos. Palavras-chave: Lipossomas; immunoliposomas; derivados de bisnaftalimidas; erlotinib; cancro pancreático; células BxPC-3. Dissertation of the Master Degree in Pharmaceutical Technology viii Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines Table of Contents DECLARATION………………………………………………………… ii AGRADECIMENTOS………………………………………………….. iii ABSTRACT……………………………………………………………... RESUMO………………………………………………………………… iv vi INDEX OF FIGURES…………………………………………………... xi INDEX OF TABLES……………………………………………………. xiii ABBREVIATIONS……………………………………………………… xiv I. Introduction……………………………………………………... 1 1.1. Nanotechnology………………………………………………. 1 1.2. Drug delivery systems……………………………………….. 2 1.2.1. Liposomes as drug carriers…………………………………. 3 1.3. Pancreatic cancer……………………………………………. 5 1.3.1. Clinical approaches for the treatment of pancreatic cancer…………………………………………………………. 6 1.3.1.1. Bisnaphthalimides as potential anticancer drugs……… 7 1.3.2. Targeted therapy with tyrosine kinase inhibitors…………. 9 1.3.3. Targeted therapy with immunoliposomes…………………. 12 II. Aims of project…………………………………………………. 15 Dissertation of the Master Degree in Pharmaceutical Technology ix Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines III. Materials and methods………………………………………... 16 3.1. Materials………………………………………………………. 16 3.1.1. Compounds and lipids………………………………………. 16 3.1.2. Cell culture……………………………………………………. 17 3.2. Methods………………………………………………………. 17 3.2.1. In vitro cell growth inhibition analysis using Sulforhodamine B assay……………………………………. 17 3.2.2. Analysis of cell cycle profile, apoptosis and protein expression……………………………………………………. 18 3.2.2.1. Cell cycle analysis………………………………………... 18 3.2.2.2. Apoptosis analysis………………………………………. 19 3.2.3. Protein expression analysis by Western Blot…………….. 19 3.2.4. Liposomes preparation……………………………………… 20 3.2.4.1. Extrusion of the MLVs…………………………………… 20 3.2.5. Immunoliposomes preparations…………………………… 20 3.2.6. Anti-EGFR antibody quantifications………………………. 22 3.2.7. Liposomes characterization………………………………... 22 3.2.7.1. Particle size, polydispersity index and zeta potential… 22 3.2.7.2. Encapsulation efficiency………………………………… 22 3.2.7.3. Transmission electron microscopy…………………….. 23 3.3. Statistical analysis………………………………………….. 23 Dissertation of the Master Degree in Pharmaceutical Technology 1 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines I. Introduction 1.1. Nanotechnology Nanotechnology involves the creation and utilization of materials, devices or systems on the nanometer scale. This field of science is currently experiencing growing developments being expected to create innovation and to play a critical role in various biomedical applications (not only in drug delivery, but also in molecular imaging, biomarkers and biosensors) (1). For pharmaceutical industry, novel technologies, such as nanotechnology, represent a strategic tool for expanding drug markets. Interestingly, it often costs substantially less to develop new methods of administration for an existing drug (resulting in improved efficacy and bioavailability, together with reduced dosing frequency and lower side effects) than to develop novel drugs. These new ways of administration may be accomplished by incorporating the existing drug into a drug delivery system (DDS) (2). As result of the nanotechnology developments, the pharmaceutical market is changing. Those changes are due to the several benefits that the new formulations of DDS offer, such as a competitive edge after patent expiry, market extension, and reduction of the drug development budget (2). However, many DDS have not been successful in delivering drugs because of their limited ability to specifically reach the target tissue. For example, in cancer chemotherapy, several drugs affect not only tumor cells but also the normal cells. Other problems include premature drug loss through rapid clearance from the blood stream and metabolism. Therefore, a drug delivery strategy which allows to selectively target the tumor and offers a long-circulating period in the blood stream is very much needed (3). New technological approaches, such as the development of new DDS, are being studied and tested not only by pharmaceutical industry but also by academic researchers. Moreover, the development of new DDS is usually a continuum process, from early research to product development, passing through all stages of clinical trials and finally resulting in commercial exploitation (1). Dissertation of the Master Degree in Pharmaceutical Technology 2 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines 1.2. Drug delivery systems Over the past 30 years, nanosized vehicles have received considerable attention as pharmaceutical carriers with a wide range of applications, including: i) vaccine adjuvants, ii) signal enhancers/carriers in medical diagnostics and analytical biochemistry, iii) support matrix for chemical ingredients and iv) solubilize various materials. Moreover, particular attention has been given to their role as penetration enhancers in cosmetic products and as drug delivery vehicles (1). A DDS is defined as an interface between the patient and the drug, and varies according to the administration route (oral, inhalation, transdermal or intravenous), the formulation (including capsules, gels or nanocarriers such as liposomes) or even with the device used (pumps, delivery conduits, reservoirs, patches or transdermal implants) (2, 3). Each year new delivery approaches are exploited and almost every body part has already been studied as a potential route for administrating both classical and novel medicines, potentiating drug delivery to become an interdisciplinary and independent field of research. A targeted and safe drug delivery can improve the performance of classical drugs (which are already commercialized) and have implications in the development and success of new therapeutic drugs. The use of various DDS to enhance the in vivo efficiency of many drugs (and drug administration protocols) has been well established during the last decade (1). Novel DDS have been developed, promising new ways for delivering poorly soluble drugs, unstable drugs, peptides, proteins, glycoproteins, genes, among others (4). Nanocarriers are among the most studied DDS, being mainly associated with drugs which are pharmacologically potent but may not be used as a “free” drug due to: side effects inherent to the high toxicity or less efficacy inherent to the low bioavailability. Nanocarrier systems have demonstrated increased drug bioavailability and stability in the blood stream. In addition, they are also able to deliver the drug into the target tissue, using lower drug doses in order to reduce side effects (1, 5, 6). Nanocarriers able to transport active compounds include: micelles, nanospheres, nanocapsules, niosomes, dendrimers, polymeric nanoparticles, solid lipid particles and liposomes (Figure 1) (7). Dissertation of the Master Degree in Pharmaceutical Technology 3 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines Figure 1- Schematic representation of different nanotechnology-based drug delivery systems. Adapted from (8). The surface modification of pharmaceutical nanocarriers is usually performed to control biological properties of DDS and to simultaneously allow the nanocarriers to perform various therapeutical or diagnostic functions. The most important advantages of such modifications in the nanocarriers include: increased stability and half-life of the drug nanocarriers in circulation, enhanced bio-distribution and active targeting into the required pathological area, among others (9). 1.2.1. Liposomes as drug carriers Alec Bangham discovered liposomes in the 1960’s and, since then, these nanoparticles have been used as a versatile tool in biology, biochemistry and medicine (10). Liposomes are small artificial vesicles of spherical shape, formed with one or more lipid bilayers constituted by amphiphilic lipids (phospholipids) and may include cholesterol. Because of their size, hydrophobic and hydrophilic character, as well as their biocompatibility, liposomes can be efficient systems for drug delivery (3). Dissertation of the Master Degree in Pharmaceutical Technology 4 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines Liposome properties vary substantially with lipid composition, size, surface charge and the method of preparation. Depending on their size and number of bilayers, they may be classified into three classes : i) Small Unilamellar Vesicles (SUV), which are surrounded by a single lipid layer and are 40-100 nm in diameter, ii) Large Unilamellar Vesicles (LUV), which are a heterogeneous group of vesicles similar to SUVs and are surrounded by a single lipid layer, with sizes ranging from 100 nm to 1 µm and iii) Multi-Lamellar Vesicles (MLV), which consist of several lipid layers separated from one another by a layer of aqueous solution, with sizes >1 µm (11). The choice of bilayer components allows determining the fluidity and the charge of the lipid bilayer. For example, saturated phospholipids with long acyl chains form a rigid and impermeable bilayer structure, whereas the unsaturated phosphatidylcholine organic species give much more permeable and less stable lipid bilayers (12). In addition, the presence of cholesterol increases the saturation of the liposomal vesicles, and decreases the fluidity of the lipid bilayer, reducing drug loss. Therefore, cholesterol acts in the lipid bilayer as a modulator, resulting in less permeable liposomal vesicles (11). Moreover, the inclusion of positively or negatively charged lipids into the formulations provides the liposomes a surface charge, important to guarantee the colloidal stability of the formulation. The surface charge of liposomes is determined by measuring the zeta potential, and values higher than ± 30 mV guarantee a stable formulation (13). Recently, developments have been reported on the use of liposomes as drug carriers, particularly for cancer therapy (14, 15). An example of a pegylated liposomal delivery system applied to an approved cancer therapy is Doxil®, a doxorubicin-loaded pegylated liposomal formulation which resulted in an therapeutic option for various human cancers, such as breast, lung or ovarian cancer (16). Indeed, liposomes can be used to improve current cancer treatment regimens due to their capacity to entrap poorly water soluble antitumor drugs (4, 14, 15). Moreover, when an inert polymer (such as polyethylene glycol, PEG) is conjugated to the nanocarrier surface, this conjugation causes a decrease on their uptake by the mononuclear phagocytic system (MPS). This results in long-circulation and stability of the nanocarriers and consequently promotes a passive targeting towards the tumor region (15, 17). Importantly, the surface of liposomes can also be readily modified to improve its specificity for certain organs or tissues, preventing their uptake by healthy tissues. Liposomes have been surface functionalized, by binding specific ligands such as: antibodies and antibody fragments (immunoliposomes), lectins, glycoproteins, oligosaccharides, vitamins or peptides. The ligand can be attached to the liposome by covalent binding to the carrier surface or by electrostatic and hydrophobic insertion into the liposomal membrane (1). These ligands are then capable of directing the liposomes to the region of interest by an “active targeting” process. Dissertation of the Master Degree in Pharmaceutical Technology 5 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines The drugs within surface-modified liposomes have altered characteristics, increasing the bioavailability of the drug and the fraction of drug delivered within the pathological area, thus improving efficacy and/or minimizing drug toxicity (3, 12, 15). 1.3. Pancreatic cancer Pancreas is an organ of approximately 15 cm in length, with a similar shape to a flattened pear (Figure 2), responsible for the secretion of enzymes that help in food digestion, and production of hormones such as insulin. Figure 2- Representation of the human pancreas. The human pancreas is constituted by a head, the body and a tail, and is placed behind the stomach, surrounded by the liver, intestines and spleen. Adapted from (18). Pancreatic cancer (PC) is one of most malignant gastrointestinal cancers and, in its advanced stage, a deadly disease for nearly all affected patients (19). In Portugal, PC accounts for 4% of the mortality associated with cancer, having been responsible for the death of a total of 4.558 patients (70.8 % men and 29.2 % women) during the year of 2010 (18, 20). One of the major factors contributing to the high mortality rate observed in PC patients is the lack of specific markers which allow its early detection. Approximately 80% of PC cases are diagnosed at an advanced stage of the disease and in these cases patients present an intrinsic resistance to radio- and chemotherapy (21). In addition, due to its late detection only a small percentage of patients (15 - 20%) present a resectable tumor (19, 22). Dissertation of the Master Degree in Pharmaceutical Technology 6 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines 1.3.1. Clinical approaches for the treatment of pancreatic cancer Surgical resection is considered the strategy that gives significantly increased survival rates in PC. However, it may only be applied in very few cases, namely those in which the tumor appears to be localized in the pancreas without invasion of important surrounding structures (such as the mesenteric blood vessels located adjacent to the head area of the pancreas). In addition, there should be no evidence of metastatic spread to the liver, lining of the intestines, and other vital organs of the peritoneal cavity (19). Nevertheless, surgical resection alone still results in a very low median overall survival, of approximately 20 months, due to the high rate of relapse. In fact, studies on long-term survival after surgery indicate low 5 to 10 year survival rates (18% and 13 %, respectively). Therefore, surgery without additional complementary treatment is not able to improve survival of patients with PC (19, 21, 23). Improvements have been described in PC’s therapeutic approaches with the use of adjuvant systemic therapies following surgical resection. Adjuvant therapy may be achieved with chemotherapeutic agents only or in combination with radiotherapy, following pancreatic resection (22). Over the past decade, gemcitabine has been considered the reference chemotherapeutic for advanced PC, along with fluorouracil (5-FU) and platinum agents (16). However, tumor recurrence and progression after chemotherapy with gemcitabine have been reported to be as high as 58% (4, 24). Recently, the combination of gemcitabine with paclitaxel or the use of FOLFIRINOX (a combination regimen of oxaliplatin, irinotecan, 5-FU and leucovorin) without gemcitabine, have resulted in an improvement of survival rates compared to treatment with gemcitabine alone (22, 24, 25). Despite the referred improvements in the survival rates with chemo- and/or radiotherapy application after tumor resection, the survival rates are still relatively low (26). Moreover, during treatment, approximately 80% of PC patients become resistant to chemotherapeutic regimens applied to them, resulting in a median survival lower than two years (21, 27). Therefore, new therapeutic strategies are needed to overcome these problems. Currently, the association of conventional cytotoxic drugs with novel nanotechnology-based approaches has allowed enhancing the efficiency of those drugs. An example is the use of gemcitabine entrapped within pegylated liposomes, which has been tested in human PC and interestingly resulted in a significant increased inhibition of tumor growth , both in vitro and in vivo when compared to “free” gemcitabine treatment (16, 28, 29). Dissertation of the Master Degree in Pharmaceutical Technology 7 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines 1.3.1.1. Bisnaphthalimides as potential anticancer drugs DNA intercalating agents are among the most common anticancer drugs currently used in the clinic. These agents intercalate between the base pairs of the DNA (without denaturing it), therefore inhibiting DNA replication and transcription (30, 31). Naphthalimide derivatives are DNA intercalating agents, which were first designed by Braña et al., in 1973 and which have presented antitumor activity (32). The naphthalimide molecule (Figure 3) was previously designed by using active structural components of several anticancer agents, such as the glutarimide rings of cycloheximide, the basic chain of tilorone, and the β-nitronaphthalene of the aristolochic acid (33, 34). Figure 3- Molecular structure of naphthalimide monosubstitued. Adapted from (35). The intercalation of the naphthalimide planar aromatic ring between DNA base pairs distorts the DNA backbone conformation and interferes with DNA-protein interactions (31, 35, 36). Due to their potential as anticancer drugs, a large group of naphthalimides were designed by varying the side chains and ring substitutions, in order to obtain an enhanced cytotoxicity effect and solubility (31, 35). Studies where the side chain of naphthalimides was substituted by carbon, sulphur or oxygen resulted in inactive compounds. Therefore the cytotoxic activity of naphthalimides seems to depend on the presence of a basic terminal group in the side chain (35). Bis-naphthalimide compounds (Figure 4), deriving from naphthalimides, have been developed by Braña et al. by linking mononaphthalimide chromophores with a polyaminelike linker chain, containing at least two amino groups (Figure 4) (37). The required presence of the amino groups is due to the fact that it was proven to be essential for the cytotoxic activity of bis-naphthalimides (36, 38). Dissertation of the Master Degree in Pharmaceutical Technology 8 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines Despite the importance of the presence of the amino group in the linker chain for bisnaphthalimides’ cytotoxicity, several of these agents (with or without the amino substituent) were tested in vitro for their cytotoxicity towards colon cancer cell lines. Results indicated that bis-napthalimides can be more potent than the most active naphthalimide, even without the crucial presence of the amino group. This is due to the own structure of bisnaphthalimides, which per se leads to improvements in the DNA binding capacity, increasing their inherent cytotoxicity (34). Figure 4 - Representation of the standard molecular structure of bis-naphthalimide, where the “point of diversity” represents the linker chain. Adapted from (39). A series of bis-napthalimide compounds, with different substitutions in the chromophore, as well as differences in the length of the linker chain, were designed and synthesized. Several studies of novel bis-naphthalimides showed promising results for cancer therapy, by intercalation into the DNA‘s double helix via the major groove, therefore inhibiting the action of topoisomerase II (31, 34). An example of one of the most cytotoxic bisnaphthalimides, selected for Phase I and II clinical trials in Europe and USA, is elinafide (Figure 5) (34). Figure 5 - Molecular structure bis-naphthalimide elinafide. Adapted from (34). Dissertation of the Master Degree in Pharmaceutical Technology 9 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines The structural features of bis-naphthalimides which have been described as important for the enhanced activity (in human colon cancer cells), are: i) a nitro substitution in the chromophore rings, ii) two internal nitrogen atoms, or iii) an alkyl linker chain of at least three methylene groups (31, 34, 36). Moreover, the development of a novel group of derivatives, the bisnaphthalimidopropyl polyamines (BNIPs) have been primarily described by Lin and Pavlov (40). In these compounds, the natural polyamines, putrescine, spermidine and spermine (Figure 6), as well as oxa-polyamines were incorporated into the linker chain (36, 40, 41). Because BNIPs are also derivatives of naphthalimides, they also present high insolubility in aqueous solutions (41, 42). However, an increase in the number of nitrogen atoms in the linker chain has been proposed to result in an enhanced solubility (40).The incorporation of heteroatoms into the linker chain of the BNIPs has been described to increase the solubility of these compounds, therefore improving their activity and their potential as anticancer agents (40, 41). In addition, the heteroatoms incorporation in the linker chain also strongly stabilized DNA duplexes (40). Figure 6 – Molecular structure of (A) Spermidine, (B) Spermine and (C) Putrescine. Adapted from (43). An example that showed the potential of BNIPs as antimour agents in PC is the study in which BNIPDaoct (a BNIP derivative) was loaded into polyallylamine and cholesteryl (CH5- PAA) sell-assembling polymers and showed potent anticancer activity both in vitro (in the BxPC-3 cell line) and in vivo (using xenograft mice) (44). Dissertation of the Master Degree in Pharmaceutical Technology 10 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines 1.3.2. Targeted therapy with tyrosine kinase inhibitors Active and passive targeting can bring selectivity and specificity to drug delivery. The ideal anticancer strategy would selectively kill tumor cells without affecting normal cells. Cancer therapy has been evolving towards this ideal, through the introduction of targeted therapies, which focus on unique molecules present in tumors or proteins whose expression or function are enriched within the neoplastic tissue. These molecules are commonly designated by molecular targets (1). Molecular-targeted therapy is regarded as an exciting research hotspot in the treatment of tumors because of its high specificity and minimal adverse effects. In the case of PC, new targeted therapeutic strategies have been recently developed. These strategies include the use of small inhibitor molecules or monoclonal antibodies (mAbs) (or their fragments), which are capable of inhibiting the tumor progression (16, 45). Tyrosine kinase inhibitors (TKIs) are small molecules that compete with the ATP binding site of the catalytic domain of several tyrosine kinases (TKs) (46). Since the early 1980’s, epidermal growth factor receptor (EGFR) aberrant expression and activation has been reported in a wide range of human malignancies, such as PC (Table 1) (47, 48). This 170 kDa transmembrane glycoprotein has ligand-dependent intracellular TK activity, being one of the four members of the receptor tyrosine kinase family (ErbB /HER family) constituted by: i) EGFR (ErbB-1), ii) HER-2/neu (ErbB-2), iii) HER-3 (ErbB-3) and iv) HER-4 (ErbB-4). It is present in most cell types, though not hematopoietic cells (49, 50). Table 1 - Expression of EGFR in human cancers. Adapted from (48). Type of tumor % EGFR expression Head and neck 40 - 80 Colorectal 25 - 100 Gastric 33 - 81 Pancreatic 30 - 50 Ovarian 35 - 70 Breast 15 - 37 Prostate 40 - 90 Activation of the EGFR-TK enzyme (epidermal growth factor receptor of the tyrosine kinase) results in its auto-phosphorylation, which drives signal transduction pathways leading to tumor growth and malignant progression. EGFR promotes the activation of TKs resulting in uncontrolled tissue growth, cellular migration and differentiation, as well as Dissertation of the Master Degree in Pharmaceutical Technology 17 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines 3.1.2. Cell culture The human pancreatic cancer cell line, BxPC-3 (American Type Culture Collection – ATCC® CRL-1687™, USA) was cultured and maintained as adherent cell culture in RPMI- 1640 with Ultraglutamine I medium (Lonza, Belgium), supplemented with 5% (v/v) heat inactivated fetal bovine serum (FBS, Gibco, USA) for sulforhodamine B assay or 10% (v/v) FBS for the remaining assays. Cells were incubated at 37 ºC, in a humidified 5% CO2 atmosphere within a CB 210 incubator (Binder, Germany). Once 80 % confluence was reached, cells were trypsinized. For this, cell medium was discarded, cells were washed and further incubated with trypsin (TrypLE™ Express; Gibco, USA), for 10 min at 37 ºC. Trypsin was inactivated with medium supplemented with FBS. Cell suspension was centrifuged for 5 min at 134 x g in a High Speed 350-R centrifuge (MPW, Poland). Cell pellets were re-suspended in fresh medium. Cells were diluted (1:10), transferred into a new flask and further incubated. For the experiments, cells were plated into 96- or 6-well plates at different densities, according to the experiment (which will be explained later on in section 3.2.). Cell number and viability were routinely assessed by counting number of cells in a Neubauer chamber (Haemocytometer; Marienfeld, Germany) under the TS100 (Nikon, USA) inverted microscope. For this, cell suspension was diluted (1:1) with trypan blue solution 0.4 % (Sigma-Aldrich, UK). 3.2. Methods 3.2.1. In vitro cell growth inhibition analysis using Sulforhodamine B assay The Sulforhodamine B (SRB) assay was carried out as previously described (69, 70). Briefly, cells were plated at 1 x 105 cells per well in 96-wells plate and incubated for 24 h. Cells were then treated with appropriate dilutions of compound stocks, prepared in medium with 5% FBS just prior to the assay. Erlotinib was diluted within a concentration range of 10-0.625 μM, BNIPDaCHM was diluted in a concentration range from 2-0.125 μM and NPA within a concentration range of 100-6.25 μM. The effect of DMSO was also evaluated in the growth of this cell line by treating cells with the maximum concentration of DMSO used (0.07 % for erlotinib, 0.03 % for BNIPDaCHM and 1.25 % for NPA). Erlotinib and BNIPDaCHM incorporated in liposomes were diluted within an adequate concentration range from 2 - 0.125 µM were added to BxPC-3 cells (final lipid concentration range from 249 to 16 µg/mL for erlotinib-loaded liposomes and final lipid concentration range 350 to 22 µg/mL for BNIPDaCHM-loaded liposomes). After 48h treatment, cells were fixed in situ with 10% ice-cold trichloroacetic acid (TCA,VWR, Belgium) and incubated on ice for 1 h. Dissertation of the Master Degree in Pharmaceutical Technology 18 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines Wells were then washed 3 times with distilled water and allowed to air-dry at room temperature. SRB solution (0.4% wt/vol in 1% acetic acid) was added to the wells and incubated for 30 min. Plates were then rinsed four times with 1% (vol/vol) acetic acid to remove the unbound dye and left to air-dry at room temperature. Tris-base solution 10 mM (pH 10.5) was added to solubilize the bounded SRB. Following a minimum period of 15 min, optical density was measured at 510 nm in a Synergy HT microplate reader (BioTek, USA). Dose-response curves were generated from the plotted results and the IC50 (corresponding to half maximal inhibitory concentration) was calculated. OD values were plotted against the concentration values and the IC50 determined by linear regression, using the equation . 3.2.2. Analysis of cell cycle profile, apoptosis and protein expression Cells were plated at 3 x 105 cells per well (in 6 well plates) in 10% FBS supplemented medium and incubated for 24 h. Cells were then treated with medium only (blank cells), with compounds/drug:BNIPDaCHM [at its IC50 (1.3 ± 0.05 µM)] and erlotinib [at its IC50 (1.7 ± 0.06) and twice this concentration (2xIC50; 3.4 µM)]. The effect of DMSO on the growth of the cell line was evaluated by treating cells with the maximum concentration of DMSO used (0.05 % for erlotinib and 0.01 % for BNIPDaCHM). Following 48 h, cell pellets were then further processed according to the following procedures. 3.2.2.1. Cell cycle analysis Cell pellets were fixed in 70% ice-cold ethanol for at least 12 hours at 4 ºC. Prior to analysis, pellets were re-suspended in PBS containing 0.1 mg/mL RNase A and 5 µg/mL propidium iodide solution (Sigma-Aldrich, Germany). Cellular DNA content was analyzed by flow cytometry using a FACScalibur (BD Biosciences, USA) flow cytometer plotting at least 10.000 events per sample. The analysis of cell cycle distribution was subsequently carried out using the FlowJo 7.6.5 software (Tree Star, Inc., Ashland, USA) after cell debris and aggregates exclusion. Dissertation of the Master Degree in Pharmaceutical Technology 19 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines 3.2.2.2. Apoptosis analysis For apoptosis analysis, the Human Annexin V-FITC/PI apoptosis detection kit (Bender MedSystems, Austria) was used according to manufacturer’s instructions. Briefly, cell pellets were re-suspended using 400 µL of binding buffer (previously diluted in water) and kept on ice. Annexin V-FITC (5 µL) was added to 195 µL of cell suspension which was then incubated in the dark for 10 min. Propidium iodide (10 µL) was then added to the samples and further incubated on ice for 2 min. Annexin V-FITC/ propidium iodide staining was analysed by flow cytometry, using a FACScalibur (BD Biosciences, USA) flow cytometer plotting at least 10.000 events per sample. Data was analysed using FlowJo 7.6.5 software (Tree Star, Inc., Ashland, USA). 3.2.3. Protein expression analysis by Western Blot For protein expression analysis, cells were lysed with Winman’s buffer (1% NP-40, 0.1 M Tris-HCl pH 8.0, 0.15 M NaCl and 5 mM EDTA) complemented with protease inhibitor, for 45 min at 4ºC, with agitation. The lysates were centrifuged for 10 min at 18188 x g at 4 ºC, to exclude cellular debris. Protein content was quantified using the “DC protein assay kit” (BioRad, USA), according to the manufacturer. Total protein extracts (20 µg) were subjected to SDS-PAGE electrophoresis in 12% Bis-Tris gel and then transferred electrophoretically to a Hybond-C Extra nitrocellulose membrane (Amersham Biosciences, UK). The membrane was stained with Ponceau solution (BioRad, USA), dried and stored at room temperature until analysis. Following incubation in blocking solution [5% non-fat dry, in 0.05% Tween 20 in Tris buffered saline solution (TBS-T)], the membrane was incubated with the following antibodies: goat anti-actin (1:2000, Santa Cruz Biotechnology), PARP-1 (1:2000, Santa Cruz Biotechnology) or Bcl-2 (1:100, Dako). Membranes were then washed and further incubated with one of the following secondary antibodies from Santa Cruz Biotechnology diluted 1:2000: anti-goat IgG, anti-rabbit IgG or anti-mouse IgG. Signal was detected using Amersham™ ECL Western Blotting Detection Reagents (GE Healthcare), the Amersham Hyperfilm ECL (GE Healthcare) and the Kodak GBX developer and fixer (Sigma). Dissertation of the Master Degree in Pharmaceutical Technology 20 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines 3.2.4. Liposomes preparation Liposomes were prepared by the film hydration method (10). Lipids eggPC in chloroform (100 mg/mL) and eggPE-PEG and Chol were dissolved in chloroform at a 60:20:20 molar ratio. Empty liposomes and liposomes loaded with BNIPDaCHM or erlotinib had a final lipid concentration of 5 mg/mL. Briefly, eggPE-PEG and Chol, were weighted in a glass vial, using an ABS 80-4 (Kern & Sohn GmbH, Germany) analytical balance. EggPC in chloroform was added to the glass vial, to obtain a final lipid concentration of 5 mg/mL. Erlotinib or BNIPDaCHM at 100 µM final concentration were added to the corresponding vials. Empty liposomes were prepared using the same procedure, without the addition of any compound. The solvent was slowly evaporated in a heating block at 37ºC under a gentle stream of nitrogen in a hood, for approximately 30 min. Removal of the solvent produced the deposition of lipids as a film on the bottom and walls of the glass vial. The lipidic film was hydrated with PBS pH 7.4, under vortex, in order to have a 5 mg/mL final lipid concentration. 3.2.4.1. Extrusion of the MLVs The extruder LiposoFast (Avestin,Germany) was assembled as specified by the manufacturer initially with the polycarbonate membrane with the highest diameter pore (400 nm), followed by the 200 nm. Hydrated liposomes were passed 20 times through each filter. 3.2.5. Immunoliposomes preparation Carbodiimide conjugation technique reacts with amine and carboxylate groups that are present in antibody molecules with abundance. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), is a water soluble carbodiimide and may form active ester functional groups with carboxylate groups using the compound N- hydroxysulfosuccinimide (NHS). NHS esters are hydrophilic active groups that react rapidly with amines on the target molecules (Figure 10) (56, 64). EDC/NHS-coupled reactions are highly efficient and usually increase the yield of conjugation, in comparison with conjugations using solely EDC (65). Dissertation of the Master Degree in Pharmaceutical Technology 21 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines . Fig.10 - Representation of the chemical reaction EDC/NHS antibody activation. Adapted from (71). For conjugation procedures, an initial antibody/liposome molar ratio of 1:1000 was defined. Erlotinib-loaded and BNIPDaCHM-loaded liposomes and empty liposomes were prepared and extruded, as described previously in 2.2.4., to a final lipid concentration of 10 mg/mL. To perform the conjugation of the anti-human EGFR (anti-EGFR) antibody (Biolegend, USA) with the liposomes, the carboxylate group of the anti-EGFR was activated with N-(3- Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and poly(N- isopropylacrylamide),N-hydroxysuccinimide (NHS) (both from Sigma-Aldrich, USA). For that EDC (2.67 mg/mL) and NHS (6.67 mg/mL) were added to the activation solution with the anti-EGFR (267 µg/mL), and incubated for 2 h at room temperature, with agitation. Excess reagents were removed by centrifugation, for 25 min at 20.000 x g at 4ºC, performed twice using a 5417 R centrifuge (Eppendorf, Germany). The activated anti- EGFR was re-suspended with phosphate buffer saline (PBS) added to each liposome formulation and left incubating overnight at room temperature, with agitation. To separate free activated anti-EGFR antibodies from the functionalized liposomes, the preparation was centrifuged three times under the previous conditions, and the supernatants were kept to further quantification. The liposomes were re-suspended in PBS (pH 7.4) with a 33 mg/mL final lipid concentration, and stored at 4 ºC. Dissertation of the Master Degree in Pharmaceutical Technology 22 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines 3.2.6. Anti-EGFR antibody quantification Quantification of the antibody was performed using a Bradford assay (BioRad, USA), accordingly to the manufacturer’s instructions. Samples were read at OD690nm and, by interpolation in the standard curve, the amount of protein was determined. 3.2.7. Liposomes characterization 3.2.7.1. Particle size, polydispersity index and zeta potential Liposome formulations were diluted 1:10 in distilled water. Each liposome formulation was analyzed by dynamic light scattering (DLS) using a Zetasyzer Nano ZS (Malvern, UK) to determine the particle size, polydispersity index and zeta potential. Measurements were made in triplicate at 25 ºC. 3.2.7.2. Encapsulation efficiency Erlotinib stock solution (2150 µg/mL) was diluted with acetonitrile to obtain standard solutions of concentrations ranging from 1000 to 31.25 ng/mL. The fluorescence intensity of the standard solutions was measured using a FluoroMax-4 spectrofluorometer (Horiba Scientific, France), at 470 nm following an excitation at 247 nm, with a slit of 5 nm. The intensity of the blank solution (acetonitrile) was also measured. BNIPDaCHM stock solution (10 mM) was diluted with 0.01 % Triton X-100 in PBS (pH 7.4) to obtain standard solutions of concentrations ranging from 0.5 - 0.05 μM. The fluorescence intensity of the standard solutions was measured on the spectrofluorometer at 397 nm following an excitation at 350 nm, with a slit of 10 nm. The intensity of the blank solution (0.01 % Triton X-100 in PBS (pH 7.4)) was also measured. After the standard curves were determined for both compounds, liposome formulations with erlotinib were diluted 1:200 with acetonitrile and liposome formulations entrapping BNIPDaCHM were diluted 1:200 with the surfactant [0.01%Triton X-100 in PBS (pH 7.4)], and the samples were read in the spectrofluorometer, under the previous described conditions. By correlation with the standard curves obtained, the encapsulation efficiency of each formulation read was determined. Dissertation of the Master Degree in Pharmaceutical Technology 23 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines 3.2.7.3. Transmission electron microscopy Liposome formulations were observed by transmission electron microscopy (TEM, JEM- 1400 (Jeol, Japan). For this, 20-30 μL of liposomes formulation were placed on carbon coated copper grid, for 1 min, and removed with the help of filter paper. The sample was stained with a drop of uranyl acetate, for 1 min at room temperature. Uranyl acetate excess was removed with filter paper. Samples on the carbon coated copper grid were observed in transmission electron microscope, with 50.000x and 80.000x magnifications. 3.3. Statistical analysis All experimental data are presented as means ± SE. Statistical analysis was conducted using the t-student test. Analysis were performed by comparing treatment with the compounds with blanks, except for the Annexin V/FITC assay in which the analysis was performed by comparing treatment with compounds with the controls. Data was considered statistically significant when P < 0.05. Dissertation of the Master Degree in Pharmaceutical Technology 24 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines IV. Results 4.1. Effect of BNIPDaCHM, NPA and erlotinib on growth inhibition of human pancreatic cells In order to assess the effect of the compounds/drug studied (BNIPDaCHM, NPA and erlotinib) in the in vitro growth of the BxPC-3 cells, the SRB assay was used. This assay measures the amount of protein in the cells, allowing to indirectly analyze cell number. Results after 48 h of treatment (Figure 11), clearly indicate that both BNIP compounds, BNIPDaCHM and NPA, inhibited cell growth, in a dose-dependent effect. Interestingly, regarding erlotinib, a dose-dependent decrease in cell growth is only observed up to 4 µM concentrations, above which a plateau is reached. Figure 11 - Analysis of the effect of (A) erlotinib, (B) BNIPDaCHM and (C) NPA on BxPC3 cell growth determined by sulforhodamine B assay. Results are presented as a percentage of cell growth in relation to blank cells (treated with medium only) and are mean ± SE of at least three independent experiments in quadruplicate. The determined IC50 values (Table 1) showed that, from the compounds studied, BNIPDaCHM was the most potent with an IC50 value of 1.3 ± 0.1 µM, followed by the drug erlotinib with an IC50 value of 1.7 ± 0.1 µM. NPA was found to be the compound that presented less activity, with an IC50 of 70.3 ± 11.1µM. B A A Dissertation of the Master Degree in Pharmaceutical Technology 25 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines Table 2 - IC50 values of erlotinib, BNIPDaCHM and NPA in BxPC-3 cells following 48 h treatment IC50 (µM) BxPC-3 Erlotinib BNIPDaCHM NPA 1.7 ± 0.1 1.3 ± 0.1 70.3 ± 11.1 Data are mean ± SE of at least three independent experiments in quadruplicate. Based on these results, erlotinib and BNIPDaCHM were selected to be further studied regarding their mechanism of action and also to be loaded into a drug delivery system based on liposomes. In order to confirm the effect of erlotinib and BNIPDaCHM, BxPC-3 viable cell number was analyzed using the trypan exclusion assay. For this, the following concentrations were used: BNIPDaCHM at its IC50 (1.3 ± 0.05 µM), erlotinib at its IC50 (1.7 ± 0.06) and twice this concentration (2xIC50; 3.4 µM). The effect of DMSO on the growth of the cell line was also evaluated by treating cells with the maximum concentration of DMSO used in the treatments with compound or drug. Results obtained after 48 h treatment (Figure 12) confirmed that both compounds caused a decrease in BxPC-3 viable cell number. Indeed a decrease to 44.7 ± 2.1% was observed in cells treated with the IC50 of BNIPDaCHM. In the cells treated with erlotinib, a decrease to 57.6 ± 2.8% was observed with the IC50 concentration and to 28.8 ± 1.7%with 2xIC50 concentration (Figure 12). Figure 12 - Analysis of the effect of erlotinib and BNIPDaCHM on BxPC-3: viable cell number determined by trypan blue exclusion assay. Results are presented as percentage of viable cells in relation to blank cells (treated with medium only) and are the mean ±SE of three independent experiments. Dissertation of the Master Degree in Pharmaceutical Technology 26 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines 4.2. Effect of erlotinib and BNIPDaCHM on the BxPC-3 cell cycle profile To initiate the elucidation of the mechanism of action of erlotinib and BNIPDaCHM, the cell cycle profile of BxPC-3 cells was analyzed by flow cytometry 48 h after treatment with the compound or drug. Results showed that treatment with the IC50 concentration of BNIPDaCHM (1.3 ± 0.1 µM) caused a statistically significant decrease (of approximately 13%) in the percentage of cells in the G1 phase of the cell cycle (P < 0.05) (Figure 13). The effect of the IC50 of erlotinib (1.7 ± 0.1µM) was less pronounced, with a smaller decrease (of only 10%) in the percentage of cells in G1 phase being observed following 48 h of treatment. However, when increasing the concentration of erlotinib to 2xIC50, a statistically significant decrease (of approximately 15%) in the G1 population was observed (P <0.05). Figure 13 - Cell cycle profile of BxPC-3 cells treated with erlotinib and BNIPDaCHM. Results are the mean±SE of four independent experiments (except for the DMSO of erlotinib and 2xIC50 of erlotinib treatments which are the mean of 3 experiments). * P<0.05 compared to blank values In addition, following 48h treatment of cells with the IC50 concentration of BNIPDaCHM and with 2xIC50 concentration of erlotinib, a slight increase in the sub-G1 peak (of 3% and 5%, respectively) was observed, which might be suggestive of induction of apoptosis. * * Dissertation of the Master Degree in Pharmaceutical Technology 33 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines In addition, immunoliposome particles presented spherical unilamellar morphology. A ring around the liposomal surface of all formulations could be observed, showing the anti- EGFR coating. Figure 17 – Imaginga of immunoliposomes obtained by TEM. Empty immunoliposomes (A), immunoliposomes with BNIPDaCHM (B) and immunoliposomes with erlotinib (C). 4.8. In vitro cell growth inhibition analysis for empty immunoliposomes and erlotinib and BNIPDaCHM loaded immunoliposomes on BxPC-3 cells In order to assess the effect of the compound/drug loaded in anti-EGFR immunoliposomes on BxPC-3 cells, the SRB assay was performed. Empty immunoliposomes were tested, to certify the non-toxicity of the carriers with the cell line BxPC-3 (Figure 18). Results demonstrated that immunoliposomes when tested up to 1 mg/mL did not affect cell growth (observing 97.3% of cell growth in relation to blank). Nevertheless higher concentrations caused a slight decrease in the percentage of cell growth. In fact, treatment with 2 mg/mL reduced the cell growth to 87.1%, while for the highest concentration (4 mg/mL) the mean percentage of cell growth obtained was 70.1 ± 2.1%. Dissertation of the Master Degree in Pharmaceutical Technology 34 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines Figure 18 - Effect of empty immunoliposomes on BxPC-3 viable cells number determined by the sulforhodamine B assay. Results are presented as a percentage of cell growth in relation to blank cells (treated with medium only) and are from one experiment in quadruplicate. The activity of erlotinib and BNIPDaCHM loaded on anti-EGFR immunoliposomes was also analyzed by SRB assay. Results from one preliminary experiment (Table 7) showed a decrease in the IC50 values for both entrapped erlotinib and BNIPDaCHM. BNIPDaCHM- loaded immunoliposomes presented an IC50 concentration of 0.9 µM and erlotinib-loaded immunoliposomes presented an IC50 of 1.2 µM, both of which are lower than the values obtained with non-functionalized liposomes (non-targeted liposomes with BNIPDaCHM presented IC50 of 1.5 ± 0.1 µM and liposomes with erlotinib presented IC50 of 1.8 ± 0.1 µM). These experiments need to be repeated in order to confirm the results. Table 7 - IC50 values of erlotinib and BNIPDaCHM entrapped immunoliposomes IC50 (µM) Erlotinib BNIPDaCHM eggPC:PE-PEG:Chol (60:20:20) anti-EGFR 1.2 0.9 Data is mean of IC50 quadruplicates, from one experiment only. Dissertation of the Master Degree in Pharmaceutical Technology 35 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines IV. Discussion In the present work the effect of two BNIP derivative compounds, BNIPDaCHM and NPA, and of the TKI, erlotinib, were studied on the BxPC-3 pancreatic cancer cell line. BNIPs have been reported as DNA-binding compounds, with potential for cancer therapy, in particular for NSCLC and breast cancer (38, 73, 74). Previous studies reported that in vitro exposure of a pancreatic cell line to a BNIP derivative (BNIPDaoct) was more effective than gemcitabine (44). The cytotoxicity assays carried out for the BNIP derivatives used in this study clearly indicated that NPA was the less active compound (IC50 of 70.3 ± 11.1 µM), in comparison with BNIPDaCHM (IC50 of 1.3 ± 0.1 µM). These results are in agreement with previous observations revealing that the presence of two naphthalimidopropyl moieties influences the biological activity of BNIP compounds in cancer cells (42, 75). NPA has only one naphthalimidopropyl moiety and consequently is less cytotoxic, as previously described for NSCLC cell lines (42, 75). Structural derivatives features on BNIPs are responsible for enhanced biological activity and solubility, such as the bisnaphthalimidopropyl functionality, an alkyl linker chain length ideally between 8-10 carbons, and the presence of heteroatoms (more than 2 nitrogen atoms) (40-42, 76). Erlotinib was the first biological drug approved by FDA (Food and Drug Administration) for the treatment of advanced pancreatic cancer, combined with the conventional chemotherapeutic agent gemcitabine, presenting improvements on the survival rates of patients (16, 54). This drug is a tyrosine kinase inhibitor, being an EGFR inhibitor, and is also used for other types of cancer such as ovarian, breast and NSCLC (77-79). In vitro assays of erlotinib on the BxPC-3 cell line confirmed its biological activity in this cell line (IC50 of 1.7 ± 0.06 µM). Others have obtained similar results for the same cell line (IC50 = 1.26 µM) (21). The more active of the two tested compound/drug on BxPC-3 cells, BNIPDaCHM and the EGFR inhibitor erlotinib, respectively, were selected to further study their cellular effect. Both affected the cell cycle profile of BxPC-3 cells. BNIPDaCHM and erlotinib elicited a significant (P < 0.05) decrease of 13 and 15%, respectively, in the G1 phase of BxPC-3, in agreement to what has been previously published, in the same cell line for erlotinib and in breast cancer cell line in the case of BNIPDaCHM (21, 75). Results also showed a very small increase in the sub-G1 peak following treatment with BNIPDaCHM at its IC50 and with erlotinib at its 2xIC50 (3 and 5%, respectively), which was suggestive of cell death by apoptosis. Interestingly, previous reports indicated that both erlotinib and BNIPDaCHM increased the sub-G1 peak (21, 75). Dissertation of the Master Degree in Pharmaceutical Technology 36 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines Therefore, the levels of apoptotic cell death were verified for both compounds, by flow cytometry analysis following Annexin V/PI staining. Results showed a slight increase in cell death following treatment with BNIPDaCHM (16.7 ± 1.8%), in comparison with the control (10.8 ± 1.7%). This is in agreement with previous reports, where BNIPDaCHM clearly increased the level of apoptosis in breast cancer cell line (75). In addition, a slight increase in cell death was observed following treatment with erlotinib (IC50 to 18.9 ± 1.7% and for 2xIC50 to 16.3 ± 1.7%) comparatively with the control (11.7 ± 1.8%). However, this effect was only statistical significant for treatment with the IC50 concentration of erlotinib (P < 0.05). Previous reports from other groups have also indicated that erlotinib induced cell death by apoptosis in these cells and also in a hepatocellular cancer cell line (21, 50). The expression levels of apoptosis-related proteins, PARP and Bcl-2 were also analyzed following treatment with both the compounds studied. A slight decrease on the expression of PARP was observed in cells treated with BNIPDaCHM and erlotinib, however these results were not very clear. Regarding the expression of Bcl-2 protein, preliminary results from this study seem to indicate a very small increase in the levels of this anti-apoptotic protein following treatment with both compounds in relation to control cells. Nevertheless, these results need to be further confirmed. In this work, erlotinib and BNIPDaCHM (the most active of both compounds tested), were further incorporated into liposomes. Entrapping the compound/drug in liposomal carriers presents advantages in vitro as well as in vivo, due to the ability of liposomes to retain the entrapped compound while promoting a sustained release of the active substance (1). In addition, because both BNIPDaCHM and erlotinib are lipophilic, liposome entrapment would help their administration in vivo and enhance the drug stability after administration (80). The average size of all produced liposomal formulations was below 200nm [empty liposomes (163.0 ± 0.74nm) < liposomes with erlotinib (174.9 ± 10.3nm) < liposomes with BNIPDaCHM (188.9 ± 13.4nm)]. It is described that nanoparticles with sizes up to 200nm are more stable in vivo, as MPS does not uptake them so easily from the blood stream (11). The determined PDI for the produced liposomal formulations were in average below 0.2 and it is described that PDI values between 0.1-0.2 indicate the presence of a homogenous size distribution (3, 72). Low PDI values are important to ascertain the colloidal stability of the liposomal formulations, since higher PDI values (≥0.5) indicate polydisperse formulations, which may result in particle aggregates (81). Dissertation of the Master Degree in Pharmaceutical Technology 37 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines The encapsulation efficiency obtained for the produced liposomal formulations with BNIPDaCHM and erlotinib were 28.5 ± 1.6 % and 40.6 ± 4.2 %, respectively. The results were promising, particularly for erlotinib, even though not as high as it was expected. Other preparation techniques should be tested in order to enhance the encapsulation efficiency. The liposomes observation by TEM showed spherical unilamellar vesicles with sizes below 200 nm, which are in agreement with the results obtained by DLS. The in vitro cytotoxic activity of BNIPDaCHM and erlotinib incorporated in the liposomes was assessed on BxPC-3 cells. Results showed that the IC50 concentrations obtained with BNIPDaCHM-loaded liposomes (IC50 1.5 ± 0.1) increased slightly in comparison with the “free” compound (1.3 ± 0.05 µM). Even though the IC50 values for BNIPDaCHM-loaded liposomes and for “free” BNIPDaCHM were rather similar, they were considered statistically significant (P <0.05). Erlotinib-loaded liposomes presented a slight increase in the IC50 concentration, with no statistical significance (IC50 1.8 ± 0.1). Literature refers to liposomes as a drug delivery vehicle that can allow a sustained release of entrapped compounds (1, 82). The slight increase in the IC50 of the BNIPDaCHM-loaded liposomes may suggest a more sustained release of the compound into the BxPC-3 cells, allowed by the liposomes; however more assays would need to be undertaken to verify this hypothesis. Immunoliposomes are an attractive strategy to enhance therapeutic approaches, by targeting specifically tumor tissues for delivery of entrapped drugs, and thereby not affecting the normal tissues which are sensitive to the toxic effects of the compounds. The coupling yield of the human anti-EGFR antibody to the drug-loaded liposomes was in average 50%, in agreement with previously published studies using the carbodiimide coupling technique (56). The immunoliposomes were characterized in terms of size, PDI, morphology and encapsulation efficiency. In general, for all formulations, it was observed an increase on the size and PDI values with the functionalization of the liposomes with the EGFR antibody. Preliminary experiments indicate that the sizes of the immunoliposomes with erlotinib were increased, in comparison with the other immune formulations. In addition, it was the only formulation that presented a PDI value ≥ 0.5, indicating a polydisperse formulation. For the immune formulations without any compound or incorporating BNIPDaCHM, a slight increase in their sizes was observed in comparison with the non-functionalized liposomes (BNIPDaCHM-loaded immunoliposomes 228.0 nm and empty immunoliposomes 353.4 nm). Dissertation of the Master Degree in Pharmaceutical Technology 38 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines Previous reports indicated that the conjugation of antibodies in the surface of liposomes can result in an increase of the liposomal size to sizes higher than 200 nm (83). The observation of immunoliposomes by TEM showed spherical unilamellar morphology and the coupling of the antibody on the liposomal surface. TEM imaging also confirmed that immunoliposomes with erlotinib were a polydisperse formulation with particles bigger than 200nm. The in vitro cytotoxicity of the BNIPDaCHM and erlotinib incorporated in the immunoliposomes was assessed on BxPC-3. Preliminary results showed a decrease in the IC50 values, for both BNIPDaCHM and erlotinib following incorporation in the immunoliposomes (IC50 1.2 µM for erlotinib-loaded immunoliposomes and IC50 0.9 µM for BNIPDaCHM-loaded immunoliposomes) in relation to the non-functionalized liposomes. This decrease on the IC50 concentration for both erlotinib and BNIPDaCHM entrapped into immunoliposomes may be due to combination of the compounds with an anti-EGFR antibody. Indeed, other authors have published the advantages of combining two distinct classes of EGFR inhibitors (small molecules TKIs and antibodies anti-EGFR), increasing their antitumor activity and overcoming the tumor resistance to TKIs. These previously published studies were undertaken in vitro using head and neck, prostate and NSCLC cancer cell lines (53). Previous in vivo reports indicate that the association of a DNA-intercalating drug (Doxorubicin) with anti-EGFR immunoliposomes increased its antitumor activity in human breast cancer xenograft mice (58, 60). In the present study, further studies for both BNIPDaCHM and erlotinib are needed, to assess possible alterations to their activity due to the anti-EGFR antibody presence. Dissertation of the Master Degree in Pharmaceutical Technology 39 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines V. Conclusions In the present work, BNIPDaCHM (a BNIP derivative compound) and erlotinib (Tarceva®) were shown to significantly affect the viability of BxPC-3 cells. Both compound and drug were shown to affect the cell cycle profile of these cells, decreasing the percentage of cells in the G1 phase of the cell cycle. In addition, a slight increase in apoptosis was observed in BxPC-3 cells following treatment with BNIPDaCHM or erlotinib. Liposomes prepared by the lipid film hydration method resulted in acceptable size of particles for further in vivo testing; the encapsulation efficiency obtained through this preparation technique was also promising even though not good, having been in average higher than 20 %. Preliminary experiments indicate that erlotinib and BNIPDaCHM incorporation in anti- EGFR immunoliposomes resulted in a decrease of the IC50 values for both erlotinib and BNIPDaCHM. These results suggest that the combination of an anti-EGFR antibody with a small molecule EGFR inhibitor or a DNA-intercalating compound enhance the biological activity for both compounds. However further studies need to be undertaken to confirm the enhanced anti-tumor activity of these immunoliposomes. Dissertation of the Master Degree in Pharmaceutical Technology 40 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines Bibliography 1. Torchilin VP. Multifunctional Pharmaceutical Nanocarriers: Promises and Problems. Nanotechnologies for the Life Sciences: Wiley-VCH Verlag GmbH & Co. KGaA; 2007. 2. Martins Ochubiojo E, Ifeoma Chinwude O, Ekaete Ibanga A, Sabinus Ifeanyi O. Nanotechnology in Drug Delivery. 2012. 3. Lasic DD. Liposomes: from physics to applications: Elsevier; 1993. 4. Ana Catarina Pinto. Combination Chemotherapy in Cancer: Principles, Evaluation and Drug Delivery Strategies, Current Cancer Treatment - Novel Beyond Conventional Approaches. In: InTech, editor.2011. 5. Vega-Villa KR, Takemoto JK, Yáñez JA, Remsberg CM, Forrest ML, Davies NM. Clinical toxicities of nanocarrier systems. Advanced Drug Delivery Reviews. 2008;60(8):929-38. 6. Alexis F, Rhee J-W, Richie JP, Radovic-Moreno AF, Langer R, Farokhzad OC. New frontiers in nanotechnology for cancer treatment. Urologic Oncology: Seminars and Original Investigations. 2008;26(1):74-85. 7. Torchlin VP. Liposomes: Practical approach. 2nd edition ed. Edition n, editor: Oxford University Press; 2003. 380 p. 8. Nature. [cited 2013 22-08]; Available from: www.nature.com. 9. Mayer LD, Cullis PR, Bally MB. Chapter 4.2 - Designing therapeutically optimized liposomal anticancer delivery systems: Lessons from conventional liposomes. In: Lasic DD, Papahadjopoulos D, editors. Medical Applications of Liposomes. Amsterdam: Elsevier Science B.V.; 1998. p. 231-57. 10. Bangham AD, Standish MM, Watkins JC. Diffusion of univalent ions across the lamellae of swollen phospholipids. Journal of Molecular Biology. 1965;13(1):238-IN27. 11. A.J. Domb YT. Particles for Pharmaceutical Applications. Ranch tS, editor: American Scientific Publishers; 2007 2007. 12. Sahoo SK, Labhasetwar V. Nanotech approaches to drug delivery and imaging. Drug Discov Today. 2003;8(24):1112-20. Epub 2003/12/18. 13. Kirby BJ, Hasselbrink EF. Zeta potential of microfluidic substrates: 1. Theory, experimental techniques, and effects on separations. ELECTROPHORESIS. 2004;25(2):187-202. 14. Torchilin VP. Recent advances with liposomes as pharmaceutical carriers. Nature reviews Drug discovery. 2005;4(2):145-60. Epub 2005/02/03. 15. Sávia Caldeira de Araújo L, Cristiane dos Santos G, Talita Guieiro Ribeiro R, Diêgo dos Santos F, Elaine Amaral L, Mônica Cristina O. Liposomes as Carriers of Anticancer Drugs. 2013. 16. Yu X, Zhang Y, Chen C, Yao Q, Li M. Targeted drug delivery in pancreatic cancer. Biochimica et biophysica acta. 2010;1805(1):97-104. Epub 2009/10/27. 17. Lasic DD, Martin FJ, Gabizon A, Huang SK, Papahadjopoulos D. Sterically stabilized liposomes: a hypothesis on the molecular origin of the extended circulation times. Biochimica et Biophysica Acta (BBA) - Biomembranes. 1991;1070(1):187-92. 18. POP. Portal de Oncologia Português. [cited 2013 24-08]; Available from: http://www.pop.eu.com/portal. Dissertation of the Master Degree in Pharmaceutical Technology 41 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines 19. Heinemann V, Haas M, Boeck S. Systemic treatment of advanced pancreatic cancer. Cancer Treat Rev. 2012;38(7):843-53. Epub 2012/01/10. 20. INE. Instituto Nacional de Estatística [cited 2013 20-07]; Available from: http://www.ine.pt/xportal. 21. Ioannou N, Dalgleish AG, Seddon AM, Mackintosh D, Guertler U, Solca F, et al. Anti-tumour activity of afatinib, an irreversible ErbB family blocker, in human pancreatic tumour cells. British journal of cancer. 2011;105(10):1554-62. Epub 2011/10/06. 22. Tamburrino A, Piro G, Carbone C, Tortora G, MELISI D. Mechanisms of resistance to chemotherapeutic and antiangiogenic drugs as novel targets for pancreatic cancer therapy. Frontiers in Pharmacology. 2013;4. 23. Belli C, Cereda S, Anand S, Reni M. Neoadjuvant therapy in resectable pancreatic cancer: a critical review. Cancer Treat Rev. 2013;39(5):518-24. Epub 2012/11/06. 24. Giuliani F, Di Maio M, Colucci G, Perrone F. Conventional chemotherapy of advanced pancreatic cancer. Current drug targets. 2012;13(6):795-801. Epub 2012/03/31. 25. Conroy T, Desseigne F, Ychou M, Bouche O, Guimbaud R, Becouarn Y, et al. FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. The New England journal of medicine. 2011;364(19):1817-25. Epub 2011/05/13. 26. Michael ELM. Medical Therapy of Pancreatic Cancer: Current Status and Future Targets, Pancreatic Cancer - Clinical Management, Prof. Sanjay Srivastava (Ed.)2012. 27. Vikas Bhardwaj SMT, James C.K. Lai and Alok Bhushan. Failure of Pancreatic Cancer Chemotherapy: Consequences of Drug Resistance Mechanisms. In: Srivastava SK, editor.: InTech; 2012. 28. Yu X, Zhang Y, Chen C, Yao Q, Li M. Targeted drug delivery in pancreatic cancer. Biochimica et Biophysica Acta (BBA) - Reviews on Cancer. 2010;1805(1):97-104. 29. Paolino D, Cosco D, Racanicchi L, Trapasso E, Celia C, Iannone M, et al. Gemcitabine-loaded PEGylated unilamellar liposomes vs GEMZAR®: Biodistribution, pharmacokinetic features and in vivo antitumor activity. Journal of Controlled Release. 2010;144(2):144-50. 30. Palchaudhuri R, Hergenrother PJ. DNA as a target for anticancer compounds: methods to determine the mode of binding and the mechanism of action. Current Opinion in Biotechnology. 2007;18(6):497-503. 31. antitumour activity of new dendritic polyamines–(imide–DNA-intercalator) conjugates: potent Lck inhibitors. European Journal of Medicinal Chemistry. 2002;37(7):541-51. 32. Brana MF, Castellano JM, Roldan CM, Santos A, Vazquez D, Jimenez A. Synthesis and mode(s) of action of a new series of imide derivatives of 3-nitro-1,8 naphthalic acid. Cancer chemotherapy and pharmacology. 1980;4(1):61-6. Epub 1980/01/01. 33. Tan S, Yin H, Chen Z, Qian X, Xu Y. Oxo-heterocyclic fused naphthalimides as antitumor agents: Synthesis and biological evaluation. European Journal of Medicinal Chemistry. 2013;62(0):130-8. 34. Bailly C, Braña M, Waring MJ. Sequence-Selective Intercalation of Antitumour Bis- Naphthalimides into DNA. European Journal of Biochemistry. 1996;240(1):195-208. 35. Brana MF, Ramos A. Naphthalimides as anti-cancer agents: synthesis and biological activity. Current medicinal chemistry Anti-cancer agents. 2001;1(3):237-55. Epub 2003/04/08. Dissertation of the Master Degree in Pharmaceutical Technology 42 Antitumor activity of liposomal formulations of Bis-naphthalimide derivatives and Erlotinib: in vitro study in pancreatic cancer cell lines 36. Bestwick CS, Ralton LD, Milne L, Kong Thoo Lin P, Duthie SJ. The influence of bisnaphthalimidopropyl polyamines on DNA instability and repair in Caco-2 colon epithelial cells. Cell biology and toxicology. 2011;27(6):455-63. Epub 2011/08/16. 37. Brana MF, Castellano JM, Moran M, Perez de Vega MJ, Romerdahl CR, Qian XD, et al. Bisnaphthalimides: a new class of antitumor agents. Anti-cancer drug design. 1993;8(4):257-68. Epub 1993/08/01. 38. Ralton LD, Bestwick CS, Milne L, Duthie S, Kong Thoo Lin P. Bisnaphthalimidopropyl spermidine induces apoptosis within colon carcinoma cells. Chemico-Biological Interactions. 2009;177(1):1-6. 39. IPA. Intellectual Property in Australia. [cited 2013 18-09]; Available from: http://www.ipaustralia.com.au/applicant/universidade-do-porto/patents/AU2007273886/. 40. Rodilla V, Pavlov V, Hion D, Lin PKT. Cytotoxic and DNA binding properties of new bisnaphthalimido polyamine derivatives. Toxicology. 2000;148(1):72-3. 41. Dance A-M, Ralton L, Fuller Z, Milne L, Duthie S, Bestwick CS, et al. Synthesis and biological activities of bisnaphthalimido polyamines derivatives: cytotoxicity, DNA binding, DNA damage and drug localization in breast cancer MCF 7 cells. Biochemical Pharmacology. 2005;69(1):19-27. 42. Barron GA, Bermano G, Gordon A, Kong Thoo Lin P. Synthesis, cytotoxicity and DNA- binding of novel bisnaphthalimidopropyl derivatives in breast cancer MDA-MB-231 cells. European Journal of Medicinal Chemistry. 2010;45(4):1430-7. 43. Pavlov V, Kong Thoo Lin P, Rodilla V. Cytotoxicity, DNA binding and localisation of novel bis-naphthalimidopropyl polyamine derivatives. Chemico-Biological Interactions. 2001;137(1):15- 24. 44. Hoskins C, Ouaissi M, Lima SC, Cheng WP, Loureirio I, Mas E, et al. In vitro and in vivo anticancer activity of a novel nano-sized formulation based on self-assembling polymers against pancreatic cancer. Pharm Res. 2010;27(12):2694-703. Epub 2010/09/28. 45. Taciane Ladislau KPM, Renata D Daltoé, Isabella S Guimarães,Rangel and Alice L Herlinger. Target Cancer Therapy, Cancer Treatment - Conventional and Innovative Approaches. Rangel PL, editor: InTech; ,2013. 46. Arora A, Scholar EM. Role of Tyrosine Kinase Inhibitors in Cancer Therapy. Journal of Pharmacology and Experimental Therapeutics. 2005. 47. Tzeng C-WD, Frolov A, Frolova N, Jhala NC, Howard JH, Buchsbaum DJ, et al. Epidermal growth factor receptor (EGFR) is highly conserved in pancreatic cancer. Surgery. 2007;141(4):464- 9. 48. Vlahovic G, Crawford J. Activation of Tyrosine Kinases in Cancer. The Oncologist. 2003;8(6):531-8. 49. Raymond E, Faivre S, Armand JP. Epidermal Growth Factor Receptor Tyrosine Kinase as a Target for Anticancer Therapy. Drugs. 2000;60(2):15-23. 50. Huether A, Höpfner M, Sutter AP, Schuppan D, Scherübl H. Erlotinib induces cell cycle arrest and apoptosis in hepatocellular cancer cells and enhances chemosensitivity towards cytostatics. Journal of Hepatology. 2005;43(4):661-9. 51. Tzeng C-WD, Frolov A, Frolova N, Jhala NC, Howard JH, Vickers SM, et al. EGFR Genomic Gain and Aberrant Pathway Signaling in Pancreatic Cancer Patients. Journal of Surgical Research. 2007;143(1):20-6.