Nanotechnology approaches for improvoved vasoactive intestinal peptide based-drug delivery systems

Rebecca Klippstein Martín CABIMER (Andalusian Centre for Molecular Biology and Regenerative Medicine)-University of Seville Direction and Supervision: Prof. Dr. D. Pozo Perez October, 2013 DIPLOMA THESIS N N A AN NO OT TE EC CH HN NO OL LO OG GY Y A A P PP PR RO OA AC CH HE ES S F FO OR R I IM MP PR RO OV VE ED D B BA AS SE ED D- -D DR RU UG G D DE EL LI IV VE ER RY Y S SY YS ST TE EM MS S O OF F V VA AS SO OA AC CT TI IV VE E I IN NT TE ES ST TI IN NA AL L P PE EP PT TI ID DE E 2 “The scientist is not a person who gives the right answers, he's one who asks the right questions.” Claude Lévi-Strauss NANOTECHNOLOGY APPROACHES FOR IMPROVED BASED-DRUG DELIVERY SYSTEMS OF VASOACTIVE INTESTINAL PEPTIDE 4 1 1. Nanoparticles for biomedical applications 11 Table of contents I. Introduction 9 1.1 Nanomedicine and its actual impact 11 1.2 Drug delivery systems 14 1.3 Types of nanoparticles 21 1.3.1 Metallic nanoparticles 25 1.3.2 Liposomes 29 2. Vasoactive intestinal peptide 35 2.1 Functions of VIP in the central nervous system 37 2.2 Functions of VIP in the immune system 41 2.3 VIP relevance to cancer biology 43 2.4 VIP as a therapeutic agent 46 3. VIP-Engineered Nanoparticles 49 3.1 Rationale for VIP applications 49 3.2 Types of VIP Nanoparticles 52 3.2.1 VIP as a drug for therapeutic applications 52 3.2.2 VIP as a targeting agent to a specific tissue 56 3.3 Clinical Potential of VIP 58 3.4 Current and Future Developments 59 II. Objectives 63 2 Section 1: Vasoactive intestinal peptide metallic nanoparticles for biomedical applications I. Summary and Rationale 67 II. Materials and Methods 69 1. Preparation of vasoactive intestinal peptide nanoparticles 71 1.1 AuNPs synthesis 71 1.2 VIP functionalization 71 1.3 VIP quantification 71 2. Characterization of VIP-metallic nanoparticles 72 2.1 AFM analysis 72 2.2 TEM imaging 72 3. Viability assays 72 3.1 MTT assay 72 4. Enzymatic degradation studies 73 4.1 Peptide degradation assay 73 4.2 Evaluation of protective effect by HPLC analytical method 74 4.3 Intracellular cAMP determinations. 74 III. Results and Discussion 76 1. Characterization of VIP-metallic nanoparticles 77 1.1 Nanoparticle functionalization 78 1.2 Size and surface characterisation: AFM, TEM and DLS 82 1.3 Cytotoxicity of nanoparticles 82 2. In vitro assays 82 2.1 Enzymatic degradation studies 82 2.2Ppetide quantication and cell response to protected VIP-AuNPs 86 3 IV. Conclusions 91 Section 2: Vasoactive intestinal peptide liposomes for targeted drug delivery I. Summary and Rationale 95 II. Materials and Methods 97 1. Prostate cancer cell lines 99 1.1 VPAC1 gene expression analysis by PCR 99 1.2 VPAC1 protein expresion analysis by WB 100 2. Preparation doxorubicin encapsulated VIP-liposomes 101 2.1 Liposome formulation 101 2.2 Doxorubicin encapsulation 102 2.3 VIP functionalization 102 3. Characterization of VIPfunctionalised liposomes 103 3.1 Cryo-TEM and DLS 103 3.2 Quantification of VIP functionalized liposomes 103 3.3 Liposome stability studies in HBS and serum 104 4. Cellular uptake studies 104 5. Cytotoxicity assays 105 5.1 Optical Microscopy imaging 105 5.2 MTT and LDH assays 105 4 6. Cell cycle analysis: Apoptosis studies 106 7. In vivo bio-distribution studies 106 7.1 Prostate canceer xenograft tumour model 106 7.2 Preparation of fluorescent liposomes 107 7.3 In vivo imging 108 7.4 Ex vivo tumour imaging 108 III. Results and Discussion 109 1. Prostate cancer cell lines characterization 111 1.1 VPAC1 gene expression study by PCR 111 1.2 VPAC1 protein expresion study by WB 112 2. Characterization of VIP-liposomes 114 2.1 Size and surface characterisation: cryo-TEM and DLS 114 2.2 Peptide functionalization quantification 115 3. In vitro assays of doxorubicin encapsulated liposomes 117 3.1 Doxorubicin encapsulation efficiency and stability studies 117 3.2 Cellular uptake of doxorubicin encapsulated VIP-liposomes by fluorescence microscopy 119 3.3 Viability studies 121 3.3.1 Optical Microscopy imaging 121 3.3.2 LDH assay and MTT assays 128 3.4 Apoptosis and cell cycle analysis 132 5 3. In vivo studies of fluorescent VIP-liposomes 135 3.1 Biodistribution studies in mouse xenograft models 135 3.2 Ex vivo tumour fluorescence quantification 137 IV. Conclusions 141 V. Appendices 143 1. Abbreviations 145 2. References 147 VI. Aknowledgements 167 INTRODUCTION 12 results) (Rosi and Mirkin 2005), specific targeting to the action site (Drechsler, Erdogan et al. 2004), efficient drug delivery into the target cell (Drechsler, Erdogan et al. 2004), or in vivo real-time monitoring of cellular events (Morawski, Lanza et al. 2005). Such combinatorial NPs may eventually provide the means to achieve “personalised medicine” by tailoring NP functionality to individual responses. In this context, development of bio-functional NPs could dramatically improve already existing NP characteristics and provide a tool to achieve better therapies (Jain 2005). Table 1. European Technology Platform Nanomedicine strategic research. The table shows the importance of smart nano-systems for therapeutic treatments, advanced diagnosis and intelligent delivery to overcome different challenges in cancer, diabetes and neurodegenerative, cardiovascular and inflammatory diseases. An important initiative, led by industry, has been set up together with the European Commission. A group of 53 European stakeholders, composed of industrial and academic experts, has established a European Technology Platform on Nanomedicine (ETPN)1 1 White Paper to the Horizon 2020 Framework Programme for Research and Innovation . The first task of this high level group was to create a vision INTRODUCTION 13 document for this highly future-oriented area of nanotechnology-based healthcare in which experts describe an extrapolation of needs and possibilities until 2020. As it has been highlighted by the ETPN in the white paper recently disclosed for research and innovation, nanotechnology is one of the six Key Enabling Technologies (KETs) that has a significant impact on many different medical developments in three main areas: therapeutics, diagnostics/imaging and regenerative medicine1 (Table 1). Over the last decade the strategic research agendas defined by the nanomedicine community were mainly based on technology and clinical demands. The research and development (R&D) projects implemented under these research priorities in FP6 and FP7 have successfully delivered a lot of new nanomedicines but few products on the market. In consequence, the next level of the strategic development of the ETPN is to emphasise the introduction of “innovation” into the Agenda by improving the translation of nanotechnology R&D into medical applications. Innovation applied to nanomedicine means enabling personalised medicine through stratification of patients by nano-based diagnostic tests or imaging agents, which is a new and different approach to current medical practice. Another example is the combination of a diagnostic test and a therapy within one type of NP thus making some clinical protocols simpler. However, for such innovations to reach the patient, close and well informed interactions between all actors in the nanomedicine research and development chain (academic, industrial, public and private partners) are mandatory, including special structures to actively manage communication and collaboration between all stakeholders, including regulatory bodies to effectively translate and commercialise ideas. One of the main challenges to be addressed by current nanotechnology research and management is to improve the crosstalk between different academic disciplines, different industries (pharmaceutical, medical devices and diagnostics), INTRODUCTION 14 clinical organisations and regulatory agencies to help structure the development of nanomedicine and to provide channels for early stage clinical proof of concept1 Nanomedicines offer the possibility of improving the therapeutic effect of existing drugs at a relatively low cost and risk compared to other areas making it more reliable to invest in the R&D. The global market for nanomaterials products is expected to grow from a global volume of €200 billion in 2009 to €2 trilion by 2015 . 2. In addition, vendors have received funding from various governments, for example, the US, the UK, China and Germany have already invested over US$ 67 billion towards nanotechnology funding. Corporate research and various other forms of private funding are expected to invest almost US$ 250 billion in nanotechnology by 2015. With the market already valued at over US$ 81 billion, additional support from government and private funding has provided the opportunity for many vendors to begin new research projects which will help increase the market growth for the upcoming years3Competitiveness and Innovation Framework Programme (CIP) . Furthermore, , which complements the Research Framework Programme and supports "Entrepreneurship and Innovation" is also running from 2007 to 2013 and has a budget of approximately 3.6 billion Euros4 NP based drug delivery systems are of great importance because they are able to improve the pharmacokinetics and biodistribution of a drug and provide controlled release kinetics to the target site. Drug targeting to specific organs and tissues has become one of the critical endeavours of the new century. The search for new drug delivery systems and new modes of action involves a multidisciplinary scientific . 1.2 Drug delivery systems 1 White Paper to the Horizon 2020 Framework Programme for Research and Innovation 2 European Commission, Nanotechnology: http://ec.europa.eu/nanotechnology 3 Global Nanomedicine Market Report 2012-2016 4 CORDIS, EU Funding Opportunities INTRODUCTION 15 approach to provide major advantages in improving the therapeutic effects and bioavailability of the drug (Baldeschwieler 1997; Beija, Salvayre et al. 2012). An ideal drug carrier should have high drug-loading capacity, low toxicity, favourable drug release kinetics, biocompatibility, and tissue-specific targeting capability. Targeted drug delivery is the ability to direct the drug-loaded system to the site of interest (Huynh, Roger et al. 2010), whereas passive targeting is a preferential accumulation of the drug in a specific tissue. Active targeting involves the surface functionalization of NPs with ligands that are selectively recognized by molecules on the surface of cells of interest (Kumar Khanna 2012). Nanotechnology medical developments over the coming years will have a wide variety of uses and could potentially increase life expectancy (Freitas 2009). Nanomedicines are already moving from passive structures to active structures, through more targeted drug therapies or “smart drugs.” These new drug therapies have already been shown to cause fewer side effects and be more effective than traditional therapies. In the future, nanotechnology will also improve the synthesis of molecular systems that may be similar to living systems. These molecular structures could be the basis for the regeneration or replacement of body parts that are currently lost due to infection, accident, or disease (Verma, Domb et al. 2011). The number of products approaching the Food and Drugs Administration (FDA) approval and review processes will likely grow as time moves forward and as new nanotechnology medical applications are developed. In the present, there is a dramatic increase in the number of reports on the use of NPs in medical applications. However, there is a general need in terms of comprenhensive characterization of the published NP systems, which requires an additional effort towards interdisciplinary research at the nanoscience/pharmaceutical interface. While, it is beyond the capability of many research groups to perform detailed physicochemical characterization, tracking analysis, pharmacokinetics and INTRODUCTION 16 circulatory half-life studies, the scientific community needs methods of standardization for physicochemical characterization and biological evaluation which is required for clinical translation to the field, not only in clinical testing, but also to reduce the number of conflicting data already published in the scientific literature (Howard and Peer 2013). Nanomedicine has the potential to impact significantly on key elements related to the diagnosis and treatment of life-threatening diseases. In this sense, drug delivery in the field of oncology has received a successful input after the introduction of several novel nanomedicine products into clinical trials due to the lack of specific anticancer treatments (Liu, Solomon et al. 2013; Rink, Plebanek et al. 2013). Currently, there is a lot of research going on to design novel nanodevices detecting and treating cancer at its earliest stages by tracking its location within the body and delivering anticancer drugs specifically to malignant cells. Regarding conventional anticancer treatments, they are toxic to both tumour and normal cells, thus the efficacy of chemotherapy is often limited by the side effects of the drugs, the need of higher doses and repeated administrations (Juliano 2013). Therefore, the oncology area has felt the biggest impact from nanomedicine because of the exploitation of the enhanced permeability and retention (EPR) effect in tumour tissues. These newly formed vessels in the tumour are usually abnormal in form and architecture. They consist of poorly-aligned defective endothelial cells with wide fenestrations, lacking a smooth muscle layer, or innervation and with a wider lumen (McDonald and Baluk 2002). The NPs can go through these fenestrations passively and get in contact with the tumour tissue more easily. With the discovery of the EPR effect, passive targeting of chemotherapeutics to solid tumour tissues is an achievable objective given specific particle sizes and chemical characteristics. As its name implies, the EPR effect is the selective accumulation of macromolecules in solid tumour tissue and the retention of those macromolecules within the tissue for a prolonged time due to increased leakage of tumour blood vessels and decreased INTRODUCTION 17 effective lymphatic drainage (Maeda, Fang et al. 2003). Figure 1 summarizes the characteristics of normal and tumour tissues, which can affect the passage and retention of NPs. Figure 1. Enhanced permeation and retention effect (EPR). Upper figure shows the vasculature of normal tissue. This vasculature is not fenestrated and only permits the passage of small molecules. Whereas the lower figure shows the tumour vasculature which is poorly aligned and has wide fenestrations. This permits the leakage of NPs and its accumulation in the tumour site and the following therapeutic effect on the cancer cells. Besides some pathologically-related situations that facilitate the passive transfer of a limited numbers of NPs, multiple obstacles are encountered on the way to their target which affect their bioavailability and must face primary anatomical barriers such INTRODUCTION 18 as the lung epithelium as well as internal barriers including the blood–brain barrier (BBB), the blood–testis barrier (BTB), the blood retina barriers (BRB) and mucosal barriers (Schleh, Semmler-Behnke et al. 2012; Pietroiusti, Campagnolo et al. 2013). The appropriate delivery system for different compounds would be one that protects them from the biological environment, while facilitates their transport though biological barriers. Thus, in order to understand the potential hazard of NPs towards these critical organs, one must understand the nature of biological barriers and how NPs may cross or interact with cellular components of these barriers, leading to detrimental effects. Importantly, each of these NP properties will also likely influence the adsorption of biomolecules onto the surface of NPs, and the bio-corona formed may influence the distribution and toxicity potential of NPs (Pietroiusti, Campagnolo et al. 2013). As a relevant example, over the past few years, many efforts have been made to understand the mechanisms of the NP uptake into the brain. This mechanism appears to be receptor-mediated endocytosis in brain capillary endothelial cells. Modification of the NP surface with covalently attached targeting ligands or by coating with certain surfactants enabling the adsorption of specific plasma proteins are necessary for this receptor-mediated uptake (Wohlfart, Gelperina et al. 2012). Research in designing and engineering long-circulating NPs, so-called ‘stealth’ NPs, has been attracting increasing interest as a new platform for targeted drug delivery, especially in chemotherapy. In particular, the modification of NPs surfaces with poly(ethylene glycol) (PEG) derivatives has shown a decreased uptake of NPs by mononuclear phagocyte system cells and, hence, an increased circulation time, which is an advantage in cancer biology by allowing passive accumulation in the tumour. In the last few years, the new-generation technique of grafting ligands on the NP surface in order to target and penetrate specific cancer cells has been developed. Since ligandreceptor interactions can be highly selective, active targeting allows a more precise and specific treatment of the site of interest (Emerich and Thanos 2007). Surface modification of drug carriers with bioactive molecules can be adsorbed, coated, conjugated or linked to them interact with cell receptors and have demonstrate a INTRODUCTION 19 selective affinity for a specific cell or tissue type and can subsequently enhance drug uptake (Huynh, Roger et al. 2010). For the utilization of functionalized NPs several considerations have to be taken, as the use of targeting ligands can enhance the distribution to secondary target sites of non-intended tissues (Kaminskas, Boyd et al. 2011). In fact, the disadvantage of using non-antibody ligands is their non-selective expression (Allen 2002). But, on the other hand, immunoconjugates pose problems related to immunogenicity and the retention in the reticuloendothelial system (RES). For example, after intravenous injection occurs, NPs are cleared from the plasma within a few minutes due to opsonisation and subsequent phagocytosis by the cells of the RES (Bennewitz and Saltzman 2009). Macrophages in the RES located in the liver and the spleen vividly take up particles bound with serum proteins. In this case, surface modification can reduce the opsonization by minimizing the clearance and leading to improved pharmacokinetic properties. The most common example is PEG, a hydrophilic polymer, which constrains the binding of plasma proteins and prevents aggregation induced by salts and proteins in the serum (Taratula, Garbuzenko et al. 2009). This approach has been used in a variety of NP systems to improve the circulation half-life, and therefore enhancing a key feature related to drug delivery. The surface modification of the NP can also incorporate specific coatings to change the lipophilicity/hydrophobicity profile and prevent the uptake by immune cells to improve cell recognition. This fact prevents opsonisation and recognition from phagocytes and thus avoids undesired immune responses. In addition, PEG can also reduce the access of enzymes and therefore reduce their degradation (Kaminskas, Boyd et al. 2011). In fact, in vivo NPs coated with PEG increase their circulation time from several minutes to many thousand and enhance residence times up to 200-fold in humans (Park, Fong et al. 2009; Gaillard 2010; Habgood and Ek 2010). Therefore, the two most important aspects of NP drug delivery must be: INTRODUCTION 20 1. The coating and functionalization of the NPs for specific targeting to the tissue. The coating of the NPs can modify the circulation time and the pharmacokinetics of the NPs. In addition, functionalization with proteins or antibodies can enhance the delivery of the drug and reduce non-specific associated toxicity. 2. The time release of the drug to prevent non-specific toxicity. Here, the drug must not diffuse out of the particle while it is still in the circulatory system, and must remain encapsulated until the NP reaches the target. Once it gets in contact with the target the loaded drug must be released. In summary, site-specific targeting drug delivery is important in the therapeutic modulation of effective drug dose and disease control. The particle size and surface characteristics (for passive or active targeting) can be selected, the release of the drug can be controlled and different routes of administration can be used according to the treatment (Figure 2). Figure 2. Smart drugs: NP drug delivery for personalised medicine. Traditional drugs are spread after administration throughout the body due to the lack of specificity. In contrast, NP INTRODUCTION 21 specific drug delivery targets tissues reducing the need of high drug doses and producing as a result less systemic toxicity. 1.2 Types of nanoparticles Extensive libraries of NPs, composed of an assortment of different sizes, shapes, and materials, and with various chemical and surface properties, have already been constructed (Schutz, Juillerat-Jeanneret et al. 2013). The field of nanotechnology is under constant growth and new additions continue to complete these libraries. There are a wide variety of NPs which can be multi-task by combining different functionalities in a single stable construct, for example incorporate a drug, a contrast agent and a targeting molecule in the same system (Figure 3). Some of their basic properties and current known uses in biotechnology, particularly in nanomedicine will be described. During the last 30 years, there has been a great improvement in the synthesis and characterization of nanomaterials. Basically, NPs are classified in two main groups depending on the type of material, organic and inorganic. Special attention will be paid to metallic NPs and liposomes, due to the experimental work developed during the current PhD project. INTRODUCTION 28 or oscillating magnetic field stimulation (Reynoso, Lee et al. 2013) and also have the potential to act simultaneously as therapeutic and diagnostic agents, in what is known as emerging theranostic platforms based NPs (Yoo, Lee et al. 2011). In the past decade, metal colloids have been the subject of attention as reflected in a considerable increase in the number of books and reviews. AuNPs are the focus of intense research for biomedical applications, for example for DNA detection (Mirkin, Letsinger et al. 1996), to target cancer cells mediated by peptide functionalization (Kang, Mackey et al. 2010) or deliver drugs like doxorubicin via folate receptor (Asadishad 2010). Aurimune (CYT-6091) is an example of tumour necrosis factor (TNF)-alpha bound to PEG-coated gold NPs (~27 nm) developed by CytImmune Sciences, Inc. for solid tumour therapy (Paciotti, Myer et al. 2004). TNF-alpha is a potent cytokine with antitumor cytotoxicity which requires incorporation into a nanocarrier formulation to reduce systemic toxicity. The results show that NP formulations delayed the tumour growth with local heating using a mammary tumour xenograft mouse model. Furthermore, El Sayed et al. have established the use of gold NPs for cancer imaging by selectively transporting AuNPs into the cancer cell nucleus by conjugating arginine-glycine-aspartic acid peptide (known to target integrin receptors on the cell surface) and a nuclear localization signal peptide which enables the translocation to the nucleus to 30 nm AuNPs via PEG (Nakielny and Dreyfuss 1999; Xue, Atakilit et al. 2001; Gao, Shi et al. 2005). These NPs enable cancer cell specific targeting and the authors demonstrate the specific targeting to the cytoplasm and nuclei of cancer cells over those of normal cells (Kang, Mackey et al. 2010). Gold is one of the most used noble metals in biomedical science, but silver NPs (AgNPs) have as much attention as gold because of their proved lack of toxicity and anti-bacterial properties (Fernandez-Montesinos, Castillo et al. 2009). In addition, AgNPs have unique optical, electrical and biological properties that have attracted significant attention due to their potential use in many applications, such as catalysis, biosensing, drug delivery and nanodevice fabrication (Menon, Jadeja et al. 2013). They INTRODUCTION 29 have received considerable attention in biomedical imaging using SERS. In fact, the SP and large effective scattering cross-section of individual AgNPs make them ideal candidates for molecular labelling (Schultz, Smith et al. 2000). As mentioned before, capped AgNPs have many biomedical applications due to its excellent biocompatibility and antibacterial properties. It has been reported that AgNPs interact with virus, bacteria, and the immune system (Lara, Garza-Trevino et al. 2011). Due to their ability to prevent infections, there are several clinical trials in progress with AgNPs. Antibacterial hand gel and catheters made of AgNPs are being tested to compare their antimicrobial effects against conventional ones (Antonelli, De Pascale et al. 2012). In fact, Furno and Coworkers have developed biomaterials by impregnating silicone coated with silver oxide NPs (Furno, Morley et al. 2004). These novel biomaterials were developed with an aim to reduce the antibacterial infections and for the first time the methodology allows silver impregnation as opposed to coating of medical polymers. Even though these particles are not as widely preferred as compared to AuNPs, they have made a tremendous impact on medical science. The interesting property of the noble metals is that they will be continuously used as newer applications and more protocols are being developed. 1.2.1 Liposomes Liposomes are vesicles containing phospholipid bilayers surrounding an aqueous core, whose diameters can vary from 20 nm to 1 μm. Research on liposome formulations has progressed from conventional vesicles to new generation liposomes. Specifically, the major advantage is that they can encapsulate both hydrophilic and hydrophobic molecules, in their core and membrane respectively (Lopez-Davila, Seifalian et al. 2012). Liposomes are characterized in terms of size, surface charge and number of bilayers. They have been used for more than 30 years as vehicles to improve the delivery of various drugs (Brenner 1989), such as anticancer drugs (doxorubicin), antibiotics (anthracycline, amphotericin B) or vaccines (Baldeschwieler 1997) (Table 1). It exhibits a number of advantages in terms of amphiphilic character, INTRODUCTION 30 biocompatibility and surface modification, being a suitable candidate delivery system for biotech drugs (Rawat, Singh et al. 2006). Hydrophobic molecules can be incorporated into the lipid bilayer of liposomes, while hydrophilic compounds can be entrapped in the aqueous core as shown in Figure 4, although it has been described that liposomes tend to be more unstable when loaded with hydrophobic molecules in comparison to hydrophilic ones (Khan, Rezler et al. 2008). In addition, other formulations such as cationic liposomes, temperature sensitive liposomes and virosomes have been developed by modulating the formulation techniques and liposome composition. The preparation of liposomes are usually simple, rapid and reproducible. (Elizondo, Moreno et al. 2011). The lipids are mixed in an organic solvent, which is then evaporated to form a thin lipid film. Hydration and agitation in an aqueous buffer produces a suspension of large multilamellar vesicles (MLV). These vesicles are too large and heterogeneous and their capacity to carry drugs is small to be useful. Therefore, to prepare small unilamellar vesicles (SUV) of sizes ranging from 50 to 200 nm, sonication or filter extrusion is performed. The membrane has two different phases. One, at low temperatures (between 0°C and the lipid transition temperature) considered as a gel phase and above the transition temperature, where the membrane undergoes a fluid phase. The difference between these two phases seems to be the way in which lipids are arranged in the membrane. Above the phase transition liposomes are more malleable, for this reason redispersion in buffer and extrusion should be both made in this phase. Liposomes have been used as drug delivery systems in various therapeutic applications such as gene therapy, drug-targeting and many others, mainly because of the following advantages (Budai and Szogyi 2001): 1. They are biodegradable and non-toxic. 2. Most molecules can be incorporated into liposomes without modification at a very high concentration. INTRODUCTION 31 3. Biologically active molecules are protected by the lipid bilayer of liposomes from damage by chemicals and enzymes after injection into the blood stream of an organism. 4. Healthy tissues are also protected from the toxicity of the entrapped molecules. Conventional liposomes are composed of phospholipids and cholesterol. These liposomes are rapidly taken up either by the organs of the RES such as the spleen and liver or by phagocytic cells such as macrophages. Different liposomes have been developed to improve their stability in vivo (Cai, Wang et al. 2012; Sax and Kodama 2013). To prolong liposomes half-life in the body circulation liposomes are coated with PEG which reduces the interaction with plasma proteins or receptors. These liposomes are known as “stealth liposomes”. Liposome drug loading can be achieved either passively (the drug is encapsulated during liposome formation) or actively (after liposome formation) (Kulkarni, Betageri et al. 1995). Hydrophobic drugs, for example amphotericin B or taxol, can be directly combined into liposomes during vesicle formation. In this case, the amount of uptake depends on drug-lipid interactions. For hidrophilic drugs, the loading depends on the ability of liposomes to trap aqueous buffer containing the dissolved drug during vesicle formation. Trapping effectiveness of hidrophilic drugs is generally less than 30% due to the limited trapped volume. On the other hand, watersoluble drugs that have protonizable amine functions can be actively entrapped by employing pH gradients, which can result in trapping effectiveness close to 100% (Gubernator 2011). Liposomal formulations form one of the largest groups of clinically approved cancer drug carriers (Table 2) (Chang and Yeh 2012). The best known example is Doxil®, which is the PEG coated liposome formulation of the chemotherapeutic drug called doxorubicin. This drug works by intercalating DNA, with the most serious adverse effect being related to life-threatening heart damage. Doxil® has been INTRODUCTION 32 successful tested in clinical trials for several cancer types and has showed significant improvements in pharmacokinetics profiles, tumour accumulation and reduction in toxicity when compared to the free drug (Gabizon 2001). These results have been proved in a phase III clinical trial in patients with breast cancer, where Doxil® exhibited an equivalent efficacy to free drug. In addition, it showed a reduction of the most serious toxicity which is cardiotoxicity and the decrease of neutrophils, but had more skin and mucosal-related side effects (O'Brien, Wigler et al. 2004). In addition to their capacity for incorporating several agents such as drugs, proteins, siRNA or nanoparticles, they have the ability to conjugate targeting molecules (active targeting). Therefore, these nanocarriers have the potential to be a therapeutic and diagnostic agent (theranostics) (Landen, Chavez-Reyes et al. 2005; Cabral, Nishiyama et al. 2011; Kang, Cho et al. 2011). Another novel approach is triggered drug delivery, where the drug is released after specific stimuli are applied to the target site. Although there are many difficulties encountered in designing a nanocarrier whose triggered release is realistic and effective enough, some of them are already in clinical trials, as is the case of Thermodox®. Thermodox® is a temperature-sensitive doxorubicin liposome that was designed for the treatment of breast and liver cancers and is currently under evaluation in Phase III clinical trials (Lammers, Kiessling et al. 2012). Furthermore, other anticancer drugs besides doxorubicin have been encapsulated in liposomes such as daunorubicin or paclitaxel. In the case of daunorubicin, which is an anticancer drug used for leukemia and a wide variety of solid tumour treatments, the liposomal formulation is called DaunoXome®. These vesicles are composed of DSPC and cholesterol and are small in size (45nm). They are currently under phase III trial and some results have elucidated higher tumour uptake when measured against free drug (Forssen, Coulter et al. 1992; Gill, Espina et al. 1995; Gill, Wernz et al. 1996). Taxol® (paclitaxel) is a marketed product for the treatment of ovarian, breast, non-small cell lung cancer, and AIDS-related Kaposi´s sarcoma INTRODUCTION 33 (Mekhail and Markman 2002). The liposomal formulation of paclitaxel is being developed to potentially reduce toxicities associated with Taxol by eliminating the drug formulation component polyoxyethylated castor oil and it is named LEP-ETU (Zhang, Anyarambhatla et al. 2005). Liposomal formulations have been used for other applications besides cancer treatments, for example in vaccination protocols. The incorporation of viral membrane proteins or peptide antigens into liposomes has shown to potentiate cell-mediated and humoral immune responses, generating solid and durable immunity against the pathogen. These vaccines are reconstituted viral liposomes, constructed without the genetic information of the virus which makes them unable to replicate and cause infection (Stegmann, Morselt et al. 1987; Gluck, Mischler et al. 1992). Some examples of these vaccines are Epaxal and Inflexal V, hepatitis A virus and influenza virus vaccines respectively (Usonis, Bakasenas et al. 2003; Kanra, Marchisio et al. 2004). In this context, studies have revealed so far that virsomal techniques may not be able to give a superior protective immunity in clinic but play an important role in the prevention of morbidity and lethality associated with vaccines themselves. INTRODUCTION 34 Table 2. Liposomal drug formulations in the market. ( Based on Hsin-I Chang et al. 2012) References in Section V.2 (Table References) To date only passive targeted nanocarriers have been approved although it has been demonstrated widely in vitro that actively targeted nanocarriers show higher internalisation and as a result increased cytotoxicity of cancer cells (Lee, Lee et al. 2008; Li, Ding et al. 2009). Increasing the specificity of cancer treatment leads to a reduction in drug toxicity and its consequent side effects and as a result improves the therapeutic outcomes (Allen, Mumbengegwi et al. 2005). As an example of active targeting nanocarriers, liposomes encapsulating doxorubicin and conjugated to the INTRODUCTION 35 F(ab)2 fragment of the human antibody GAH, directed against metastatic stomach cancer is in early phases of clinical trials (Matsumura, Gotoh et al. 2004) . 2. Vasoactive Intestinal Peptide (VIP) Among the neuropeptides, the 28-amino acid vasoactive intestinal peptide (VIP) is a molecule that has evolved from being considered a mere neuropeptide/hormone into a novel agent for modifying immune function and, possibly as a cytokine-like molecule (Pozo and Delgado 2004). Originally identified by Said and Mutt in the late 60’s, VIP was originally isolated as a vasodilator and hypotensive peptide (Piper, Said et al. 1970; Said and Mutt 1970). Subsequently, its biochemistry was elucidated and within the first decade its signature features as a neuropeptide/neurotransmitter became consolidated: it is currently known to act as a neuromodulator in many organs and tissues, including heart, lung, thyroid gland, kidney, immune system, urinary tract and genital organs (Henning and Sawmiller 2001). Sustained interest in therapeutic applications of VIP include areas related to neuroprotection (Gozes, Bachar et al. 1998; Gozes and Furman 2003; Gozes 2008), inflammation and autoimmune disorders (Abad, Martinez et al. 2003; Keino, Kezuka et al. 2004; Gonzalez-Rey, Chorny et al. 2007; Hamidi, Prabhakar et al. 2008; Pozo, Anderson et al. 2009), or asthma (Onoue, Yamada et al. 2007; Said, Hamidi et al. 2010). The amino acid sequence of VIP contains homologies with many gastrointestinal hormones such as pituitary adenylate cyclase-activating peptide (PACAP) and shares some of its receptors (Miyata, Arimura et al. 1989; Miyata, Jiang et al. 1990). In agreement with its widespread distribution, VIP is involved in many physiological and pathophysiological processes related to development, growth, cancers, immune responses, circadian rhythms, control of neuronal and endocrine cells and functions of the digestive, respiratory, reproductive and cardiovascular systems. The VIP peptide is remarkably well conserved across species and is identical in human, cow, pig, rat, dog, INTRODUCTION 36 and goat (Mutt 1988). Even across species, amino acid substitutions are conservative and usually do not result in changes in bioactivity (Singh, Jagannathan et al. 2013). Two heterotrimeric G protein-coupled receptors mediate the actions of VIP, named by VPAC1 and VPAC2, according to the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification (NCIUPHAR), each of them with a unique expression pattern (Harmar, Arimura et al. 1998). VIP and PACAP share receptors VPAC1 and VPAC2, but PAC1 receptor is specific for another peptide called PACAP (Harmar, Fahrenkrug et al. 2012). Each of these receptors is coupled primarily to G proteins, and activate adenylyl cyclase and protein kinase A (PKA), but other pathways are often activated or inhibited in some cells in parallel or downstream of cyclic adenosine monophosphate (cAMP), including pathways involving exchange proteins activated by cAMP (EPACs) (Ster, De Bock et al. 2007), nitric oxide (Murthy, Zhang et al. 1993), phospholipase C (Spengler, Waeber et al. 1993), phosphatidylinositol 3-kinase (Straub and Sharp 1996), MAP kinases (Barrie, Clohessy et al. 1997; Lelievre, Pineau et al. 1998), Jak/STAT, and NFkB (Delgado and Ganea 2000). The physical sites of interaction between VIP and its VPAC1 receptor remained elusive until the development of extensive photoaffinity experiments showing that the side chains of VIP in position 6, 22, 24 and 28 are in direct contact with different amino acids of the receptor VPAC1 N-ted (Tan, Couvineau et al. 2003; Tan, Couvineau et al. 2004; Ceraudo, Murail et al. 2008). Elucidation of VIP structure by nuclear magnetic resonance (NMR) revealed that most of the 28 amino acid sequence has an a-helical structure (sequence 7–28) with the exception of the N-terminal 1–5 sequence, which has no defined structure in solution when unbound to the receptor (Figure 6). The development of a structural model of the VPAC1 receptor N-ted has made it possible to localize the binding site of VIP. The N-ted structure contains two antiparallel b sheets and is stabilized by three disulphide bonds between residues Cys50 and Cys72, Cys63 and Cys105 and Cys86 (Figure 6). This finding is in good agreement with a INTRODUCTION 37 speculative, but largely accepted, mechanism for peptide–ligand interaction with class B GPCRs, which is referred to a ‘two-site’ binding model (Hoare 2005). In this model the central and C-terminal α-helical segments of the ligand are trapped by the N-ted domain of the receptor. In this respect, the integrity of the α-helical conformation seems crucial for the binding to their receptors. Although numerous structure and activity studies have elucidated the molecular interactions between VIP and their receptors, the determination of the structure of VIP receptors is still a challenge and more information will provide and facilitate the emergence of new potent drugs. Figure 6. VIP molecule interaction with VPAC1 receptor (Couvineau and Laburthe 2012). The N-ted structure of VPAC1 contains two antiparallel b sheets and is stabilized by three disulphide bonds. The central and C-terminal α-helical segments of VIP are trapped by the Nted domain of the receptor. 2.2 Functions of VIP in the central nervous system VIP and its receptors are expressed in a wide variety of brain regions. The widespread distribution of VIP, PACAP and their receptors in both, the central nervous system (CNS) and peripheral nervous system (PNS) suggests an important role in the INTRODUCTION 44 by using [123I]VIP as a radioligand and compared the results with computerized tomography (CT). In this study the VIP scan indicated the presence of disease before CT in four patients (Raderer, Kurtaran et al. 1998). Moreover, several studies have reported an effect of VIP and PACAP analogues on tumour growth in animal tumour models, mediated by specific receptors (Moody, Zia et al. 1993; Maruno, Absood et al. 1998). Therefore, VIP and the related peptide PACAP may be of great potential importance for oncology. Recently, a high incidence of PAC1 was found in human gliomas, neuroblastomas, and pituitary adenomas (Robberecht, Vertongen et al. 1993; Robberecht, Woussen-Colle et al. 1994; Vertongen, Devalck et al. 1996), whereas VPAC1 was identified in pancreatic, colorectal, prostate and breast cancers (Jiang, Kopras et al. 1997; Reubi, Laderach et al. 2000). Figure 7. Tumour targeting approach by VIP-NPs and the incidence of VIP-receptors in different human tumours. INTRODUCTION 45 In particular, human prostate carcinomas have been analysed in order to better understand the differences between human normal and malignant prostate tissue. A study from Collado et al. (Collado, Carmena et al. 2005) showed that the level of expression of VIP receptors was about two times higher in adenocarcinoma samples than in normal tissue, measured by a polymerase chain reaction (PCR) method and by enzyme immune-analysis. In addition, Garcia-Fernandez et al detected VPAC1, VPAC2 and PAC1 proteins in solubilized human normal and tumoral prostate membranes by immunoblot and analyzed by immunohistochemistry the presence of VPAC1, VPAC2 and PAC1 (Garcia-Fernandez, Solano et al. 2003). The receptors were mainly located in the epithelial layer of prostate glands and also the presence of PAC1 was observed in some dispersed cells of the stroma (Garcia-Fernandez, Solano et al. 2003). The functionality of these receptors were also confirmed in both, normal and tumoral tissues by VIP stimulation of adenylyl cyclase activity. Moreover, Reubi performed radio-labelled binding experiments in prostate tumours with 125I-VIP and showed 2.5 times more VIP binding sites during malignant transformation (Reubi 1996). Prostate cancer is the most commonly diagnosed cancer and second most common cause of cancer death 1,2 1 . There is currently no consensus on the optimal management of highrisk prostate cancer because there are different primary modalities available, such as surgery or radiation. In addition there is unstandardized timing of different therapies which makes comparisons of efficacy problematic. Increased understanding into the mechanisms leading to the formation of advanced metastatic disease has increased the development of agents to target these pathways. The early diagnosis of prostate cancer is not reliable in all cases and usually men with localized cancer have few or no symptoms at all . The most common test for prostate cancer screening is Prostate Cancer Antigen blood (PSA) test, which measures abnormal levels of PSA in blood. However, there are additional reasons for having an elevated PSA level, and some men http://health.nih.gov/search_results.aspx?terms=Prostate+Cancer 2 http://www.who.int/gho/ncd/mortality_morbidity/cancer_text/en/ INTRODUCTION 46 who have prostate cancer do not have elevated PSA. In addition, the potential complications and harmful side effects of treatments for early prostate cancer, such as surgery and radiation therapy include urinary, problems with bowel function, erectile dysfunction and infection 1,2 Small peptides are valuable tools for clinical applications as they offer many distinct advantages over other bioactive molecules like proteins and monoclonal antibodies ( . For these reasons other alternatives are being studied and other diagnoses methods are being evaluated. The studies mentioned above support the notion that this family of receptors are highly expressed in cancerous tissues and that they are maintained in a functional condition during malignant transformation in the human prostate gland. Therefore, they could be considered as an option for effective diagnosis and targeted drug delivery. 2.5 VIP as a therapeutic agent Okarvi 2008). Small peptides can be easily synthesized and manipulated to optimize their affinity for a particular receptor and to display a more specific biodistribution pattern. Moreover, peptides have the ability to tolerate the harsh conditions of chemical modifications and/or radiolabeling (Heppeler, Froidevaux et al. 2000). Despite the advantages, the difficulty with peptides is often their short biologic half-life, because of their rapid proteolysis in plasma by endogenous peptidases and proteases. A few years ago, from a therapeutic perspective, VIP was identified as a potential bioactive agent for various diseases as mentioned in sections 2.1, 2.2 and 2.3. However, the effective translation of preclinical studies related to VIP to clinical realities faces several major challenges, most of them are common place for other neuropeptides. Thus, one of the major issues for the use of VIP as a therapeutic agent is that once it has been released into the body it is quickly degraded by enzymes, 1 http://health.nih.gov/search_results.aspx?terms=Prostate+Cancer 2 http://www.who.int/gho/ncd/mortality_morbidity/cancer_text/en/ INTRODUCTION 47 leaving the peptide with a very short half-life. In recent years, many studies have been made to elucidate VIP´s beneficial effects, but the native peptide has poor metabolic stability and therefore reduced biological activity (Chapter, White et al. 2010). The VPAC receptors, in particular VPAC1, are very promising targets for the development of therapeutic molecules, but first VIP obstacles have to be solved. While new peptide derivatives specifically targeting VPAC receptor subtypes are now available, however, their short half-life and the inconvenience related to their administration routes make them difficult to use in human therapy. Protein degradation by endogenous proteases of peptides leads to limited bioavailability. This remains as one of the key issues in drug discovery related programs, without a general solution up to date. To tackle these limitations, two main approaches are under way. A first strategy involves the use of covalent and noncovalent reversible inhibitors of specific proteases. A representative example is the case of inhibitors of dipeptidyl peptidase IV (CD26) that inactivates incretin neuropeptides aimed at the treatment of type 2 diabetes or cancer progress (Karagiannis, Paschos et al. 2012; Nisal, Kela et al. 2012). Unfortunately, although in advanced phase clinical trials, this approach can show safety issues (kidney impairment, immune defects or skin lesions) or limited efficacy which result in contradictory approvals from US or EU regulatory agencies (Drucker, Sherman et al. 2011; Monami, Dicembrini et al. 2011). These precautions regarding long-term treatments with specific proteases inhibitors are reflecting the increasing and complex world of the protease degradome where a given protease is often involved in different key physiological processes increasing the chances of adverse effects (Quesada, Ordonez et al. 2009; Turk, Turk du et al. 2012). The second strategy involves the development of small, non-peptide mimic ligands, but since endogenous ligands are often large molecules (20-40 amino acid residues or more) with large corresponding binding pockets within the receptor and diffuse pharmacophoric domains, the resulting mimic molecules often have reduced affinity and selectivity compared to INTRODUCTION 48 their endogenous ligand counterparts (Hoyer and Bartfai 2012). Therefore, neuropeptide applications are still in need of alternative solutions to reduce protein degradation without biological assets impairment. . It is to be hoped that recent advances of our knowledge of the structure of the VPAC receptor binding site and more generally of the class B GPCR ligand requirements will lead shortly to the design of non-peptide receptor agonists and/or antagonists. So far, the development of VIP agonists still has to overcome serious limitations, such as poor stability and short duration of action due to kidney clearance, resulting in high dose administration (Gozes, Lilling et al. 1996).For example, BAY 55-9837, a VPAC-2 selective agonist had several limitations with respect to its degradation and low efficacy (Pan, Li et al. 2007). Other alternatives have been studied such as BAY Q9Q28 showing better stability and high affinity to the receptor (Pan, Li et al. 2007). Furthermore, covalent attachment of long chain PEG molecules is another method used to increase the life span of a peptide by increasing its overall stability and solubility. Most importantly, the sustained plasma concentration of PEGylated peptides can reduce the side effects by reducing the levels of the drug. However, success in peptide PEGylation has been limited by the propensity of the large polymers to interfere with peptide function. In this context, nanotechnological approaches could also be another option. VIP functionalization to NPs could increase their half-life, increasing its efficacy and decreasing the dose needed for treatments. Such molecules would be of considerable interest in the therapy of human diseases, in particular inflammatory, neurodegenerative and cancer-related conditions. For the clinical application of VIP, studies aimed at developing an appropriate delivery system of VIP will be a key direction of future research, and the combination of VIP with nanoparticle features might provide novel insight into the therapeutic potential of VIP and its functionalization to nanoparticles. INTRODUCTION 49 3. VIP-Engineered Nanoparticles 3.1 Rationale for VIP applications Many efforts have been made to obtain highly potent VIP analogues (Igarashi, Ito et al. 2005) focusing on the improvement of stability to create drug candidates for the treatment of several diseases including asthma (Bolin, Michalewsky et al. 1995), neurodegenerative diseases (Gozes 2008), impotence (Gozes, Reshef et al. 1994), septic shock (Delgado and Ganea 2001; Lv, Tang et al. 2009), diabetes (Yung, Dela Cruz et al. 2003) or as a tumour imaging agent (Kothari, Prasad et al. 2007). However, none of these analogues has yet reached the clinical stage. Different VIP ligand modifications have been performed in order to affect the binding to the cognate receptors (VPAC1 and VPAC2). Tandem extensions and additional branching methodologies have been carried out on the C-terminal domain, in an attempt to amplify VPAC1 binding. However, these alterations at the C-terminus showed no significant difference in the activation of the VPAC1 receptor to cAMP production when compared to unmodified VIP (Dangoor, Rubinraut et al. 2007). Nonetheless, manipulations at the C-terminal region of VIP have been shown to affect discrimination between VPAC1 and VPAC2 receptors (Caraglia, Carteni et al. 2008). While modifications of the N-terminus have shown increased affinity for the VPAC2 receptor (Langer, Gregoire et al. 2004). Major drawbacks include internal degradation, as well as a difficult balanced equilibrium between potential side effects and low availability of the peptide at the disease site when systemic peptide doses have to be necessarily increased. In this sense, peptide encapsulation and/or functionalization using NPs are important applications that could solve the limitation of the therapeutic use of peptides owing to their short half-lives caused by enzymatic degradation, catalytic antibodies, and spontaneous hydrolysis in biological fluids. The fact that VIP is so attractive for therapeutic use by itself leads to the study of nano-applications such as a peptide delivery system which may solve the problem of drug break-down by digestive acids and enzymes before they reach their targets INTRODUCTION 50 (Domschke, Domschke et al. 1978). The half-life of VIP needs to be substantially prolonged in biological fluids to be employed in therapeutics with increased effectiveness, as VIP-based drug design is hampered by the instability of the peptide and has limited bioavailability. Therefore, NP encapsulation protects the peptide contained from being broken down too early and has a slow release mechanism which allows a gradual delivery of its contents. It can be predicted that the future of drug delivery involves smart systems which maintain the drug at a desired therapeutic level in the body and avoid the need of frequent administration. Additionally, it would be desirable to use a NP that acts only on a unique receptor or biological site of interest with the possibility of being loaded with a drug at different concentrations. For this reason, VIP has been studied as a method of transport to target cells. A variety of primary human tumours such as breast, prostate, urinary bladder, colon, pancreas and lung cancer, among others, express large numbers of high-affinity receptors for VIP (Reubi 1996; Reubi, Laderach et al. 2000) which may represent the molecular basis for NP applications in cancer. One of the main reasons for the use of peptides and peptide receptors in cancer is the possibility of its targeting. VIP could actively target different cancer cells leading to the delivery of drug compounds or imaging agents to cancer cells that overexpress VIP receptors (Reubi 2003). Moreover, the VIP-NPs-mediates high sensitivity and specificity to detect cancer cells offer the potential for early diagnosis and treatment. This is important as the earlier the diagnosis, the less the cost of patient care, and what is the most important, the higher the chances of treatment success that is normally lower in disease at an advanced stage. INTRODUCTION 51 Figure 8. Flow-chart depicting the VIP-based approach for targeted cancer. Tumours leaky vasculature is commonly used for NP passive targeting, whereas VIP-NPs can use this approach and in addition take advantage of the effects of active targeting to cells that overexpress VIP receptors and as a result increase the specificity of treatments. The rationale behind VIP-functionalized NPs is the fact that the active targeting has a higher specificity for the tumour cells, which results as an enhanced therapeutic effect when compared to the passive targeting. Moreover, the NPs reach the tumour by the leaky vasculature as well as the passive targeting due to their size, but have more affinity for the cells. INTRODUCTION 52 Receptor scintigraphy using radiolabeled peptides for the localization of tumours and their metastases as well as for radiotherapy is used with a clinical impact at the diagnostic and therapeutic level which emerges as an important treatment option. Therefore, VIP-functionalized NPs can be used as a drug delivery systems to a specific cancer tissues well as for in vivo cell labelling and image acquisition. For example, it has been reported that NPs, such as superparamagnetic iron oxide NPs, could be an option as contrast agents for targeted magnetic resonance imaging, which offers a high potential for diagnosis (Schlorf, Meincke et al. 2010; Liao, Wang et al. 2011). As a summary, two main applications can be developed with VIP engineered NPs which will be described in more detail in the next section: 1. VIP encapsulated in NPs or attached to its surface in order to act as a drug itself for therapeutic applications. 2. VIP functionalised NPs as a targeting agent to transport drugs to a specific tissue site. 3.2Types of VIP Engineered Nanoparticles 3.2.1 VIP as a drug for therapeutic applications. Drug delivery of protein and peptide-based drugs, which represent a growing and important therapeutic class, is hampered by these drugs' very short half-lives. High susceptibility towards enzymatic degradation necessitates frequent drug administration followed by poor adherence to therapy. Furthermore, from a quality control and safety point of view, avoidance of degradation and structural transition during manufacturing and/or long-term storage is also considered necessary for developing peptide/proteins-loaded formulation systems. Drug delivery plays a crucial role in the improvement of therapeutic agents since many drugs have unfavourable drawbacks if applied directly. As well as the chemical and metabolic issues, there is a possible drawback that the systemic administration of VIP or other VIP receptor agonists may cause moderate hypotension and/or other adverse effects depending on INTRODUCTION 53 the rate of infusion (Morice, Unwin et al. 1983). Therefore, developing NPs which can work as carriers for a controlled delivery of a peptide is a promising option to improve its availability by reducing its side effects and enhancing its efficacy. Several studies have been developed to establish whether the encapsulation of VIP into NPs enhances its effects or whether its attachment to a surface leads to an appropriate NP transport. Different NPs have been used to encapsulate VIP, such as liposomes (Gao, Noda et al. 1994; Stark, Andreae et al. 2008), biodegradable protamine oligonucleotide NPs (Wernig, Griesbacher et al. 2008) or poly (ethylene glycol)-poly (lactic acid) NPs (Gao, Wu et al. 2007). Depending on the location where the peptide needs to be delivered, the nature of the NP and/or surface ligands are different. Liposomes are widely used for drug delivery due to their unique properties (Lasic 1998). They are biodegradable, typically made from natural molecules and non-immunogenic, which makes them even more attractive in view of their ability to encapsulate a variety of molecules, including VIP. Stark and co-workers demonstrated that the encapsulation of VIP into liposomes protects the peptide from proteolytic degradation while maintaining its biological activity (Stark, Andreae et al. 2008). Furthermore it has been reported that VIP liposome encapsulation enhances its vasoactive effects on systemic arterial blood pressure (Gao, Noda et al. 1994) while another study showed the delayed release of VIP when administered into liposomes within hyaluronic acid gel for uveitis (Lajavardi, Camelo et al. 2009). However, although liposomes can be highly effective as VIP carriers in some cases, this is not the case of VIP transport to the brain. For this reason, other options have been engineered, such as glucose targeted niosomes-nonionic surfactant-based liposomesallowing an efficient delivery of intact VIP to the brain by crossing the bloodbrain barrier following intravenous administration (Dufes, Gaillard et al. 2004) or poly (ethyleneglycol)-poly (lactic acid) nanoparticles modified with wheat germ agglutinin that enhanced VIP transport to the brain by intranasal administration(Gao, Wu et al. 2007). The aim of this study was to investigate the potential of this VIP formulation as a possible delivery system to the brain for the INTRODUCTION 60 size, surface charge and surface chemistry will require robust characterization with overlapping assays. Although still far from VIP-enabled nanoproducts in the market, the areas of healthcare where VIP nanotechnology can make their greatest contributions are cancer research, inflammatory disorders, neurodegenerative diseases and molecular imaging. Undoubtedly, these carriers provide the hope to treat and diagnose several of the mentioned diseases. However, there are some issues that need to be understood in order to ensure their safety and effectiveness. Nevertheless, in the future, novel VIP-NPs could offer new perspectives for the treatment of diseases and new applications will be developed for personalized molecular medicine. 61 62 OBJECTIVES 63 The specific purpose of this project is to study the feasibility as a proof of concept of nanotechnolgy approaches for VIP improved based-drug delivery systems. Two main questions have been addressed using VIP engineered NPs: 1. VIP functionalized gold NPs. In this case, VIP could act as a drug/diagnostic molecule for theranostic applications. 2. VIP functionalised liposomes as a targeting agent to transport drugs to a specific tissue site. The rationale behind the first part of this study is to increase the half-life of VIP as it needs to be substantially prolonged in biological fluids to be employed in therapeutics. VIP-based drug design is hampered by the instability of the peptide and has limited bioavailability. For this reason VIP was functionlized to gold nanoparticles to investigate a possible enzyme protection and the mechanisms involved in this process. In the second part of this study, VIP was used as a directing agent of a drugloaded liposome to prostate cancer cells, and was compared with a non-targeted liposome. The aim of this study was to assess the potential of VIP as a ligand for prostate cancer and evaluate the efficacy of the treatment 64 65 66 SECTION 1: SUMMARY AND RATIONALE 67 Lead optimization is one of the most critical steps in drug development in which nanotechnology-based applications offer great potential. Within the molecules in need of lead optimization, ligands for G protein-coupled receptors (GPCRs) are of particular concern, with at least 40% of drugs currently in the market thought to modulate GPCRs. Neuropeptides are a class of ligands for GPCRs. There have been several important improvements in the development of neuropeptide therapeutics with limited success, mainly due to poor bioavailability after protease degradation. Thus, the development of small, non-peptide mimic ligands results in molecules that often have reduced affinity and selectivity compared to their endogenous ligand counterparts. Likewise, the development of inhibitors of specific proteases increases the chances of adverse effects. In this sense, as a paradigm of other peptide-based therapeutics, the neuropeptide VIP is still not available for treating clinical problems despite its potential. For this reason, we looked for an alternative strategy that simultaneously: a) targets the protease substrate (neuropeptide) instead of the protease, and b) makes use of the entire neuropeptide molecule to retain its full biological activity. Remarkably, although it has been hypothesized that surface functionalization of proteins and bioactive peptides on noble metallic nanoclusters might protect from protease degradation, so far there are no formal proofs in this sense. Our aim is to prove that coating gold NPs with the neuropeptide VIP impairs the hydrolytic activity of extracellular proteases, leading to VIP-mediated functional responses after harsh conditions resembling the extracellular circulating proteases milieu. Combining physical and chemical characterization to determine size, dispersion and homogeneity of VIP AuNPs, by AFM and TEM analysis and quantifying the amount of peptide. This is the first study to address the potential protection from protease degradation upon AuNPs functionalization of a given peptide. Besides the implications in the field of neuropeptides, our study places the concept of surface functionalization in the broader perspective of proteins escaping from SECTION 1: SUMMARY AND RATIONALE 68 extracellular proteases, which could represent a major driven force and an added value to steer the research in the field of engineering NPs. 69 76 SECTION 1: RESULTS AND DISCUSSION 77 1. Characterization of VIP-metallic nanoparticles 1.1 Nanoparticle functionalization Since AuNPs have proper biocompatibility features in terms of reduced in vivo NP destabilization (Bar-Ilan, Albrecht et al. 2009), safety (Kittler, Greulich et al. 2010), and well-tolerant behaviour after systemically delivered in humans,(Davis, Zuckerman et al. 2010) we decided to surface functionalize AuNPs with VIP (Figure 10). Figure 10. Synthetic approach to obtain tiopronin-capped gold NPs functionalized with VIP and PEG. To perform this functionalization, in a first step, tiopronin capped AuNPs were obtained by reduction of HAuCl4 in the presence of tiopronin. After this, VIP was SECTION 1:RESULTS AND DISCUSSION 78 covalently coupled through its amino terminal end via carbodiimide-mediated amideforming reactions with the free carboxyl groups of the tiopronin monolayer after their activation with EDC/NHS. Finally, to prevent nonspecific adsorption of proteins, the remaining activated carboxylated groups, not used to anchor VIP molecules, were masked by reaction with an amino-polyethylene glycol (PEG) derivative of 750 daltons. This functionalization process finally rendered VIP-functionalized AuNPs where VIP C-terminus remains free. VIP has a diffuse pharmacophoric profile, and recent evidences indicate a major role of the C-terminal and central parts of VIP (Couvineau, Ceraudo et al. 2012; Harmar, Fahrenkrug et al. 2012). In this sense, our VIP AuNPs could be suitable to functional coupling to VPAC1 receptors. The method for the synthesis of VIP AuNPs described herein produced NPs with final VIP concentrations ≈1.5 uM, depending on the batch, with an average ratio of 2 VIP molecules/AuNP. This concentration range is ideal for VIP biological applications, as the Kdand EC50 values for VIP receptors are in the nanomolar range. 1.2 Size and surface characterisation: AFM, TEM and DLS The resulting AuNPs were analyzed by transmission electron microscopy (TEM). A representative TEM image of these gold NPs is shown in Figure 11A and the statistics shown in Figure 11B is indicative of a monodisperse distribution of particle diameter, with a mean value equal to 3 nm, whereas the minimum and maximum values are 0.5 nm and 5 nm, respectively. SECTION 1: RESULTS AND DISCUSSION 79 Figure 11. Characterization of AuNPs by TEM.(A) TEM image of AuNPs, scale bar; 50 nm. The inset shows a higher magnification image, scale bar; 5 nm. (B) Statistical analysis of colloid diameter, as evaluated from TEM image. A representative TM-AFM image of AuNPs and VIP AuNPs deposited on a freshly cleaved mica surface is shown in Figure 12 A and B respectively. Single colloids appear stably bound to the mica, and display homogeneous lateral and vertical dimension in the case of AuNPs nanoparticles and show an increased size in the case of VIP AuNPs, due to the surface functionalization. An estimate of particle diameter can be provided by the vertical size of the colloid above the substrate. Particle diameter is approximately 2.5 nm for AuNPs and 3.5 nm in the case of VIP NPs, in close agreement with values obtained from our TEM characterization. Also, the lateral size is monodisperse but subject to significantly broadening because of the well known tip convolution effect (Howald, Haefke et al. 1994). Taken together, we have developed a reliable method to synthesize homogeneous, stable, and properly characterised VIP AuNPs in order to test our working hyphothesis, i.e, the protection from protease degradation upon nanoparticle functionalization. SECTION 1:RESULTS AND DISCUSSION 80 Figure 12. A) Characterization of AuNPs by AFM. Up-left: Tapping mode topography; UpRight: measurement of NPs height (indicated in the topographic image); Down-left: 3D AFM image; Down-right: statistical analysis of colloids height evaluated from AFM image. SECTION 1: RESULTS AND DISCUSSION 81 Figure 12. B) Characterization of Au-PEG/VIP NPs by AFM. Up-left: Tapping mode topography; Up-Right: measurement of NPs height (indicated in the topographic image); Down-left: 3D AFM image; Down-right: statistical analysis of colloids height evaluated from AFM image. SECTION 1:RESULTS AND DISCUSSION 82 1.3 Cytotoxicity studies In order to evaluate if VIP-AuNPs could be used for the treatment of several diseases, cytotoxicity studies were performed. Figure 13 shows the cell viability after 24 h of incubation with AuNPs and AuNPs-PEG/VIP. Over 90 % cell viability was still obtained after 24 h with 10-7 M VIP, in the case of AuNPs-PEG/VIP and its equivalent amount of NPs in the case of AuNPs (16,6 ug). These results showed that AuNPs and AuNPs-PEG/VIP have low toxicity and could be potentially used due to its good biocompatibility as a carrier or platform for VIP therapeutic purposes. Figure 13. Evaluation of AuNPs and AuNPs-PEG/VIP cytotoxicity by MTT assay after 24h. 2. In vitro assays 2.1 Enzymatic degradation studies To evidence the relevance of the functionalization strategy on the VIP AuNP and protease interaction, we carried out a HPLC analysis of VIP degradation products. SECTION 1: RESULTS AND DISCUSSION 83 Figure 14. Functionalized AuNPs impairs hydrolytic protease activity. Experimental approaches. VIP AuNPs were characterized by AFM in order to determine size, dispersion and homogeneity. In the same characterization step VIP functionalization was quantified to determine the initial peptide content. VIP AuNPs were exposed to proteases at different time points and the remaining functionalized peptide was quantified. Furthermore, the resulting functionalized nanoparticles were incubated with cells to study the functional response by cAMP assay. Additional steps consisted in the detection by HPLC of peptides and amino acid residues that have been degraded by proteases and did not remain attached to AuNPs. Finally, these data help to verify the results of the previous step, as they are complementary. That is, the higher the content of free VIP degradation products, the lower the VIP biological activity, and viceversa. As described in details in the Material and Methods section, treatment of equal amounts of soluble VIP with a mix of the proteases trypsin and α-chymotrypsin resulted in different degradation patterns compared to VIP AuNPs (Figure 14). HPLC SECTION 1:RESULTS AND DISCUSSION 84 chromatograms of non-degraded soluble VIP and its degradation products after 10 and 30 minutes of treatment with proteases are depicted in Figure 15A, whereas representative chromatograms of VIP AuNPs degradation products after 10 and 30 min of treatment with proteases are shown in Figure 15B. The number of arrows is a figurative representation of the extension of degraded peptides detected by HPLC in the appropriate retention times (Figure 15; central and lower panels). Our strategy to evaluate VIP protection involves the assessment of free, degraded VIP molecules (see Material and Methods), and that is the reason of the absence of VIP signal in Figure 15B (upper panel), as all the neuropeptide remains surface functionalized to AuNPs in the absence of proteases. On the contrary, a strong signal corresponding to soluble, intact VIP is identified around 25 minutes retention time (Figure 15A). After protease treatment, remarkable differences are observed between VIP and VIP AuNPs degradation patterns, showing higher number of degraded peptides in the case VIP compared to VIP AuNPs (Figure 15; central and lower panels). A main degradation product of soluble VIP is observed after 10 min of protease treatment whereas after 30 min the number of degradation products increase, but none of them predominate (Figure 15A; central and lower panels). In addition, after 10 min of exposure to proteases, the peak corresponding to intact VIP disappears (Figure 15A; central panel). On the other hand, VIP AuNPs show very similar degradation patterns after 10 and 30 min of protease treatment, probably due to the existence non-detectable residues by HPLC (Figure 15A; central and lower panels). This is consistent with a situation in which NP functionalization restraints molecular interactions in context-specific manners between proteases and their substrates, reducing the number of random events. Of particular relevance is the striking reduction in the number of free peptide degradation products, even at the longest exposure with protease (30 min) shown by VIP AuNPs compared to soluble, nonfunctionalized VIP (Figure 15B; central and lower panels). Overall, the previous set of experiments demonstrated a notable degree of VIP protection from protease activity, SECTION 1: RESULTS AND DISCUSSION 85 as the hydrolytic reactions were performed under ideal enzyme reaction conditions used for in vitro evaluation of the protective effects towards other alternative strategies (Smoum, Rubinstein et al. 2006; Sun, Scharff-Poulsen et al. 2008). These observations strongly support the notion that functionalization of the VIP neuropeptide to gold nanoparticles impairs protease activity. Figure 15. HPLC analysis of Au-PEG/VIP degradation products before and after 10 and 30 min exposure to proteases. (A) Chromatograms of VIP after 0, 10 and 30 min of degradation. (B) Chromatograms of VIP AuNPs degradation products after 0, 10 and 30 min. All samples SECTION 1:CONCLUSIONS 92 Figure 19. NP functionalization protects from enzymatic degradation. VIP protection due to NP conjugation produces a functional response on VIP receptor expressing cells. 93 94 SECTION 2: SUMMARY AND RATIONALE 95 Prostate cancer (PCa) is one of the most frequently diagnosed solid tumours in men and is one of the leading causes of mortality in developed countries and with increasing rates in the developing world (Leonetti, Biroccio et al. 2007; Baade, Youlden et al. 2009). The majority of men with newly diagnosed localized prostate cancer may be eligible for active surveillance, surgery or radiation therapy either alone or in combination with androgen deprivation therapy (Stangelberger, Waldert et al. 2008). Moreover, chemotherapy may also be an option for cancers that don't respond to hormone therapy. The decision whether or not to treat localized PCa with curative intent is a patient trade-off between the expected beneficial and harmful effects in terms of patient survival and quality of life (Bangma, Roemeling et al. 2007). Conventional chemotherapy is the most applied treatment for many cancers but has low specificity and limited effectiveness due to its severe side effects. Doxorubicin (Adriamycin) is a broad-spectrum antitumor antibiotic that has been widely used for treatment of several cancers, including breast, ovarian, and prostate cancers (Singal, Li et al. 2000). The effectiveness of doxorubicin is limited due to its high toxicity and side effects, including myelosuppression, alopecia, acute nausea, vomiting, stomatitis, cumulative cardiotoxicity (Rivera 2003), and strong multidrug resistance response in tumour cells after repeated administration (Shen, Chu et al. 2008). Therefore research aims to doxorubicin targeted delivery to PCa cells could be part of the proof of concept studies to reduce repeated administrations and secondary effects as the therapeutic efficacy increases with specific treatments (Sharifi and Steinman 2002; Perrino, Schiattarella et al. 2012). The specific delivery of anticancer drugs to PCa cells has important implications for diagnosis and therapy. Biomarkers that differentiate cancerous tissues from normal tissues can be used as targets for this purpose and one of these attractive molecular targets is VIP receptors which are overexpressed in human PCa compared to normal prostate tissue (Reubi 1995; Reubi 1996) as mentioned in the introduction (section 2.3) in more detail. SECTION 2: SUMMARY AND RATIONALE 96 For this reason VIP liposomes have been synthesized to exploit VIP-R to actively target carriers to PCa and therefore improve its therapy. Particulate carriers such as liposomes with mean size of about 100 nm are passively targeted by predominantly accumulating at certain sites such as tumours due to the presence of leaky vasculature and liposomal extravasation (section 1.2, Figure 1) (Malam, Loizidou et al. 2009). The use of liposomes is recognized as a promising strategy for improving the delivery of anticancer drugs to tumours, leading to a reduction in drug toxicity and improving the therapeutic outcomes (Gosselin and Lee 2002; Allen, Mumbengegwi et al. 2005). Furthermore, VIP phospholipid liposomes were used to encapsulate doxorubicin in order to deliver it to PCa cells. The aim of this study was to assess the potential of VIP as a ligand for prostate cancer targeting by liposomal nanocarriers. Moreover, we wanted to evaluate the effect of a peptide coupling method on the cellular uptake, cytotoxicity and apoptosis of doxorubicin liposomal formulations. We also addressed in vivo experiments in a preclinical setting in order to evaluate the VIP active driven targeting of the prostate cancer cells by liposomes. 97 98 SECTION 2: MATERIALS AND METHODS 99 1.Cell lines HFF-1 (ATCC), DU145 (ATCC), PC3 and LnCAP (ICLC) human prostate cancer cells were kindly provided by Dr. M. Japon (Department of endocrine tumorigenesis and hormonal regulation of cancer, Biomedicine institute of Seville, IBIS, CSICUniversity of Seville, Spain). The cells were maintained in RPMI 1640 medium supplemented with 10 % fetal bovine serum, 1% L-glutamin, 100 IU/ml penicillin and 100 IU/ml streptomycin in a humidified atmosphere of 5 % CO2 at 37 °C. 1.1 VPAC1 gene expression study by PCR The expression level of VPAC1 and the Cyclophilin B housekeeping gene was determined in HFF-1, DU145, PC3 and LnCAP by qPCR and agarose gelelectrophoresis. RNA isolation was performed using TriPure isolation reagent (Roche, Switzerland). To quantify VPAC1 expression levels, equal amounts of cDNA were synthesized using the QuantiTect Reverse Transcription Kit (Qiagen, Valencia, CA, USA). 100 nanograms of cDNA were amplified using 12.5 uL of SYBR qpCR Master Mix (Takara), 200 nM of each primer and H20 up to 25 uL. The PCR amplification scheme was: 10 min at 95 °C followed by 40 cycles at 95°C for 15 sec, 55°C for 30 sec and 72°C for 30 sec. The oligonucleotides were as follows: VPAC1 forward 5ATGTGCAGATGATCGAGGTG-3, reverse 5-TGTAGCCGGTCTTCACAGAA-3, which yield a PCR product of 324 bp. CyclophilinB forward 5-CTTCCCCGATGAGAACTTCA-3, reverse 5-TCTTGGTGCTCTCCACCTTC-3, which yield a PCR product of 193 bp. The oligonucleotides were designed using the following sequences: for VPAC1 NCBI Reference Sequence, NM_004624.3 and for Cyclophilin B, NM_000942.4. SYBRGreen detection was followed by the generation of melting curves and visualization of the products to confirm specificity. Quantitative PCR results were obtained using the comparative Ct method. The threshold crossing value was noted for each transcript and normalized to the internal control. Experiments were performed using an ABI Prism 7900 System (Applied Biosystems), and data processing was performed using ABI SDS v2.1 software (Applied Biosystems). PCR products were SECTION 2: MATERIALS AND METHODS 100 electrophoresed on a 1.5% agarose gel stained with 0.5 g/ml ethidium bromide and visualized under UV light, followed by (700 Imaging Densitometer, Bio-Rad Laboratories, CA). 1.2 VPAC1 protein expresion study by WB HFF-1, DU145, PC3 and LnCAP cells were cultured on 6 well plates (Thermo Scientific, Nunclon Delta Surface) until 80% confluency , washed once with ice-cold PBS, and lysed in ice-cold lysis buffer (RIPA buffer: 50 mM HEPES pH. 7.4, 150 mM Sodium chloride, 1 mM EDTA 1% Nonidet P 40, 0.25% sodium deoxycholate, 0.1 % sodium dodecyl sulfate, 10 mM sodium fluoride, 50 mM sodium orthovanadate) containing Complete, EDTA free Protease inhibitor cocktail Tablets (Roche, Switzerland) for 30 minutes at 4 ºC. After centrifugation of the lysates at 21,000 g at 4 ºC for 20 minutes, the supernatants were collected and stored at −80 °C until use. The protein concentrations of these extracts were determined with the bicinchoninic acid (BCA) protein assay (Bio-rad Laboratories, California). Western blot analysis was conducted by employing the corresponding antibody. In brief, equal amounts of lysate proteins (40 μg) were loaded onto 15% SDS-PAGE gel (the loading buffer containing 0.1 M DTT) and subjected to electrophoresis at 200 V. All samples were electrophoretically transferred to nitrocellulose membrane (GE Healthcare Life Sciences, Germany). After blocking with 5% nonfat milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST) overnight at 4 ºC and washing for 5 minutes four times with TBST, the membrane was incubated with rabbit anti-human VPAC1 (1:500, Themo Scientific, USA) for 1 hour at room temperature. The membrane was washed as described before with TBST and incubated with an anti-rabbit IgG HPR conjugate secondary antibody (1:20.000, Promega, Madison, Wi, USA) for 1 h at room temperature. The antibodybound proteins were detected with the ECL chemiluminescence reagent (Millipore, USA) and the signal revealed by autoradiography on Amersham Hyperfim ECL (GE SECTION 2: MATERIALS AND METHODS 101 Healthcare, Buckinghamshire, UK). Quantification of Western Blot by performed by densitometry with ImageQuant TL software after scanning the film on ImageScanner II (GE Healthcare). The relative expression of proteins was normalized to that of loading controls. The values are expressed as percentages of the expression levels in control cells that were arbitrarily set to 100%. 2. Preparation of doxorubicin encapsulated VIP-liposomes 2.1 Liposome formulation A mixture of phospholipids DPPC, DSPC (molar ratio 6:4), DSPE-PEG2000 (5% of total phopolipids) and DSPE-PEG-Mal (5% of total phopolipids) were dissolved in chloroform:methanol (4:1 v/v) mixture. Multilamellar vesicles were prepared by reverse phase evaporation method. The organic solvent was evaporated in a 25 mL round-bottom flask using a rotavapor (Heidolph, Germany), under vacuum for 30 min at 40°C. After removal of the solvent the dried lipid film was dissolved in 3 mL of di-ethyl-ether and 3 mL of chloroform. To this solution 1.5 mL ammonium sulphate (250 mM pH 5) were added. The resulting two-phase system is sonicated (Ultrasonic cleaner, VWR) for 5 to 10 min until the mixture becomes homogeneous and does not separate for at least 30 min after sonication. The mixture is then placed on the rotary evaporator and the organic solvent is removed until the sample becomes an aqueous suspension. Moreover, the sample is left under vacuum for 30 minutes to ensure the solvent removal and achieve a final lipid concentration of 4mM. Small unilamellar vesicles (SUV) were prepared by further bath sonication at 60°C for 15 minutes. Liposomes were passed down a sephadex G-50 column equilibrated with HEPES buffered saline (HBS; 20 mM HEPES,150 mM NaCl, pH 7.4) to exchange the external buffer. The eluted liposomes had a transmembrane pH gradient, pH 5.0 inside and pH 7.4 outside, necessary for DOX encapsulation. SECTION 2: MATERIALS AND METHODS 108 unencapsulated fluorophore (calcein or AF-750) was then separated from the liposome vesicles by dyalisis and collected to a final lipid concentration of 3 mg/ml. The mean diameter of the liposomes (100 ± 20nm) was verified using a Nanosizer. 7.3 In vivo imaging Live animal fluorescence optical imaging monitored the in vivo biodistribution of fluorescently-loaded liposomes using the IVIS Caliper system (Caliper Life Sciences Corp.,Alameda, CA). Images were acquired and analyzed with Living Image software 2.5 (CaliperLife Sciences Corp.) with identical illumination settings for all images. The system settings were: binning = 4, exposure time = 5 seconds, field-of-View = 15, fStop = 2 and filters with an excitation of 705–780 nm and emission of 810–885 nm. Data are displayed in the unit value of efficiency and represent the ratio of light emitted to light incident. Mice were sedated via inhalation of isoflurane/oxygen and injected with a volume of 150 μL of liposome solutions, such as Alexa 750 encapsulated liposomes (LIP-Alexa750) and VIP functionalized ones, (VIP-LIPAlexa750) via the tail vein. In vivo images were acquired over 12 hours in four positions (ventral decubitus, right lateral decubitus, left lateral decubitus and dorsal decubitus) and fluorescence of major organs was differentiated. 7.4 Ex vivo tumour imaging Animals were sacrificed immediately following fluorescence imaging and xenograft tumours were excised. Only one untreated tumour was used for this group as a background reference in order to use the other two mice for following experiments. Images were normalized with common minimum and maximum values. The Region of Interest (ROI) were drawn on the DU145 tumours and the average signal intensity within the ROI was used for subsequent quantitative analysis. The fluorescence intensity of DU145 tumour based on in vivo NIRF imaging was presented as mean ± SD (n=3). 109 110 SECTION 2: RESULTS AND DISCUSSION 111 1. Prostate cancer cell lines characterization 1.1 VPAC1 gene expression study by qPCR The VPAC1 mRNA expression study by qPCR revealed that all of the PCa cell lines expressed VPAC1 and no mRNA was detected for HFF-1. However, the abundance of mRNA varied considerable between the different cell lines. LnCAP showed the highest VPAC1 mRNA expression, almost 100 times more than DU145 expression levels and 1000 times more than PC3 (Figure 20). DU 145 showed an intermediate expression level between LnCAP and PC3 (which showed the lowest expression levels for VPAC1). A SECTION 2: RESULTS AND DISCUSSION 112 B Figure 20. VPAC1 gene expression. A) Relative Quantification of VPAC1 versus Cyclophilin B reference gene in 4 cell lines, HFF-1, LnCAP, DU145 and PC3. B) Agarosegel showing the DNA amplified bands for Cyclophilin B and VPAC1 genes. From left to right, a water control to discard any contamination, M corresponds to the DNA markers, HFF-1 , DU145, PC3 and LnCAP cyPB and VPAC1 DNA amplification bands respectively. PCR products were separated by 1.5% agarose gel electrophoresis and stained with ethidium bromide. The PCR products were of the expected sizes of 324 bp (VPAC1) and 193 bp (CyPB). 1.2 VPAC1 protein expresion study by WB The VPAC1 protein expression study by Western Blot revealed that all of the PCa cell lines expressed VPAC1 protein. LnCAP showed the highest VPAC1 protein expression, in accordance with qPCR results. But PC3 and DU145 showed very similar amounts of VPAC1 (normalized against internal GAPDH controls), showing slightly higher amount in PC3 than in DU145 (Figure 21A). HFF-1 (non-prostatic cell line used as control) showed a weak VPAC1 signal, although it seems to be due to inespecificity, as two bands are observed close to the 55 KDa region (Figure 21B) in the Western Blot. SECTION 2: RESULTS AND DISCUSSION 113 A B Figure 21. VPAC1 protein expression in PCa cell lines. A) Normalized values of VPAC1 against internal GAPDH controls for each cell line. B) VPAC1 (62.5KDa) and GAPDH (37 KDa) proteins determined by Western Blot for each cell line. 2. Characterization of VIP-liposomes 2.1 Size and surface characterisation: DLS and cryo-TEM. SECTION 2: RESULTS AND DISCUSSION 114 In this study VIP functionalised liposomes were prepared consisting of 90 wt% DPPC and DSPC (molar ratio 6:4), 5 wt % DSPE-PEG2000 and 5 wt% DSPE PEG-Mal through the well-known reverse phase evaporation method. Dynamic light scattering (DLS) measurements showed that the size and surface potential of the formed liposomes were 120 nm ± 2.66 (Figure 22). The size was also measured by cryogenic transmission electron microscope (Cryo-TEM) (Figure 23). Figure 22. Liposome size measurements by DLS. This data shows an average size of 120 nm for three independent measurements, each one as a result of 10 readings. SECTION 2: RESULTS AND DISCUSSION 115 Figure 23. (A) Scheme of VIP functionalization via DSPE-PEG-Mal and liposome composition. (B) Cryo-TEM of VIP-liposomes (Scale bar 200nm).The image corroborates DLS results, showing an average size of approximately 120 nm. SECTION 2: RESULTS AND DISCUSSION 116 2.2 Peptide functionalization quantification. The peptide was bounded to the liposome surface by via its cysteine to the maleimide-modified termini of PEG chains as shown in Figure 23A. The quantification of the peptide was performed after the dialysis of the liposome for 24 h, to ensure the quantification of the coupled VIP-liposome. The result for VIP quantification was calculated using the plotted standard curve (Figure 24) and the amount of functionalized peptide was an average of 15 ug/mL in a 2 mM liposome solution (Figure 25). y = 0,971x -3,264 R² = 0,990 -0,5 0 0,5 1 1,5 2 2,5 2,5 3 3,5 4 4,5 55,5 6 Log (Abs) Log (ng/mL Peptide) Standard curve: Peptide Quantification Figure 24. Standard curve for functionalized VIP determination. The standard curve was plotted as fluorescence over peptide quantity (log10 fluorescence vs log10 peptide quantity). SECTION 2: RESULTS AND DISCUSSION 117 0 10 20 30 40 VIP-LIP-DOX LIP-DOX TOTAL VIP-LIP-DOX ug/mL VIP Functionalized VIP Quantification Figure 25. Quantification of functionalized VIP. Total VIP-lipdox refers to the subtraction of lip-dox to VIP functionalized liposomes due to the interference with doxorubicin’s fluorescence. 3. In vitro assays of doxorubicin encapsulated liposomes 3.1 Doxorubicin encapsulation efficiency and stability studies Liposomes encapsulated an average of 0,42 mg of DOX in a 4 mM lipid concentration. Liposomes with and without VIP and with and without doxorubicin were also prepared as controls for the following experiments. The encapsulation efficiency of the drug was determined an average of 95%. To evaluate the stability of doxorubicin encapsulated liposomes in HBS and in physical conditions such as 50% serum, liposomes and VIP-liposomes were incubated at 37°C and samples were analyzed every 15 and 30 minutes and at each hour for 5 hours. The release of the drug was measured by a fluoremeter and samples of HBS and 50% serum were used as a reference. Figure 26 depicts the stability of both formulations over time. No difference of stability was observed between the two formulations in HBS, but in serum VIPliposomes seem more stable, which can be explained by the presence of the SECTION 2: RESULTS AND DISCUSSION 124 Figure 29. DU145 cells imaging after 24 hour treatment by optical microscopy. A) Untreated, B) Doxorubicin, C) Liposome, D) LIP-DOX, E) VIP-LIP, F) VIP-LIP-DOX. SECTION 2: RESULTS AND DISCUSSION 125 Figure 30. PC3 cells imaging after 24 hour treatment by optical microscopy. A) Untreated, B) Doxorubicin, C) Liposome, D) LIP-DOX, E) VIP-LIP, F) VIP-LIP-DOX. SECTION 2: RESULTS AND DISCUSSION 126 Figure 31. LnCAP cells imaging after 48 hour treatment by optical microscopy. A) Untreated, B) Doxorubicin, C) Liposome, D) LIP-DOX, E) VIP-LIP, F) VIP-LIP-DOX. SECTION 2: RESULTS AND DISCUSSION 127 Figure 32. DU145 cells imaging after 48 hour treatment by optical microscopy. A) Untreated, B) Doxorubicin, C) Liposome, D) LIP-DOX, E) VIP-LIP, F) VIP-LIPDOX. SECTION 2: RESULTS AND DISCUSSION 128 Figure 33. PC3 cells imaging after 48 hour treatment by optical microscopy. A) Untreated, B) Doxorubicin, C) Liposome, D) LIP-DOX, E) VIP-LIP, F) VIP-LIP-DOX. 3.3.2 Citotoxicity and viability determination by LDH and MTT assay. In vitro viability and cytotoxicity was assessed by MTT and LDH assays (Figure 34 and 35). The cells were exposed to the different formulations for 24 and 48 hours. Each experiment represents the average of at least three different experiments. The cells were exposed to free doxorubicin, doxorubicin encapsulated liposomes, VIP-doxorubicin liposomes and the same liposome SECTION 2: RESULTS AND DISCUSSION 129 formulations without the drug used as controls. PC3 cell line did not show differences between the two doxorubicin liposome formulations (with and without VIP) and showed very similar cytotoxicity and viability after 24 and 48 h. By contrast, LnCAP shows higher cytotoxicity and lower viability after 24 and 48 h when treated with VIP targeted doxorubicin liposomes. As shown by PCR it seems that LnCAP express higher levels of VPAC1 receptor than DU145 and PC3. This could be the cause why a higher effect is observed after incubation with VIP-doxorubicin liposomes which enhances the cytotoxicity due to a higher uptake and also an activation of intracellular signalling due to the G-coupled receptors. It has been described that VIP can induce a cellular differentiation n LncAP cells (Gutierrez-Canas, Juarranz et al. 2005), which could sensitize the cells to the doxorubicin treatment. SECTION 2: RESULTS AND DISCUSSION 130 Figure 34. 24 h cytotoxicity studies by LDH and MTT assay on LnCAP, DU145 and PC3 prostate cancer cell lines. . Values are mean ± S.D * P<0.1, ** P<0.05, *** P<0.01 SECTION 2: RESULTS AND DISCUSSION 131 Figure 35. 48 h cytotoxicity studies by LDH and MTT assay on LnCAP, DU145 and PC3 prostate cancer cell lines. . Values are mean ± S.D * P<0.1, ** P<0.05, *** P<0.01 SECTION 2: RESULTS AND DISCUSSION 132 3.4 Apoptosis and cell cycle analysis by flow cytometry Doxorubicin inhibits cell growth and proliferation primarily through cell cycle arrest and apoptosis inducing mechanism. To further confirm the therapeutic potential of the targeted delivery system, in this case VIP-LIP-DOX, cell cycle analysis was performed using flow cytometry. Figure 35 and Figure 36 show the relative cell cycle blocking activities and the subG1 phase increase due to apoptosis at 24 and 48 hours respectively. SubG1 is used to detect cells that have lost some of their DNA in late stage of apoptosis process following endonucleases activity. Here, the number of hypodiploid cells undergoing this process can be counted in subG1 region of PI histogram. Differences upon 24 and 48 h treatments with free drug and targeted and non-targeted drug liposomes were observed. In the case of 24 h treatments, VIP-LIP-DOX treatment showed the highest cell cycle arrest in SubG1 phase in the case of LnCAP cells but not in the case of PC3 and DU145, which showed these same results but after 48 h. It seems that PC3 and DU145 are more resistan to DOX treatments and that clear differences start to be appreciated after 48 h. This is not the case for LnCAP, where notable differences are observed after 24 h treatments, and after 48 h LIP-DOX and VIP-LIP-DOX reach the same values. The results observed in the Figure 36 and Figure 37 agree with the cytotoxicity results (Figure 34 and Figure 35), showing a higher cell death effect with targeted liposomes on LNCAP cells than on PC3 and DU145 cells, which show less difference between untargeted and targeted liposomes. SECTION 2: RESULTS AND DISCUSSION 133 Figure 36. SubG1 analysis by flow cytometry after 24 hours treatment on LnCAP, DU145 and PC3 cells. 140 SECTION 2: CONCLUSIONS 141 Conclusions  We have developed a reliable method to synthesize homogenous, stable and properly characterized VIP functionalized liposomes  VIP-liposomes demonstrated significant cellular binding and uptake by VIP receptor expressing cells (PC3, DU145 and LnCAP) in contrast to unconjugated liposomes.  VIP-liposomes showed higher therapeutic efficacy in VIP receptor expressing cells than unconjugated liposomes confirmed by cytotoxicity and apoptosis studies.  In vivo biodistribution studies showed VIP-liposome accumulation in liver, kidneys and spleen, whereas unconjugated liposomes showed preference for liver and kidneys after 12 hours of intravenous injection.  In vivo biodistribution studies showed higher VIP-liposome accumulation in the tumour in comparison to unconjugated liposomes after 12 hours of intravenous injection. 142 143 144 APPENDICES: ABBREVIATIONS 145 1. Abbreviations AD Alzheimer's disease ADNP Activity Dependent Neuroprotective Protein AgNPs Silver Nanoparticles AuNPs Gold Nanoparticles BBB Blood Brain Barrier BRB Blood Retina Barrier BTB Blood Testis Barrier cAMP cyclic Adenosine Mono-Phosphate CNS Central Nervous System CT Computerized Tomography EPR Enhanced Permeability and Retention HBS Hepes Buffer Saline IL Interleukin NFkB Nuclear Factor kappa-light chainenhancer of activated B cells NMR Nuclear Magnetic Resonance NO Nitric Oxide PACAP Pituitary AdenylateCyclase-Activating Peptide PCa Prostate Cancer PCR Polymerase Chain Reaction PEG Polyehtylene Gyclol PKA Protein Kinase A APPENDICES: ABBREVIATIONS 146 PLGA Poly(lactide-co-glycolide) PNS Peripheral Nervous System PSA Prostate Specific Antigen RES Reticuloendothelial System ROI Region of Interest SERS Surface-enhanced Raman Spectroscopy SP Surface Plamosn SPR Surface Plasmon Resonance TNF Tumour Necrosis Factor Tregs Regulatory T cells VIP Vasoactive Intestinal Peptide VPAC1 Vasoactive Intestinal Peptide Receptor 1 APPENDICES: TABLE REFERENCES 147 2. References Abad, C., C. Martinez, et al. (2003). "Therapeutic effects of vasoactive intestinal peptide in the trinitrobenzene sulfonic acid mice model of Crohn's disease." Gastroenterology Abarca, K., I. Ibanez, et al. (2011). "Immunogenicity and tolerability of a paediatric presentation of a virosomal hepatitis A vaccine in Chilean children aged 1-16 years." 124(4): 961-971. Vaccine Alivisatos, A. P., K. P. Johnsson, et al. (1996). "Organization of 'nanocrystal molecules' using DNA." 29(48): 8855-8862. Nature Allen, T. M. (2002). "Ligand-targeted therapeutics in anticancer therapy." 382(6592): 609-611. Nat Rev Cancer Allen, T. M., D. R. Mumbengegwi, et al. (2005). "Anti-CD19-targeted liposomal doxorubicin improves the therapeutic efficacy in murine B-cell lymphoma and ameliorates the toxicity of liposomes with varying drug release rates." 2(10): 750-763. Clin Cancer Res Antonelli, M., G. De Pascale, et al. (2012). "Comparison of triple-lumen central venous catheters impregnated with silver nanoparticles (AgTive(R)) vs conventional catheters in intensive care unit patients." 11(9): 3567-3573. J Hosp Infect Arvizo, R. R., S. Bhattacharyya, et al. (2012). "Intrinsic therapeutic applications of noble metal nanoparticles: past, present and future." 82(2): 101-107. Chem Soc Rev Asadishad, B. V., M.; Alemzadeh, I. (2010). "Folate-Receptor-Targeted Delivery of Doxorubicin Using Polyethylene Glycol-Functionalized Gold Nanoparticles." 41(7): 2943-2970. Ind Eng Chem Res Asnicar, M. A., A. Koster, et al. (2002). "Vasoactive intestinal polypeptide/pituitary adenylate cyclase-activating peptide receptor 2 deficiency in mice results in growth retardation and increased basal metabolic rate." 49(4): 1958–1963. Endocrinology Atkinson Ralls, L., D. R. Drover, et al. (2011). "Prior epidural lidocaine alters the pharmacokinetics and drug effects of extended-release epidural morphine (DepoDur(R)) after cesarean delivery." 143(10): 3994-4006. Anesth Analg Aton, S. J., C. S. Colwell, et al. (2005). "Vasoactive intestinal polypeptide mediates circadian rhythmicity and synchrony in mammalian clock neurons." 113(2): 251-258. Nat Neurosci Auffan, M., J. Rose, et al. (2009). "Towards a definition of inorganic nanoparticles from an environmental, health and safety perspective." 8(4): 476-483. Nat Nanotechnol Baade, P. D., D. R. Youlden, et al. (2009). "International epidemiology of prostate cancer: geographical distribution and secular trends." 4(10): 634-641. Mol Nutr Food Res 53(2): 171-184. APPENDICES: TABLE REFERENCES 148 Baldeschwieler, J. S., P. (1997). "Liposomal drugs: From setbacks to success." Chemtech Bangma, C. H., S. Roemeling, et al. (2007). "Overdiagnosis and overtreatment of early detected prostate cancer." 27: 34-42. World J Urol Banner, K. H. and M. A. Trevethick (2004). "PDE4 inhibition: a novel approach for the treatment of inflammatory bowel disease." 25(1): 3-9. Trends Pharmacol Sci Bar-Ilan, O., R. M. Albrecht, et al. (2009). "Toxicity assessments of multisized gold and silver nanoparticles in zebrafish embryos." 25(8): 430436. Small Barrie, A. P., A. M. Clohessy, et al. (1997). "Pituitary adenylyl cyclase-activating peptide stimulates extracellular signal-regulated kinase 1 or 2 (ERK1/2) activity in a Ras-independent, mitogen-activated protein Kinase/ERK kinase 1 or 2-dependent manner in PC12 cells." 5(16): 1897-1910. J Biol Chem Barrientos, A. G., J. M. de la Fuente, et al. (2009). "Modulating glycosidase degradation and lectin recognition of gold glyconanoparticles." 272(32): 19666-19671. Carbohydr Res Beija, M., R. Salvayre, et al. (2012). "Colloidal systems for drug delivery: from design to therapy." 344(12): 1474-1478. Trends Biotechnol Bennewitz, M. F. and W. M. Saltzman (2009). "Nanotechnology for delivery of drugs to the brain for epilepsy." 30(9): 485-496. Neurotherapeutics Bolin, D. R., J. Michalewsky, et al. (1995). "Design and development of a vasoactive intestinal peptide analog as a novel therapeutic for bronchial asthma." 6(2): 323-336. Biopolymers Boyle, J. A. and C. E. Swenson (1999). "ABELCET treatment." 37(2): 57-66. J Clin Pharmacol Brannon-Peppas, L. and J. O. Blanchette (2004). "Nanoparticle and targeted systems for cancer therapy." 39(4): 427-428. Adv Drug Deliv Rev Brenner, D. E. (1989). "Liposomal encapsulation: making old and new drugs do new tricks." 56(11): 1649-1659. J Natl Cancer Inst Budai, M. and M. Szogyi (2001). "[Liposomes as drug carrier systems. Preparation, classification and therapeutic advantages of liposomes]." 81(19): 1436-1438. Acta Pharm Hung Cabral, H., N. Nishiyama, et al. (2011). "Supramolecular nanodevices: from design validation to theranostic nanomedicine." 71(1): 114-118. Acc Chem Res Cai, L., X. Wang, et al. (2012). "Peptide ligand and PEG-mediated long-circulating liposome targeted to FGFR overexpressing tumor in vivo." 44(10): 999-1008. Int J Nanomedicine Caraglia, M., M. Carteni, et al. (2008). "Experimental study on vasoactive intestinal peptide (VIP) and its diaminopropane bound (VIP-DAP) analog in solution." 7: 4499-4510. Amino Acids Caro, C. C., P.M.; Klippstein, R.; Pozo, D.; Zaderenko, A.P. (2010). 35(2): 275-281. Silver nanoparticles: sensing and imaging applications. APPENDICES: TABLE REFERENCES 149 Ceraudo, E., S. Murail, et al. (2008). "The vasoactive intestinal peptide (VIP) alphaHelix up to C terminus interacts with the N-terminal ectodomain of the human VIP/Pituitary adenylate cyclase-activating peptide 1 receptor: photoaffinity, molecular modeling, and dynamics." Mol Endocrinol Colwell, C. S., S. Michel, et al. (2003). "Disrupted circadian rhythms in VIPand PHIdeficient mice." 22(1): 147-155. Am J Physiol Regul Integr Comp Physiol Colwell, C. S., S. Michel, et al. (2004). "Selective deficits in the circadian light response in mice lacking PACAP." 285(5): R939-949. Am J Physiol Regul Integr Comp Physiol Collado, B., M. J. Carmena, et al. (2005). "Expression of vasoactive intestinal peptide and functional VIP receptors in human prostate cancer: antagonistic action of a growth-hormone-releasing hormone analog." 287(5): R1194-1201. Int J Oncol Couvineau, A., E. Ceraudo, et al. (2012). "The VPAC1 receptor: structure and function of a class B GPCR prototype." 26(6): 16291635. Front Endocrinol (Lausanne) Couvineau, A. and M. Laburthe (2012). "VPAC receptors: structure, molecular pharmacology and interaction with accessory proteins." 3: 139. Br J Pharmacol Cui, H., Y. Feng, et al. (2009). "Strategies of large scale synthesis of monodisperse nanoparticles." 166(1): 42-50. Recent Pat Nanotechnol Cutler, D. J., M. Haraura, et al. (2003). "The mouse VPAC2 receptor confers suprachiasmatic nuclei cellular rhythmicity and responsiveness to vasoactive intestinal polypeptide in vitro." 3(1): 32-41. Eur J Neurosci Chan, S., N. Davidson, et al. (2004). "Phase III trial of liposomal doxorubicin and cyclophosphamide compared with epirubicin and cyclophosphamide as firstline therapy for metastatic breast cancer." 17(2): 197-204. Ann Oncol Chang, H. I. and M. K. Yeh (2012). "Clinical development of liposome-based drugs: formulation, characterization, and therapeutic efficacy." 15(10): 1527-1534. Int J Nanomedicine Chao, T. C., W. S. Wang, et al. (2003). "A dose-escalating pilot study of sterically stabilized, pegylated liposomal doxorubicin (Lipo-Dox) in patients with metastatic breast cancer." 7: 49-60. Cancer Invest Chapter, M. C., C. M. White, et al. (2010). "Chemical modification of class II G protein-coupled receptor ligands: frontiers in the development of peptide analogs as neuroendocrine pharmacological therapies." 21(6): 837-847. Pharmacol Ther Chen, H. and R. Langer (1998). "Oral particulate delivery: status and future trends." 125(1): 39-54. Adv Drug Deliv Rev Chen, J. R., Y. C. Yang, et al. (2008). "Salvage chemotherapy in recurrent cervical cancer with biweekly pegylated liposomal Doxorubicin (lipo-dox)." 34(2-3): 339-350. Taiwan J Obstet Gynecol 47(3): 322-326.