Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Centre for Drug Research Division of Biopharmaceutics and Pharmacokinetics Faculty of Pharmacy University of Helsinki Finland In vitro, in vivo, and in silico investigations of polymer and lipid based nanocarriers for drug and gene delivery Julia Lehtinen ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Pharmacy of the University of Helsinki, for public examination in Auditorium 2 at Viikki Korona Information Centre, Viikinkaari 11, on 7th September 2013, at 12 noon. Helsinki 2013 Supervisors: Professor Arto Urtti, Ph.D. Centre for Drug Research Faculty of Pharmacy University of Helsinki Finland Professor Heike Bunjes, Ph.D. Institute of Pharmaceutical Technology Technische Universität Braunschweig Germany Mathias Bergman, Ph.D. Karyon Ltd. Helsinki Finland Reviewers: Docent Juha Holopainen, M.D., Ph.D. Institute of Clinical Medicine Faculty of Medicine University of Helsinki Finland Professor Stefaan de Smedt, Ph.D. Laboratory of General Biochemistry & Physical Pharmacy Faculty of Pharmaceutical Sciences University of Ghent Belgium Opponent: Academic Rector, Professor Jukka Mönkkönen, Ph.D. University of Eastern Finland Finland © Julia Lehtinen 2013 ISBN 978-952-10-9039-4 (pbk.) ISBN 978-952-10-9040-0 (PDF, http://ethesis.helsinki.fi) ISSN 1779-7372 Helsinki University Print Helsinki, Finland 2013 Abstract Nanomedicine research has expanded rapidly in the last decades. Several nanoparticle formulations are accepted in clinical use, e.g. for the treatment of cancer, infections and eye diseases, and also for diagnostics. Nanoparticle mediated drug delivery has many potential advantages over the free drug, such as better pharmacokinetic profile, lowered toxicity, and its possible use for cell-specific targeting and intracellular drug release. Therapeutic genes can also be packed into nanocarriers to protect them from enzymatic degradation and to mediate their cellular entry. The transfection efficacy of these synthetic vectors is modest when compared to viral vectors, but they are considered to be safer. Nonetheless, even though nanoparticles have so many advantages, there are many extracellular and intracellular barriers to overcome before achieving successful drug or gene delivery. The focus of this research work was the formation and physico-chemical features of lipid and polymer based nanoparticles for drug and gene delivery. In addition, two classes of cancer cell targeting approaches were evaluated in biological and physical studies. First, the effect of the polymeric gene carrier composition and structure on DNA condensation efficacy, transgene expression, and cellular toxicity was examined. The linear architecture and flexibility of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA)-based block co-polymers clearly enhanced DNA condensation and transfection efficiency. In addition, by conjugating a membrane active protein, hydrophobin (HFBI) to DNA-binding cationic dendrons, the transfection efficacy was increased compared to plain dendron. However, cationic polymer-DNA complexes are prone to disruption by polyanions such as glycosaminoglycans (GAG) in the extracellular space. We coated poly(ethyleneimine) PEI/DNA complexes with anionic lipid mixture. The coating was able to protect the contents against GAGs, and it could respond to the change of endosomal pH and release the cargo inside the cells. The next studies aimed to evaluate targeted liposomal cancer drug carriers in physicochemical studies and in cancer cell models in vitro and in mice. A promising activated endothelium targeting peptide (AETP) failed to target the liposomes to the cells. Molecular modeling revealed that hydrophobic AETP was hidden in the PEG shield of the liposomal surface thus it was not accessible for the target receptors. The last study describes applicability of pre-targeting and local intraperitoneal administration of liposomes for drug targeting to tumors located in peritoneal cavity. Epithelial growth factor receptor (EGFR)-targeted liposomes bound specifically to ovarian cancer cells in vitro. In the animal study, increased accumulation of liposomes in the xenograft tumors of the mice was seen after intraperitoneal administration compared to intravenous administration. In conclusion, the composition and architecture of nanocarriers have a crucial impact on DNA condensation, stability of the complexes and transfection efficacy. In liposomal cancer drug targeting, polyethylene glycol (PEG) shield may hinder the targeting efficiency of small molecular peptides. Intraperitoneal administration of liposomal drugs seems to be promising route for targeting to tumors located in the peritoneal cavity. 3 Acknowledgements This study was carried out at the University of Kuopio, in the Department of Pharmaceutics, (currently University of Eastern Finland, School of Pharmacy) during the years 2003-2005 and it was continued at the University of Helsinki, Division of Biopharmaceutics and Pharmacokinetics and the Centre for Drug Research (CDR) during the years 2006-2013. This work has been financially supported by the Academy of Finland, the Association of Finnish Pharmacies, the Finnish Cultural Foundation, the Finnish Pharmaceutical Society, the National Agency of Technology (TEKES Finland), the Science Foundation of Orion-Farmos, and the University of Helsinki. All financial support is greatly acknowledged. I want to express my deepest gratitude to my principal supervisor, Professor Arto Urtti for his continuous support and optimistic attitude during these years. I am also very grateful to my other supervisors, Professor Heike Bunjes for introducing me to the world of nanoparticles and Mathias Bergman, Ph.D., for his skilful advice and guidance in peptide targeting. Professor Stefaan de Smedt and Docent Juha Holopainen are greatly acknowledged for critical reading of this dissertation and for their valuable comments. I am honored that Professor Jukka Mönkkönen has accepted the invitation to be my opponent in the public defense of this thesis. I would like to thank the current and former Deans of Faculty of Pharmacy in Kuopio and in Helsinki, and Heads of Department of Pharmaceutics and Heads of Division of Biopharmaceutics and Pharmacokinetics for providing excellent working facilities. I wish to warmly thank my co-authors: Anu Alhoranta, M.Sc., Kim Bergström, Ph.D., Alex Bunker, Ph.D., Annukka Hiltunen, M.Sc., Zanna Hyvönen, Ph.D., Professor Olli Ikkala, Raimo Ketola, Ph.D., Mauri Kostiainen, Ph.D., Katariina Lehtinen, M.Sc., Huamin Liang, Ph.D., Aniket Magarkar, M.Sc., Ann-Marie Määttä, Ph.D., Jere Pikkarainen, Ph.D., Sari Pitkänen, M.Sc., Mari Raki, Ph.D., Tomasz Róg, Ph.D., Michał Stepniewski, M.Sc., Astrid Subrizi, M.Sc., Professor Heikki Tenhu, Päivi Uutela, Ph.D., Thomas Wirth, Ph.D. and Professor Marjo Yliperttula for their valuable contribution to this work. It has been a pleasure to collaborate with you all. I am also very grateful to Lea Pirskanen in Kuopio and Leena Pietilä in Helsinki for their skilful and friendly assistance in laboratory. I also wish to thank the personnel of Karyon ltd for welcoming me to do part of my Ph.D. work in their laboratory. My sincere thanks go to my friends and colleagues in the Faculty of Pharmacy in Kuopio, and in the CDR and Drug Delivery and Nanotechnology (DDN) group in Helsinki. Special thanks to the girls of the girls’ room: Astrid, Heidi, Johanna, Jonna, Kati-Sisko, Mari, Marika, Martina, Melina and Polina for their friendship, joyful company, and refreshing conversions. Finally, I want to warmly thank my friends and relatives for their support and presence 4 during these years. I owe my dearest gratitude to my husband and colleague Mika for his love and support but also for scientific advice, and to our wonderful children Viljam and Hilda for brightening up our everyday life. Helsinki, July 2013 Julia Lehtinen 5 Contents Abstract 3 Acknowledgements 4 List of original publications 8 Abbreviations 9 1 Introduction 11 2 Review of the literature 13 2.1 Nanoparticles as drug and gene carriers 13 2.1.1 Liposomes 13 2.1.2 Polymeric carriers 15 2.1.3 Hybrid particles 17 2.2 Challenges in efficient drug and gene delivery 18 2.2.1 Stability of the vector 18 2.2.2 Tissue distribution and elimination 19 2.2.3 Cellular uptake 20 2.2.4 Intracellular distribution and cargo release 22 2.2.5 Diffusion in cytoplasm and nuclear import 23 2.3 Targeted cancer therapy 25 2.3.1 Passive targeting 25 2.3.2 Active targeting 26 2.3.2.1 Cancer cell targeting in solid tumors 27 2.3.2.2 Targeting to the tumor vasculature 27 3 Aims of the study 30 4 Overview of the methods 31 10 Summary of the main results 34 11 General discussion 36 6 11.1 Structure-activity relationship of polymeric DNA carriers on DNA-complex formation, transfection efficacy, and toxicity 36 11.2 Lipid-coated DNA-complexes as stable gene delivery vectors 37 11.3 Hindering effect of liposomal PEG on the targeting efficiency of a small hydrophobic peptide, AETP 39 11.4 Pre-targeting and local administration of liposomes as potential approaches in tumor targeting 40 12 Conclusions 43 13 Future prospects 44 Nanoparticles – drugs of the future? 44 References 45 7 List of original publications This thesis is based on the following publications: I Anu M. Alhoranta, Julia K. Lehtinen, Arto O. Urtti, Sarah J. Butcher, Vladimir O. Aseyev and Heikki J. Tenhu. Cationic amphiphilic star and linear block copolymers: synthesis, self-assembly, and in vitro gene transfection. Biomacromolecules 2011, 12, 3213-3222 II Mauri A. Kostiainen, Géza R. Szilvay, Julia Lehtinen, David K. Smith, Markus B. Linder, Arto Urtti and Olli Ikkala. Precisely defined proteinpolymer conjugates: construction of synthetic DNA binding domains of proteins by using multivalent dendrons. ACS NANO 2007, 1, 103-113 III Julia Lehtinen, Zanna Hyvönen, Astrid Subrizi, Heike Bunjes and Arto Urtti. Glycosaminoglycan-resistant and pH-sensitive lipid-coated DNA complexes produced by detergent removal method. Journal of Controlled Release 2008, 131, 145-149 IV Julia Lehtinen, Aniket Magarkar, Michał Stepniewski, Satu Hakola, Mathias Bergman, Tomasz Róg, Marjo Yliperttula, Arto Urtti and Alex Bunker. Analysis of course of failure of new targeting peptide in PEGylated liposome: molecular modeling as a rationale design tool for nanomedicine. European Journal of Pharmaceutical Sciences 2012, 46, 121-130 V Julia Lehtinen, Mari Raki, Kim A. Bergström, Päivi Uutela, Katariina Lehtinen, Annukka Hiltunen, Jere Pikkarainen, Huamin Liang, Sari Pitkänen, Ann-Marie Määttä, Raimo A. Ketola, Marjo Yliperttula, Thomas Wirth and Arto Urtti. Pre-targeting and direct immunotargeting of liposomal drug carriers to ovarian carcinoma. PLoS ONE 2012, 7(7):e41410 The publications are referred to in the text by their roman numerals 8 Abbreviations Ab AETP αvβ3, αvβ5 bp BSA CHEMS CMC CPP CMV DMPG DNA DOPE DOTAP DPPC DSPE-PEG antibody activated endothelium targeting peptide integrins upregulated in proliferating endothelial cells base pair bovine serum albumin cholesteryl hemisuccinate critical micelle concentration cell penetrating peptide cytomegalovirus 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) deoxyribonucleic acid 1,2-dioleyl-sn-glycerol-3-phosphoethanolamine 1,2-dioleyl-3-trimethylammonium-propane 1,2-dipalmitoyl-sn-glycero-3-phosphocholine 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-amino(polyethylene glycol) DSPG 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) EGFR epidermal growth factor receptor Egg PC egg phosphatidyl choline Egg SM egg sphingomyelin EMA-DNA ethidiummonoazide EPR enhanced permeability and retention Fab´ antigen-binding fragment of antibody FACS fluorescence-activated cell sorting FITC fluorescein isothiocyanate Fmoc 9-fluorenylmethyloxycarbonyl GAG glycosaminoglycan HII inverted hexagonal structure of lipid membrane HDL high density lipoprotein HER-2 human epidermal growth factor receptor 2 HFBI hydrophobin HIV-1 human immunodeficiency virus 1 HPMA N-(2-hydroxypropyl)-methacrylamide copolymer HSA human serum albumin HSPC fully hydrogenated phosphatidyl choline HUVEC human umbilical vein endothelial cell IgG immunoglobulin G IgM immunoglobulin M Kd dissociation constant Lα multilamellar structure or liquid crystalline phase of lipid membranes Lβ gel phase of lipid membranes LCDC lipid-coated DNA complexes 9 LC-MS LDL LUV mAb miRNA MLV MPS mRNA MTT NCE n/p ONPG PAMAM PBuA PC PDMAEMA PDP PE PEG PEI PG PGA PL PLL PS RGD Rho RNA scFv siRNA SPECT-CT TAT TRF VEGFR liquid chromatography - mass spectrometry low density lipoprotein large unilamellar vesicle monoclonal antibody microRNA multilamellar vesicles mononuclear phagocyte system messenger RNA (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide new chemical entity nitrogen/phosphate ratio ortho-nitrophenyl-β-D-galactopyranoside poly(amidoamine) poly(n-butyl acrylate) phosphatidylcholine poly(2-(dimethylamino)ethyl methacrylate) N-(3´-(pyridyldithio)propionoylamino phosphatidylethanolamine polyethylene glycol poly(ethylene imine) phosphatidylglycerol poly-L-glutamic acid phospholipid poly-L-lysine polystyrene arginine, lysine and aspartic acid containing peptide rhodamine ribonucleic acid single chain variable fragment of antibody small interfering RNA single-photon emission computed tomography - computed tomography trans-acting activator of transcription time-resolved fluorescence vascular endothelial growth factor receptor 10 1 Introduction Discovery of new potent drug molecules has significantly improved the treatment of serious illnesses, such as cancer and cardiovascular diseases. However, the development of new compounds to serve as clinical drugs has become more and more difficult. This is evident by the decreasing numbers of new chemical entities (NCE) that are introduced annually for clinical use. Many new compounds face significant development challenges, such as poor water-solubility or short half-life in the blood circulation. In many cases, adverse effects do hamper treatment, particularly in the case of anti-cancer drugs. Drug delivery systems can be used to modify the drug properties, for example by increasing solubility, modulation of pharmacokinetics, and improving the safety of drug treatment. Gene therapy was launched in the 1990s as an alternative to traditional drug therapy. It presents a promising alternative for the correction of genetic deficiencies, e.g. haemophilia or cystic fibrosis, but also for the treatment of acquired diseases, such as cancer and cardiovascular conditions. Gene medicines can either induce protein translation in the target cells (gene therapy) or silence the expression of the target protein (oligonucleotide drugs, e.g. siRNA). These compounds (plasmid DNA, siRNA) cannot be delivered as such, because they undergo rapid enzymatic degradation and do not reach target tissues. The negative charges of DNA and RNA, and a large size of plasmid DNA prevent their passage through cell membranes. Nanocarriers are one possible solution to overcome the pharmacokinetic challenges in drug and gene delivery. Nanocarriers, often generally referred to as nanoparticles, are typically below one micrometer in diameter, usually consisting of lipids (e.g. liposomes, lipid-DNA complexes), polymers (e.g. polymeric nanoparticles and micelles, polymerDNA complexes), peptides, proteins and/or metallic nanoparticles. Liposomes were already developed in the 1960s by Alec Bangham and polymer nanoparticles in the 1970s by Peter Speiser. Thereafter, the field of nanomedicines has expanded tremendously: currently almost 20 000 publications are found in PubMed database with the search terms ‘nanoparticle and drug’. By formulating a drug in a nanocarrier, the solubility and the pharmacokinetic profile can be dramatically improved (Shi et al. 2010). Nanoparticles may also decrease the toxic effects of the drug from off-target sites. For example, liposomal encapsulation of doxorubicin lowered the risk of cardiotoxicity seven times compared to the free drug (O'Brien et al. 2004). Nanocarriers can be tailored to release drugs in a controlled manner or triggered by a change in environmental conditions and they can be targeted to desired cell type expressing the target protein. In addition, they can be used to deliver several drugs simultaneously as a combination therapy (Zhang et al. 2008). Complexation of DNA and RNA into small nanoparticles masks their negative charges and protects them against enzymatic degradation. When a massive molecule of plasmid DNA (mw of millions) is condensed to a nanosized particle it is more suitable for systemic delivery. In this case, nanoparticle has dual function of DNA protection from enzymatic catalysis and augmenting cellular entry. Around 40 nanoparticle formulations are now accepted in clinical use, mainly for the treatment of cancer, but also for the treatment of infections, anemia, hypercholesterolemia, 11 hepatitis, age-related macular degeneration, and in diagnostics (Duncan, Gaspar 2011). The marketed products are so-called first generation nanomedicines that are not targeted. The second generation targeted nanomedicines can bind to specific cellular antigens, but they have not reached the market. Although targeted therapeutics have a lot of potential, there are plenty of challenges and risks when more complicated formulations are designed (Cheng et al. 2012). Efficient but safe gene delivery vectors are still under development. For 500 million years, viruses have developed a very efficient way to carry genetic material into cells. Viral vectors are effective in DNA delivery, but their safety has not been totally verified. In clinical studies, viral vectors have shown severe immunological reactions and even caused patient death (Marshall 1999, Giacca, Zacchigna 2012). To date, three virus-based gene therapy products have received market authorization from regulatory agencies in China (Gendicine® and Oncorine® for the treatment of cancer) and Europe (Glybera®, for the treatment lipoprotein lipase deficiency). Although non-viral polymer- and lipid-based gene carriers lack the efficiency of the viral vectors, they are considered to be safer. In addition, they are easier to synthesize and produce in large scale, and their DNA loading capacity is higher than in the viral vectors (Kreiss et al. 1999). By learning from viruses, more efficient synthetic carriers might also be developed. In this study, the effect of the composition and architecture of polymer- and lipidbased gene carriers on DNA condensation efficacy, transgene expression and cellular toxicity was investigated. Furthermore, two types of liposomal cancer cell targeting approaches were evaluated in physical and biological studies. 12 2 Review of the literature 2.1 Nanoparticles as drug and gene carriers The family of nanocarriers includes lipid-based carriers, such as liposomes and micelles, polymer-based carriers, such as polymer conjugates, polymeric nanoparticles and dendrimers, gold nanoparticles and carbon nanotubes. The size of the nanocarriers usually varies between a few nanometers (polymer-drug conjugates, micelles and dendrimers) to some hundreds of nanometers (liposomes and polymeric nanoparticles). Examples of nanocarriers used for drug and gene delivery are presented in Figure 1. In the following review, liposomes and polymeric nanocarriers for drug and gene delivery are discussed in more detail. Figure 1 delivery. Schematic illustration of different kinds of nanoparticles used for drug and gene 2.1.1 Liposomes Liposomes are spherical, self-assembling vesicles formed by one or several lipid bilayers leaving an aqueous core inside. The lipid bilayer is composed of amphiphilic lipids, derived from or based on the structure of biological membrane lipids. The hydrophobic part of the lipid is usually formed of two hydrocarbon chains, which typically vary from 8 to 18 carbons in length and can be either saturated or non-saturated. Long and saturated acyl chains form a membrane in gel phase (Lβ), resulting in increased stability and rigidity of the liposomes. On the contrary, the use of short and/or unsaturated acyl chains results in more fluid, liquid crystalline (Lα) bilayers. Incorporation of cholesterol into the lipid bilayer minimizes the membrane permeability and improves the mechanical strength of the liposomes. Surface charge of the liposome can be affected by varying the hydrophilic head group of the lipid: being either zwitterionic (e.g. phosphatidylcholine (PC) and 13 phosphatidylethanolamine (PE)), negatively charged (e.g. phosphatidylglycerol (PG)), or positively charged (e.g. 3-trimethylammonium-propane (TAP)) (Figure 2) (Ulrich 2002). Figure 2 Chemical structures of some phospholipids (fully hydrogenated soy phosphatidylcholine (HSPC), 1,2-dioleyl-sn-glycerol-3-phosphoethanolamine (DOPE), 1,2distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DSPG), 1,2-dioleyl-3-trimethylammoniumpropane (DOTAP)) and cholesterol. In water, amphiphilic lipids tend to form bilayers since they are poorly water soluble with a critical micelle concentration (CMC) of 10-12 to 10-8 M. Spontaneously formed multilamellar vesicles (MLV) are very heterogenous in lamellarity and in size, ranging from 500 to 5000 nm. More sophisticated, small unilamellar vesicles (SUV, <100 nm) and large unilamellar vesicles (LUV, 100–800 nm) can be prepared by sonication or extrusion (Ulrich 2002, Torchilin 2005). Hydrophobic drug molecules can be entrapped passively in the liposome bilayer during the preparation of the liposomes using the aforementioned methods. Hydrophilic drugs are encapsulated in the aqueous core of the liposome (or in the aqueous phase between bilayers in the case of MLVs) using passive loading procedures, such as reverse phase evaporation (Szoka Jr., Papahadjopoulos 1978), dehydration-rehydration method (Shew, Deamer 1985), or active loading involving pH-gradient across the liposome membrane (Mayer, Bally & Cullis 1986, Hwang et al. 1999). Remote loading of doxorubicin into preformed liposomes using ammonium sulfate gradient as a driving force results in the efficient and stable entrapment of the drug (Bolotin et al. 1994). Some liposomal cancer drugs that are currently employed clinically, utilise remote loading; including Caelyx® and Myocet® loaded with doxorubicin, and Daunoxome® with daunorubicin. Cationic liposomes can be used for complexation of negatively charged DNA or RNA molecules. The formed complexes are called lipoplexes. The size of the highly cationic 14 lipoplexes varies typically between 100 and 450 nm, whereas the lipoplexes carrying a charge close to neutral are more heterogenic, varying from 350 to 1200 nm in diameter. In lipoplexes, two types of structures have been observed; multilamellar structure (Lα), where DNA is located as a monolayer between cationic membranes (Radler et al. 1997), or inverted hexagonal structure (HII), where DNA is encapsulated within cationic lipid monolayer tubes (Koltover et al. 1998) (Figure 3). To enhance gene delivery, so called helper lipids, such as 1,2-dioleyl-sn-glycerol-3-phosphoethanolamine (DOPE), are often mixed with cationic lipids to promote conversion of the lamellar phase into a hexagonal structure (Hafez, Cullis 2000). Figure 3 Schematic structures of lamellar (A) and inverted hexagonal phase (B) in the cationic lipid/DNA complexes. Modified from Morille et al. (2008). 2.1.2 Polymeric carriers Polymer based carriers can be divided into different categories by their structure; 1) polymeric nanoparticles have a structure of a capsule or matrix, 2) polymeric micelles of amphiphilic polymers with core-shell structure are spontaneously formed in water, 3) polymersomes are polymeric vesicles, membrane bilayer constructed from amphiphilic polymers, and 4) dendrimers are hyperbranched structures, composed of multiple branched monomers emerging radially from the core (Cho et al. 2008, Brinkhuis, Rutjes & Van Hest 2011). Drug is usually either linked covalently to a polymer or physically entrapped into the polymer capsule or matrix (Rawat et al. 2006). Even though natural polymers such as albumin, chitosan, and heparin have been used for the delivery of drugs and genetic material, the synthetic polymers may be preferable because they can be designed and synthesized to achieve required properties. Among various polymers tested N-(2-hydroxypropyl)-methacrylamide copolymer (HPMA), polyethylene glycol (PEG), and poly-L-glutamic acid (PGA) are examples of synthetic polymers used for drug delivery (Cho et al. 2008). Albumin-bound paclitaxel (Abraxane®) is an example of a nanoparticle formulation in clinical use for the treatment of metastatic breast cancer. Cationic polymers are able to bind and condense DNA into polyplexes which are even smaller than lipoplexes formed after condensation of DNA with cationic liposomes (Dunlap et al. 1997). Poly(ethylene imine) (PEI), poly-L-lysine (PLL), and poly(2(dimethylamino)ethyl methacrylate) (PDMAEMA) are well known polymers for gene delivery (Figure 4). Charge ratio between positive nitrogen atoms of polymer and negative 15 phosphate groups of nucleic acid (n/p ratio) is an important factor in mediation of transfection and toxicity. An excess of positive charges, n/p ratio 2.5 in the case of PEI, is needed for total DNA condensation (Boeckle et al. 2006). Increasing the n/p ratio of PEI/DNA complexes from 2 to 20 results in a decrease in a particle size from 1000 nm to 100-200 nm and a simultaneous reduction in a polydispersity (Erbacher et al. 1999). High cationic charge of polyplexes leads to enhanced transfection efficiency, but as a drawback, to higher toxicity because of the free polymer in solution (Hanzlíková et al. 2011). It has been demonstrated that polymer architecture has an impact on the DNA condensation and gene transfection properties of the polyplexes. Männistö et al. (2002) demonstrated lower transfection activity for dendritic PLL compared to linear PLL. They reasoned it might be due to unfavourable shape and orientation of dendritic amines for DNA binding. However, in the case of PDMAEMA having a star-shaped architecture, which mimics the structure of dendrimer, the polymer showed enhanced transfection efficacy and reduced cytotoxicity compared to linear PDMAEMA (Xu et al. 2009). It has been shown that the molecular weight of PEI strongly influences on the transfection efficiency (Godbey, Wu & Mikos 1999). Choosakoonkriang et al. (2003) showed that both branched and linear PEI (25 kDa) mediated higher transgene expression than smaller, branched PEI 2 kDa. Linear PEI 22 kDa (ExGen 500) has proven to be more effective than branched PEI 25 kDa in mediating transfection in lung epithelial cells both in vitro and in vivo (Wiseman et al. 2003). However, linear PEI forms large unstable aggregates in salt-containing medium that might explain its high gene delivery ability in vivo (Wightman et al. 2001). Figure 4 Chemical structures of some essential polymeric DNA carriers. Branched poly(ethylene imine) (PEI), poly-L-lysine (PLL), poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), and polyethylene glycol (PEG). 16 2.1.3 Hybrid particles To design an optimal drug or gene delivery vehicle, a combination of lipids, polymers, and proteins, forming different types of nanostructures, could be used. The materials can be linked together covalently or mixed as a physical mixture. Probably the most well-known modification of nanoparticles is the steric stabilization of the particle surface with a hydrophilic polymer, most commonly PEG. PEG is a bio-compatible, water-soluble, and chemically inert synthetic polymer. It generates a stealth effect on the surface of the particles thus reducing aggregation, enhancing the stability, and prolonging the circulation time in the body (Allen et al. 2002). The surface coverage of the particles is determined by the molecular weight of the polymer as well as the graft density. PEG molecular weight of 2000–5000 has been shown to be the most effective in prolonging circulation half-life of liposomes (Allen et al. 1991). It has been proposed that if the graft density of PEG2000 on liposomes is below 5%, it takes the shape of a “mushroom” or a half sphere, and if the density is high (> 5%), it takes an extended “brush” shape (Allen et al. 2002) (Figure 5). The inclusion of 3-10% of PEG in liposomes has been shown to prolong their circulation times (Allen et al. 1991). The surface of the nanoparticles can be functionalized with targeting ligands, such as antibodies, antibody fragments, or peptides that are able to bind to the specific cell types. For the purpose of diagnostics and imaging, different kinds of markers such as radio ligands or fluorescent markers can be used (Figure 5). Membrane-active, cationic peptides on the surface of the particles have proven to enhance intracellular delivery (Kale, Torchilin 2007, Ye et al. 2010). When the targeting ligands or membrane active peptides are added to the surface of PEGylated particles, these ligands are usually coupled to the end of the PEG chains rather than straight onto the particle surface (Hansen et al. 1995). This minimizes the interference of the PEG shield thus enabling the interaction between the ligand and the target cell or antigen (Shiokawa et al. 2005). The correct orientation of the targeting moieties is important in order to achieve efficient interaction with the receptors. Figure 5 Functionalization of a liposome. Steric stabilization with PEG2000 at < 5 mol % results in “a mushroom” shape of the PEG molecules (a), if the graft density is > 5 mol % PEG takes“a brush” shape (b). Targeting antibody (c) and cell penetrating peptide (d) coupled to the distal end of a PEG chain. Hydrophilic drugs or imaging agents can be encapsulated in the core of the liposome (e) and lipophilic ones into the liposome bilayer (f). 17 2.2 Challenges in efficient drug and gene delivery Despite the great potential of drug and gene carriers, they face multiple challenges on their way from the vial to the site of action (Figure 6). The vector has to remain stable during storage, and also in physiological conditions. It should not be cleared too fast from the blood circulation or cause immunological reactions. Intravenously administered carrier must pass from the vasculature to the target tissue, bind, and internalize to the target cells. The drug or gene should be released from the carrier at the target site and, in certain cases, be imported into the nucleus (Mastrobattista, Koning & Storm 1999, Wang, Upponi & Torchilin 2011). These issues should be taken into account during the development of new nanocarrier systems. Figure 6 Critical steps for efficient drug and gene delivery. Storage stability (1), stability and half-life in blood circulation (2), extravasation from the blood stream (3), specific binding and internalization into the target cells (4), escape from the endosomes and intracellular trafficking (5), and nuclear localization (6). 2.2.1 Stability of the vector In order to have a good nanoparticle formulation for clinical use, it should first be stable during storage. Uncoated particles are prone to aggregation, which results from many factors, such as ionic strength and pH of the solution, the initial size distribution of the particles and storage temperature (Lee, Mount & Ayazi Shamlou 2001). Charge-neutral complexes or complexes formed at low n/p ratios tend to aggregate because of hydrophobic interactions or van der Waals forces. Whereas higher surface charge reduces aggregation because of electrostatic repulsion (Tros de Ilarduya, Sun & Düzgüneş 2010). In addition to physical stability, chemical stability also has to be taken into account; for example, the lipids may be hydrolysed, resulting in lysolipids, and especially unsaturated lipids can be oxidized easily. Hydrolysis and oxidation finally lead to degradation of lipidic carriers. To enhance chemical stability, antioxidants can be added to the 18 preparation, or liposomes can be stored as lyophilized powders, but the size distribution, morphology, and entrapped cargo must be examined after reconstitution (Ulrich 2002). Reserving the stability is even more difficult in physiological conditions. In blood circulation there are serum proteins, mainly albumin, but also lipoproteins (high- and low density lipoproteins, HDL and LDL) and many other proteins which may interact with polymeric and lipidic particles. They can alter the complex diameter and zeta potential, especially in the case of cationic complexes, and lead to premature release of encapsulated material (Zelphati et al. 1998). Extracellular space and cell surface contain negatively charged glycosaminoglycans (GAGs), components of connective tissues that are often covalently linked to protein in the form of proteoglycans. Sulphated GAGs, such as chondroitin sulphate and heparan sulphate are able to block the transfection of cationic polyplexes and lipoplexes (Ruponen, Ylä-Herttuala & Urtti 1999, Ruponen et al. 2004). To prevent aggregation and premature disruption in vivo, the carrier system should be neutral in charge, and the particle size and shape should be optimal (Tao et al. 2011). Interestingly, polymeric nanoparticles, having shapes like long cylinders (Geng et al. 2007) or elliptical discs (Muro et al. 2008), have demonstrated longer blood circulation times than their spherical counterparts. Most of the current nanocarriers, however, are cylindrical in shape, probably because of ease of manufacture. PEG-shield on the surface of the particles can mask the possible charges and form a hydrated steric barrier against aggregation (Tirosh et al. 1998, Erbacher et al. 1999). 2.2.2 Tissue distribution and elimination To be able to find their targets in the body, therapeutic particles should remain long enough in the blood circulation. Still, only a small fraction of the dose can reach the tumor. In mice, 24 h from intravenous injection of PEGylated liposomes, roughly, only 0.5–5% of the injected dose has internalized the tumor xenograft, 10–20% still remains in the blood circulation, 10–20% is up taken by the liver, and 2–5% is up taken by the spleen (Chang et al. 2007, Chow et al. 2009, Lee et al. 2010). The defence mechanisms of the body react rapidly against foreign material. The mononuclear phagocyte system (MPS) consists of phagocytic cells in spleen, liver (Kuppfer cells), lungs, and lymph nodes. Especially cationic or hydrophobic particles can interact with serum proteins, be opsonised, and removed from the blood circulation by MPS (Allen et al. 1991, Dash et al. 1999). This can be seen as high accumulation of carrier systems in liver, spleen, and lungs. Aggregation of the complexes may also lead to embolization in the lungs, which is obviously life threatening (Morille et al. 2008). Opsonization can also activate the complement system that induces phagocytosis and initiates inflammatory responses against the foreign particles (Müller-Eberhard 1988). Vauthier et al. (2011) reported binding of bovine serum albumin (BSA), fibrinogen, and a complement activating protein, C3, on the nanoparticles consisting of poly(isobutylcyanoacrylate)-dextran co-polymers. Adsorption of BSA on the nanoparticles following C3 protein binding activated the complement cascade, while fibrinogen induced aggregation of the particles. There are several approaches to reduce MPS recognition of the particles after i.v. administration, referred in Harasym, Bally & Tardi (1998). The first method is to modify 19 the particle surface properties, e.g. by incorporation of hydrophilic polymers and size (favourably 50–150 nm) to inhibit the recognition of the immune system. In the second approach, the phagocytic cells are saturated by increasing the dose of the particles beyond the level that is required for therapy (>100 mg lipid/kg in murine studies). Certain drugs, such as liposomal doxorubicin, can also be used to block the phagocytic system. From these methods, the first option is more relevant because of its safety. Coating the liposome surface with inert, hydrophilic polymers (e.g. PEG) provides steric stabilization against interactions with opsonic factors. PEGylated liposomes show longer circulation times and reduced uptake by MPS compared to conventional, non-PEGylated ones (Allen et al. 1991, Lu et al. 2004). PEG forms a highly hydrated shield around the liposome that has been thought to sterically inhibit both electrostatic and hydrophobic interactions with the serum proteins (Mastrobattista, Koning & Storm 1999, Ogris et al. 1999). However, there is increasing evidence suggesting that PEG does not inhibit plasma protein binding on the liposome surface (Moghimi, Szebeni 2003, Dos Santos et al. 2007). In fact, PEG may even enhance complement activation via binding of immunoglobulin M (IgM) and IgG (Moghimi, Szebeni 2003). Instead, the mechanism for prolonged circulation time provided by PEG could be prevention of aggregation of the liposomes (Dos Santos et al. 2007). Attachment of targeting ligands to the nanoparticle surface may lower the stability and alter the pharmacokinetics of the carrier. Especially antibody-coupled carriers are rapidly recognized by the immune system and cleared from the blood circulation. The higher the antibody density on the particles, the faster the clearance (Aragnol, Leserman 1986). Harding et al.´s (1997) pharmacokinetic study with repeated injections of antibodycoupled liposomes showed even more rapid clearance after second and third injection compared to initial administration, evidencing immunogenicity of the formulation. Interestingly, they also demonstrated that antibodies coupled to liposomes are more immunogenic than free antibodies. Using smaller antibody fragments (Fab´, scFv) instead of the whole antibody molecule, the half-life can be prolonged to almost the same level with PEGylated, non-targeted liposomes (Maruyama et al. 1997, Pastorino et al. 2003b). A prolonged circulation time is prerequisite for efficient accumulation of the particles to the target site. The mechanism of the liposomal accumulation from blood circulation into the tumors is discussed in section 2.3.1. 2.2.3 Cellular uptake When the nanocarrier reaches the target tissue, for example tumor, it is facing the next barrier, the cell membrane. There are two main routes for cellular uptake: endocytic and non-endocytic. Endocytic cell uptake can occur via several pathways: clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis, or phagocytosis (Hillaireau, Couvreur 2009). Fusion and penetration of the nanocarriers through the cellular membrane are examples of non-endocytic pathways (Xiang et al. 2012). It has been shown that endocytosis (Figure 7) is the predominant route for internalization of polymeric and lipidic nanoparticles (Wang, Upponi & Torchilin 2011). Cationic particles trigger endocytosis by interacting non-specifically with the negatively charged cell surface via cell membrane associated proteoglycans (Mislick, Baldeschwieler 20 1996, Mounkes et al. 1998) or other anionic components on the cellular membrane. On the other hand, it has been shown that cell-surface glycosaminoglycans can also inhibit cellular uptake and gene expression of cationic DNA complexes (Ruponen et al. 2004). Figure 7 Cellular uptake mechanisms of drug-loaded nanoparticles. Non-specific adsorption and internalization via endocytosis (a), target-specific binding followed by receptor-mediated endocytosis (b). If drug carrier binds to non-internalizing receptor, drug can be released outside of the cell (c). Lipidic carriers can fuse with the cell membrane (d) or exchange the lipid components with the cell membrane (e), leading to drug release inside the cell. Modified from Torchilin (2005). Receptor-mediated endocytosis can take place after specific binding of the targeting ligand to its receptor. This is a very important mechanism by which the cells take up nutrients and regulatory proteins, and it is nowadays also utilized in drug and gene delivery. Binding to the receptor does not automatically mean rapid internalization into the cell. In the case that the drug carrier is targeted to a non-internalizing receptor, the carrier should release the drug outside the cell after which the free drug is taken up by the host cell and also by the neighbouring cells (Figure 7). This kind of “bystander effect” might be preferable in solid tumors where diffusion of large carrier systems is limited or all of the cancer cells do not express the targeted antigens (Mastrobattista, Koning & Storm 1999, Sapra, Allen 2003). However, liposomal drug carriers endocytosed via receptor binding have been shown to have enhanced antitumoral efficacy over the carriers bound to noninternalized receptors (Chuang et al. 2010). Non-endocytic pathways are preferable for non-viral gene delivery because the destructive effect of lysosomes is then usually avoided (Morille et al. 2008). To enhance the internalization of drug and gene carriers, cationic membrane active peptides can be coupled to the particle surface. These cell penetrating peptides (CPP), for example transacting activator of transcription (TAT) peptide from HIV-1, can mediate intracellular 21 delivery via endocytic, and according to some studies, also via non-endocytic pathways (Torchilin et al. 2001, Bolhassani 2011). Nonetheless, the non-endocytic mechanism of cellular penetration of CPPs can be considered to be ambiguous. In the study of Subrizi et al. (2012), only the endocytic uptake mechanism for CPPs could be seen. Lipidic carriers are able to fuse with the cellular membrane and directly release the contents to the cytoplasm before entering the endocytic pathways. However, the main uptake route for lipoplexes is the endocytic pathway, whilst fusion plays an important role in releasing the DNA in endosomes (Hafez, Maurer & Cullis 2001, Xiang et al. 2012). 2.2.4 Intracellular distribution and cargo release Following internalization via endocytic pathway, endosome capture and subsequent lysosomal degradation are the major obstacles to efficient gene delivery. To be effective, the vector, or at least its contents must be released from the endosome before its maturation into the lysosome. DNA and RNA degrade easily in the lysosomal compartments by hydrolytic enzymes. The endosomal release should happen rather fast since after endocytosis, the endosomal vesicles mature into lysosomes in 10–20 min (Simões et al. 2004). For polymer-based vectors, two possible escape mechanisms have been proposed. The first one, physical disruption of the negatively charged endosomal membrane via interaction with cationic polymers has been suggested by Zhang, Smith (2000). They noticed that high generation poly(amidoamine) (PAMAM) dendrimers were much more effective than PLL in inducing lipid mixing and leakage of the contents. This escape mechanism seems to depend also on the composition of the cellular membrane (cell type). The other, better known mechanism, “proton sponge” effect can be applied by PEI, PDMAEMA and PAMAM which contain protonable secondary and tertiary amines having pKa-value of 5–7, near to endosomal pH (Boussif et al. 1995). The proton-sponge hypothesis is based on high buffering capacity of the polymers. Increase in the endosomal pH causes transportation of protons into the endosome that then results in an influx of counter ions (Cl-). This promotes osmotic swelling and finally rupture of the endosomal membrane (Boussif et al. 1995, Sonawane, Szoka & Verkman 2003). Cationic lipid-based carriers are able to destabilize the anionic endosomal membrane via electrostatic interactions. So called flip-flop-mechanism has been described by Xu, Szoka (1996) and Zelphati, Szoka (1996). After endocytosis, the cationic complex destabilizes endosomal membrane resulting in flip-flop of anionic lipids. The anionic lipids diffuse into the complex, forming a charge neutral ion pair with cationic lipids. As a consequence, entrapped nucleic acids dissociate from the complex and are released into the cytoplasm. Destabilization of the endosomal membrane can also occur after lipid phase transition. Helper lipid, DOPE, as discussed earlier, is able to acquire an inverted hexagonal phase (HII) which is unstable and rapidly fuses and releases DNA or drug upon adhering to endosomal vesicles (Koltover et al. 1998, Mönkkönen, Urtti 1998). To release the DNA or the drug in a controlled manner at the desired target site, delivery systems that are sensitive to a certain signal have been developed. The pH inside the endosomes is 5–6, which is more acidic compared to its environment. The low pH can 22 trigger the release of cargo from pH-sensitive carriers. A combination of DOPE/cholesteryl hemisuccinate (CHEMS) is widely used in pH-sensitive formulations (Kirchmeier et al. 2001, Simões et al. 2001, Shi et al. 2002b, Fattal, Couvreur & Dubernet 2004). In an acidic environment, anionic CHEMS becomes protonated, and this neutral form induces formation of fusogenic hexagonal phase with DOPE. Whereas, at neutral or alkaline pH, CHEMS stabilizes DOPE into a more stable lamellar phase (Hafez, Cullis 2000). In addition to pH, increased temperature and enzymatic activity have been utilized to trigger drug release. In tumors, the temperature is slightly higher than in healthy tissues, but in practice the temperature difference is so small that it makes the controlled release challenging. Drug release from thermosensitive carriers can be triggered by using localized external heating. Kullberg, Mann & Owens (2009) used external heating up to 42 °C to trigger calcein release from temperature-sensitive 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC)-based immunoliposomes. The drug release is based on a sharp gel to liquid crystalline phase transition of thermosensitive lipids at a certain temperature. Paasonen et al. (2007) created a liposomal system where the contents of the liposomes were released by UV-light induced heating of the gold nanoparticles incorporated into the liposome bilayer. Local heating of the gold nanoparticles resulted in leakage of thermosensitive liposomes. Enzymatically active carriers, for example human serum albumin (HSA) nanoparticles, have shown degradation and drug release in the presence of physically existing enzymes: trypsin, proteinase K, protease, pepsin, and intracellular enzyme cathepsin B (Langer et al. 2008). 2.2.5 Diffusion in cytoplasm and nuclear import Some drugs can act in the cytoplasm, but others, such as plasmid DNA and many cytostatic drugs, must enter the nucleus to reach the site of action (Table 1). After escape from the endosomes, the drug then faces the challenges of intracellular trafficking and nuclear localization. For large molecular weight DNA in particular, these are difficult barriers to overcome. Mobility of DNA in cytoplasm is slow because of the tight network of cytoskeletal filaments, the presence of cell organelles, and high protein concentration (Lechardeur, Verkman & Lukacs 2005). The diffusion rate of DNA depends strongly on the size of the molecule. Plasmid DNA, containing 1 000–10 000 base pairs (bp), diffuses much slower than DNA or RNA molecules under 250 bp (Dauty, Verkman 2005). Slow diffusion makes DNA an easy target for cytoplasmic nucleases (Lechardeur, Verkman & Lukacs 2005). 23 Table 1 Examples of target sites inside the cell of different therapeutic agents. Therapeutic agent Genetic drugs plasmid DNA oligonucleotides (siRNA, miRNA) Small molecular anticancer drugs doxorubicin paclitaxel camptothecin Site of action in the cell nucleus mRNA, cytoplasm/nucleus DNA, nucleus microtubules, cytoplasm DNA enzyme topoisomerase I, nucleus If DNA remains complexed in the cytosol, the resistance against nucleases can be increased (Lechardeur et al. 1999). Pollard et al. (1998) showed that 1% of cytoplasmic DNA/PEI complexes entered the nucleus, which was 10 times more than uptake of free plasmid DNA. Since passive diffusion into the nucleus via nuclear pore complexes is limited for particles less than 10 nm in diameter, the nuclear entry of plasmid DNA can occur mainly during cell division when the nuclear envelope is reformed (Görlich, Mattaj 1996). Toropainen et al. (2007) demonstrated substantially higher transgene expression in dividing human corneal epithelial (HCE) cells compared to differentiated HCE cells after transfection with PEI/DNA and DOTAP/DOPE/DNA complexes. More specifically, higher nuclear accumulation has been seen in the cells which are close to mitosis phase compared to the cells in post-mitotic phase (Gap 1) (Männistö et al. 2007). Männistö et al. (2007) also demonstrated that despite the high amount of imported transgene in the nucleus, only 10-6 to 10-4 parts were totally released from the carrier and thus available for transcription. Release of DNA from the carrier is thus a critical step since premature disassembly can lead to DNA degradation while incomplete release impairs gene expression. 24 2.3 Targeted cancer therapy Anti-cancer drugs are usually toxic for the cell, which is why they are undesirable in healthy tissues. With targeted nanocarrier systems, the drug concentration could be increased at the tumor site (ElBayoumi, Torchilin 2009) and thus the spreading of the harmful drug to the normal tissues may be reduced. Targeting also improves internalization of the drug into cancer cells (Mamot et al. 2003, Dubey et al. 2004). Tumor targeting can be divided into two types; passive and active (Figure 8). Figure 8 Passive targeting and active targeting of nanoparticles. A. Passive targeting is based on leaky vasculature of the tumor and long circulation time of nanocarriers. B. In active targeting, nanocarriers can be targeted to the receptors overexpressed either on tumor cells (1) or on angiogenic endothelial cells (2). Modified from Danhier, Feron & Preat (2010). 2.3.1 Passive targeting When tumor volume reaches 1–2 mm3, it starts to form new blood vessels in order to bring oxygen and nutrients to the growing cells (Feron 2004). This blood vessel formation is called angiogenesis. The morphology of tumor vasculature differs from the normal vessels. The tumor vessel endothelium is malformed and leaky; having 100–600 nm gaps between the endothelial cells, whereas normal endothelial cells form a continuous, uniform monolayer (Yuan et al. 1995, Hashizume et al. 2000). Pericytes, the cells surrounding the endothelial cells, are also malformed in angiogenic tumor vessels (Morikawa et al. 2002). Thus, small 50–200 nm particles can enter the tumor. Moreover, due to a non-functional, or absent, lymphatic drainage system, nanoparticles can be also retained in the tumor interstitium. This phenomenon is called the “Enhanced permeability and retention” (EPR) effect and is utilized in passive targeting of nanoparticles into tumor (Maruyama 2011). Because of the EPR effect, it is possible to achieve even 10–50 fold local concentrations of nanoparticles in tumor compared to normal tissues (Iyer et al. 2006). The properties of the nanocarriers can influence on the EPR effect. The carriers should have a long half-life in blood in order to have enough time for efficient tumor 25 accumulation. Optimally, the size of the particle should be more than 10 nm to avoid filtration through the kidneys, but under 100 nm to avoid a capture by the liver (Danhier, Feron & Preat 2010). In addition, they should be sterically stabilized to avoid aggregation and rapid recognition by the MPS system. The tumor environment also provides some barriers for successful therapy, such as heterogenous blood flow, increased interstitial fluid pressure, and large transport distances in the tumor interstitium (Jain 1990, Harrington et al. 2000). Harrington et al. (2000) demonstrated the influence of tumor size on the uptake of PEGylated liposomes. In large tumors, the uptake of liposomes is probably reduced due to higher osmotic pressure and because of a relatively low vascular volume, reflecting areas of poor perfusion or even necrotic areas. 2.3.2 Active targeting To mediate active tumor targeting, cancer cell specific targeting ligands are attached to the surface of the nanocarrier. The chosen ligand actively binds to the receptors that are either selectively expressed or overexpressed in cancerous cells compared to normal cells (Sapra, Allen 2003). The most commonly used ligands for liposome and nanoparticle targeting are: monoclonal antibodies (mAb) (ElBayoumi, Torchilin 2009), fragments of the antibodies (Fab or svFc) (Pastorino et al. 2003b, Iyer et al. 2011), growth factors (Lee et al. 2010), peptides (Moreira et al. 2001, Temming et al. 2005, Xiong et al. 2005), small molecule ligands (such as folate and transferrin) (Gabizon et al. 1999, Voinea et al. 2002, Riviere et al. 2011), sugars (such as galactosamine, lactose, and trivalent galactose) (David et al. 2004), and aptamers (Tong et al. 2010) (Table 2). To attach the ligands on the sterically stabilized liposomes, the ligands are preferably coupled to the termini of the PEG chains. When the ligands are attached on the bilayer of the liposome, the PEG may serve as a steric hindrance for both ligand coupling and later on for binding to the receptors, especially in the case of small molecular weight ligands (Sapra, Allen 2003). The end group of the PEG-spacer can be functionalized for the chemical ligand coupling, for example with maleimide (Kirpotin et al. 1997) or N-(3´(pyridyldithio)propionoylamino (PDP) (Allen et al. 1995) for the thiol-containing ligands, or with biotin for avidin-coupled ligands (Loughrey, Bally & Cullis 1987). The amount of targeting ligands attached on the liposomes is crucial since excessive ligand density leads to rapid clearance of the liposomes, while insufficient ligand density fails to facilitate satisfactory targeting efficiency. Only 10–20 molecules of whole targeting antibody or Fab´ fragments per liposome are required for sufficient internalization to the target cell (Park et al. 1997, Iden, Allen 2001). For antibody density in excess of 35 molecules/liposome, an increased rate of clearance has been reported (Allen et al. 1995). In the case of small peptides, even 200–500 peptide molecules/liposome did not cause highly elevated blood clearance compared to PEGylated, non-targeted, liposomes (Zalipsky et al. 1995). Active tumor targeting could be achieved by direct targeting, where the targeting ligands are coupled straight on the drug carrier, or by a pre-targeting (multistep) approach. In the pre-targeting method, ligands are not covalently linked to the carrier system; 26 instead, the target-specific ligand is administered as a first step. Once ligand has bound to the target receptor, the ligand-binding drug-containing nanoparticles are administered. Pre-targeting is commonly based either on biotin-avidin binding, that shows extremely high affinity of Kd ~ 1015 M-1, (Weber et al. 1989, Lesch et al. 2010) or on bispecific antibodies (Sharkey et al. 2003). Pre-targeting has been utilized in targeting of polymeric nanoparticles (Nobs et al. 2006, Pulkkinen et al. 2008) and liposomes (Xiao et al. 2002, Pan et al. 2008). 2.3.2.1 Cancer cell targeting in solid tumors To reach the cancer cells throughout the solid tumor, nanocarriers should extravasate from blood circulation to tumor interstitial space and diffuse evenly around. Moreover, the target receptors should be expressed homogenously on all targeted cells. The most targeted receptors in solid tumors are: 1) human epidermal growth factor receptors (EGFR and HER-2), overexpressed in many tumor types, e.g. in breast, colon, ovarian, pancreatic, head and neck cancer, and non-small cell lung cancer; 2) transferrin receptor, which participates in iron transfer, expressed in tumor cells 100-fold more than in normal cells; and 3) folate receptor, which takes care of folic acid intake and is also overexpressed in many human cancer types (Danhier, Feron & Preat 2010). Even though a high affinity to the target receptor is desirable, it can also limit the tumor penetration properties of the nanocarriers. Adams et al. (2001) showed that with low affinity (Kd = 3.2 x 10-7M), anti HER2/neu scFv exhibited broad diffusion from the vasculature to the tumor, whereas the high affinity scFv (Kd = 1.5 x 10-11M) failed to traverse more than 2-3 cell diameters. This study was done with labelled scFv, (molecular weight 27 kDa) without nanocarrier. After coupling these high affinity ligands onto nanoparticles or liposomes (molecular weight of millions), even more restricted spreading to the tumor would be expected. Interestingly, Sugahara et al. (2010) showed that coadministration of non-conjugated tumor-penetrating peptide (iRGD) improved tumor tissue penetration and therapeutic efficacy of free doxorubicin, nanoparticles (Abraxane®), doxorubicin liposomes, and antibody trastuzumab. The mechanism of tumor penetration for iRGD is distinct from the passive EPR-effect, since it is receptor-mediated and energy-dependent. 2.3.2.2 Targeting to the tumor vasculature Tumor vasculature targeting aims to obstruct the blood supply of the tumor. That leads to a lack of nutrients and oxygen, in turn causing tumor cell starvation and death. When compared to tumor cell targeting, vascular targeting has some advantages: 1) direct accessibility to the endothelial cells from blood circulation avoiding the problems related to poor extravasation and tumor tissue penetration; 2) high efficacy, since one tumor capillary supplies hundreds of tumor cells; 3) avoidance of drug resistance, because endothelial cells are genetically stable compared to tumor cells that may become resistant 27 to the therapy; 4) broad applicability, since most solid tumors are dependent on neovascularization (Mastrobattista, Koning & Storm 1999, Feron 2004). Besides during tumor growth, angiogenic vessels are formed also in some nonmalignant conditions such as in atherosclerosis, wound healing, psoriasis, and in certain eye diseases, e.g. in the wet form of age-related macular degeneration and neovascularisation of the cornea. Angiogenic vessels express markers such as vascular endothelial growth factor receptor (VEGFR) and integrins (αvβ3 and αvβ5) that are not present in the resting blood vessels of normal tissues (Ruoslahti 2002). The integrins are also upregulated in different tumor cells, including metastic melanoma cells (Conforti et al. 1992, Seftor, Seftor & Hendrix 1999). Integrins can be specifically recognized by RGD-peptide, consisting of arginine, glycine, and aspartic acid. RGD-peptide was found by screening of phage display peptide libraries (Pasqualini, Koivunen & Ruoslahti 1997) and it is one of the most studied tumor vasculature homing peptides. 28 Table 2 Nanocarrier-based targeted therapeutics in clinical development and examples of preclinical studies. Targeting ligand Clinical trials stomach cancer specific GAH mAb anti-transferrin receptor scFv human transferrin human transferrin peptide Preclinical studies cancer cell specific mAb 2C5 Fab´ fragment of cetuximab mesothelioma targeting scFv (M1) epidermal growth factor A10 aptamer folate cyclic RGDpeptide NGR-peptide Fab´ fragment of anti-VEGFR-2 mAb antidisialoganglioside and NGR-peptide Formulation Target Study phase Reference PEGylated liposomal doxorubicin (MCC465) liposomal p53 plasmid DNA (SGT-53) liposomal oxaliplatin MBP-426) siRNA loaded nanoparticles (CALAA-01) docetaxel-loaded polymeric nanoparticles (BIND014) tumor antigen in stomach cancer I Reviewed in (Cheng et al. 2012) transferrin receptor I transferrin receptor I/II transferrin receptor I prostate specific antigen I PEGylated liposomal doxorubicin cancer cell surface bound nucleosomes (ElBayoumi, Torchilin 2009) PEGylated liposomal doxorubin/vinorelbin PEGylated 111Inlabeled liposomes epidermal growth factor receptor surface antigens on human mesothelioma tumor cells (Mamot et al. 2005) (Iyer et al. 2011) 111 In-labeled polymeric micelles polymer-paclitaxel conjugates PEGylated liposomal doxorubicin PEGylated liposomal 5-fluorouracil epidermal growth factor receptor prostate-specific membrane antigen folate receptor (Lee et al. 2010) PEGylated liposomal doxorubicin PEGylated liposomal doxorubicin PEGylated liposomal doxorubicin angiogenic endothelial cell marker aminopeptidase N vascular endothelial growth factor receptor disialoganglioside receptor and aminopeptidase N αvβ3 integrins 29 (Tong et al. 2010) (Riviere et al. 2011) (Dubey et al. 2004) (Pastorino et al. 2003a) (Roth et al. 2007) (Pastorino et al. 2006) 3 Aims of the study The general objective of this study was to develop and evaluate lipid and polymer based nanocarriers for targeted drug and gene delivery using in vitro, in vivo, and in silico methods. The specific aims were: 1. To investigate the effects of the architecture and flexibility of cationic amphiphilic star and linear PDMAEMA-based block copolymers on DNA complex formation, in vitro transfection efficiency, and cytotoxicity. 2. To determine the DNA binding ability, in vitro transfection efficiency, and cytotoxicity of novel BSA- and hydrophobin (HFBI)-dendron conjugates. 3. To develop an extracellularly stable gene delivery vector that can release its contents at the acidic endosomal pH. 4. To investigate a targeted liposomal drug delivery system when novel activated endothelium targeted peptide (AETP) is used as a targeting ligand. 5. To explore an epidermal growth factor receptor (EGFR) targeted liposomes using direct targeting and pre-targeting approaches. 30 4 Overview of the methods The materials and prepared formulations for gene delivery (I – III) and for targeted liposomal drug delivery (IV, V) are summarized in Table 3. The cell lines used in the original publications are shown in Table 4. General methods of physicochemical and biological studies are shown in Tables 5 and 6, respectively. In addition, a computational study combining molecular dynamics simulation and ligand-protein docking was performed (IV). Materials and methods are described in detail in the publications. Table 3 Summary of materials and prepared formulations in the publications. Material/Formulation plasmid DNA (pCMVβ) encoding βgalactosidase Polymeric materials PDMAEMA block copolymers: (PS-PDMAEMA)6, (PBuA-PDMAEMA)6, PDMAEMA-PS-PDMAEMA, PDMAEMA-PBuA-PDMAEMA protein-polyamine dendron conjugates: HFBI, HFBI-G1, HFBI-G2, BSA, BSA-G1, BSA-G2 other polymers: PEI 25K, PLL Lipidic materials DOPE, CHEMS, Egg PC, Egg SM, DMPG, cholesterol HSPC, cholesterol, DSPE-PEG2000, DSPE-PEG2000-maleimide, DSPE-PEG2000-biotin Targeting ligands AETP cetuximab Formulations cationic polyplexes lipid-coated polyplexes calcein containing liposomes PEGylated liposomes Source/Preparation method amplification in E.coli, purification by column separation Publication I - III synthesized, purified and characterized in the Department of Chemistry, Laboratory of Polymer Chemistry, University of Helsinki I synthesized, purified and characterized in the Department of Engineering, Physics, and Mathematics, and Center for New Materials, Helsinki University of Technology commercially available II commercially available III commercially available IV, V phage display and peptide synthesis using Fmoc chemistry in Karyon Ltd., Finland commercially available IV self-assembling detergent removal reverse-phase evaporation extrusion I - III III III IV, V 31 I - III V AETP-targeted liposomes cetuximab-targeted liposomes doxorubicin- liposomes extrusion + post-insertion method of AETP extrusion + biotin-avidin-biotin binding of cetuximab extrusion + remote-loading of doxorubicin IV V IV, V CMV = cytomegalovirus, PDAMEMA = poly(2-(dimethylamino)ethyl methacrylate), PS = polystyrene , PBuA = poly(n-butyl acrylate), HFBI = hydrophobin, BSA = bovine serum albumin, G1 = first generation, G2 = second generation, PEI = poly(ethylene imine), PLL = poly-L-lysine, DOPE = 1,2-dioleyl-sn-glycerol-3phosphoethanolamine, CHEMS = cholesteryl hemisuccinate, Egg PC = egg phosphatidyl choline, Egg SM = egg sphingomyelin, DMPG = 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol), HSPC = fully hydrogenated phosphatidyl choline, DSPE-PEG2000 = 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[amino(polyethylene glycol)-2000], AETP = activated endothelium targeting peptide, Fmoc = 9fluorenylmethyloxycarbonyl Table 4 Cell line ARPE-19 C8161 CV1 primary HUVEC KS1767 SKOV-3 SKOV3.ip1 SVEC4-10 Table 5 The cells used in the publications. Type retinal pigment epithelial cell line melanoma cell line kidney fibroblast cell line umbilical vein endothelial cells Species human human monkey human Publication I IV I-III, V IV kaposi’s sarcoma cell line ovarian adenocarcinoma cell line ovarian adenocarcinoma cell line lymph node endothelial cell line human human human mouse IV V V IV Physicochemical characterization methods used in the publications. Study objective particle size DNA-complex integrity pH-sensitivity phospholipid (liposome) concentration liposomal drug concentration ligand coupling efficiency Method dynamic light scattering ethidium bromide intercalation assay calcein-dequenching assay fluorescence Probe ethidium bromide calcein fluorescein-PE Publication I, III - V I-III absorbance doxorubicin IV, V fluorescence tryptophan IV, V 32 III IV, V Table 6 Biological methods used in the publications. Study objective in vitro studies cytotoxicity/therapeutic activity of DNA-complexes and liposomes transfection efficacy Method Probe Publication formazan II - IV Alamar Blue -assay resorufin I, V ONPG-assay ONPG I - III cellular affinity/uptake of the formulations in vivo studies pharmacokinetics: half-life of liposomes biodistribution of liposomes FACS-analysis EMA-DNA fluorescein III IV - V TRF Europium IV TRF confocal microscopy LC-MS analysis SPECT-CT and gammacounting Europium Rho/FITC doxorubicin 99m Technetium IV IV V V MTT-assay ® MTT = 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, ONPG = ortho-nitrophenyl-β-Dgalactopyranoside, FACS = fluorescence-activated cell sorting, EMA-DNA = ethidiummonoazide, TRF = timeresolved fluorescence, Rho = rhodamin, FITC = fluorescein isothiocyanate, LC-MS = liquid chromatography mass spectrometry, SPECT-CT = single-photon emission computed tomography - computed tomography 33 10 Summary of the main results The main results of the experimental data and molecular modeling are presented in Table 7. Table 7 Summary of the main results. Gene delivery vectors DNA-binding properties Publication All PDMAEMA block co-polymers bound DNA. Only (PS- I PDMAEMA)6 was not able to condense DNA totally. HFBI-G1, HFBI-G2 and BSA-G2 showed the highest binding II capacity towards DNA. LCDCs condensed DNA completely. After treatment with III anionic dextran sulphate, LCDCs prepared by detergent dialysis method were stable at least for 24 h, while LCDCs prepared by ethanol injection method had released ~50% of DNA at 24 h time point. pH-sensitivity LCDCs were stable at neutral pH, but they were able to fuse with calcein-containing endosome-mimicking liposomes at endosomal pH (5-6). Transfection Linear PDMAEMA-PBuA-PDMAEMA was able to transfect ARPEefficacy in 19 cells with efficacy comparable to PEI, and CV-1 cells with vitro efficacy of 1/10 of that of PEI. The other carriers showed some transfection efficacy in the following order: PDMAEMA-PSPDMAEMA ≈ (PBuA-PDMAEMA)6 > (PS-PDMAEMA)6. Of the protein-dendron conjugates, only HFBI-G2 was able to transfect CV-1 cells. Transfection efficacy was only 1/20 of that of PEI. Transfection efficiency of LCDCs was ~80% of that of PEI/DNA polyplexes in the absence of serum. In the presence 10% serum, the transgene expression level decreased dramatically. Cytotoxicity All PDMAEMA block co-polymers showed over 80% viability in in vitro ARPE-19 and CV-1 cells at low n/p ratios (0.5–4). With increasing n/p ratio, the cell viability decreased gradually. All protein-dendron conjugates showed over 80% viability in CV-1 cells at n/p low ratios (0.125–4). HFBI-G1 and HFBI-G2 were observed to be slightly cytotoxic at higher n/p ratios. Cancer-targeted liposomes Cellular AETP did not increase the cellular affinity of liposomes in HUVECaffinity in cells. vitro EGFR-targeted liposomes had 22–38 times higher affinity towards SKOV-3 cells and 13–17 times higher affinity towards CV-1 cells compared to affinity of non-targeted liposomes. Competition with 34 III I II III I II IV V Cytotoxic activity in vitro Pharmacokine tics in vivo Cellular affinity in vivo Uptake in tumor Orientation of the targeting ligands in silico Affinity towards serum albumin in silico free cetuximab decreased the affinity of the targeted liposomes to the level of the non-targeted liposomes. Pre-targeting showed lower cellular association than direct targeting. AETP did not increase the cytotoxic efficacy of doxorubicin-loaded liposomes in HUVEC or SVEC4-10 cells. EGFR-targeted doxorubicin-loaded liposomes showed slightly higher cytotoxicity than non-targeted liposomes in SKOV-3 cells. However, free DXR was significantly more cytotoxic than both of the liposome types. AETP-targeted liposomes showed similar elimination half-life (~7– 10 h) to non-targeted liposomes (~6–7 h). Significant differences in co-localization of liposomes and endothelial cells could not be seen between the AETP-targeted and non-targeted liposomes in confocal microscopy. No significant difference in tumoral uptake between AETP-targeted and non-targeted liposomes could be seen. After direct targeting or pre-targeting, no significant difference in uptake in tumor between EGFR-targeted and non-targeted liposomes could be seen at 24 h time point. However, intraperitoneal administration of the liposomes, targeted or not, led to faster and higher tumoral accumulation of the liposomes than intravenous administration. Computational modeling revealed that both AETP and RGD peptides located in the PEG region of the PEGylated liposomes. However, AETP was more covered by the PEG chains, while RGD was more exposed to the solvent. Protein-ligand docking showed HSA having 12 times stronger binding affinity to PEG and 14 times stronger affinity to AETP than to RGD peptide. 35 IV V IV IV IV V IV IV 11 General discussion 11.1 Structure-activity relationship of polymeric DNA carriers on DNA-complex formation, transfection efficacy, and toxicity To be able to condense the DNA, polymer should have a sufficient density of positive charges. On the other hand, high cationic charge density is often related to increased toxicity. Electrostatic interactions with the plasma membrane are known to damage the cell (Lv et al. 2006). An additional critical step of gene delivery is escape from the endosomes. Because cationic polymers lack hydrophobic parts, they cannot destabilize the endosomal membrane. However, some polymers do protonate at endosomal pH (e.g. PEI, PAMAM or PDMAEMA) and they can act as swelling proton sponges. To modify the properties of the cationic polymers, they can be synthesized in different lengths, with different architecture (linear or branched), with different substitutions or additions of functional groups (Tros de Ilarduya, Sun & Düzgünes 2010). Nonetheless, it seems that there is no universal rule regarding how the architecture of a polymer effects its properties as a gene delivery vehicle. To investigate the effect of polymer composition and architecture on the ability to condense DNA and mediate transgene expression, two different star block copolymers and corresponding linear triblock copolymers were synthesized. The hydrophobic core of starlike polymers was composed of either glassy polystyrene (PS) or rubbery poly(nbutylacrylate) (PBuA). The outer block consisted of hydrophilic and cationic PDMAEMA. All of the synthesized polymers were able to form spherical core-shell micelles in aqueous solutions. The linear polymers and star-like (PBuA-PDMAEMA)6 could condense plasmid DNA completely, while glassy (PS-PDMAEMA)6 formed loose DNA complexes. The gene transfection efficiency was highest for linear PDMAEMA-PBuA-PDMAEMA at n/p ratio 2 and 4, and lowest for star-like (PS-PDMAEMA)6. Cytotoxicity was increased at charge ratios of n/p 8 and higher, (PS-DMAEMA)6 being least toxic. The poor transfection efficiency of star-like (PS-PDMAEMA)6 may arise from glassy polystyrene in the core of the star polymers which stiffens the structure of the polymer and may restrict the interaction with plasmid DNA. On the contrary, rubbery pBuA core may not limit the contact of PDMAEMA arms and the DNA. Linear polymer chain is more flexible making the structure more favorable for DNA condensation and transfection compared to star-like structures. In addition, the amine groups are more available for DNA binding in the linear form of the polymer. Linear architecture and rubbery pBuA core seem to be favorable features for mediating transfection. The positive effect of linear architecture was also shown in the case of poly-L-lysine polymers (Männistö et al. 2002). This is the first known investigation into the effects of rubbery and glassy states on gene transfer. This study has revealed evidence that polymer architecture and composition effect gene packing ability and transfection efficiency of the studied block copolymers. Accordingly, linear PDMAEMA-PBuA-PDMAEMA showed transfection efficiency close to PEI 25 kDa in a retinal pigment epithelial cell line in vitro. However, similar to all 36 cationic polyplexes, PDMAEMA-based polyplexes are also liable to deactivation by negatively charged serum proteins and other polyanions in vivo. In another study, protein-dendron conjugates, consisting of either BSA or hydrophobin (HFBI) protein and first-generation (G1) or second-generation (G2) polyamine dendrons, were synthesized. BSA is a rather large (66.4 kDa) biocompatible protein that exhibits long circulation times, while HFBI is a small (8.7 kDa) surface-active protein that is also well known for its safety. Since both of the proteins lack DNA binding motifs, cationic polyamine dendrons were conjugated to them to achieve sufficient DNA binding. All of the dendron-conjugated proteins showed enhanced DNA binding over the naked proteins. HFBI-conjugates expressed stronger affinity to DNA than corresponding BSA-conjugates, especially BSA-G1. This is not surprising because the dendron (~1 kDa) is relatively small compared to the size of BSA and plasmid DNA. HFBI-G2 was the only conjugate with enhanced transfection activity, even though BSA-G2 could bind to DNA with a similar affinity. This could be due to the amphiphilic nature of HFBI that may lead to a favorable interaction with cell membrane structures. Despite the successful use of BSA as a drug nanocarrier, it was not able to mediate transfection when conjugated with dendrons. It has been shown earlier that the small polyamine dendrons are relatively inefficient in mediating transfection on their own, without endosomal disrupting agent, chloroquine (Hardy et al. 2006). Nonetheless, in combination with hydrophobic HFBI, enhanced transgene activity without increased toxicity could be achieved. The transfection efficiency of HFBI-G2 was, however, fairly low compared to PEI 25 kDa. It has been shown that by increasing the size of the PAMAM dendrimers, higher transfection could be achieved (Haensler, Szoka 1993). This may also be the case for HFBI-dendrons. The endosomal escape of HFBI-dendrons would, however, most likely persist as a bottleneck because of deficiency of protonable tertiary amines in the structure of polyamine dendrons. The concept of protein-dendron conjugates worked in the sense of efficient DNA condensation, but the transfection efficiency was very modest compared to PEI 25 kDa. The protein-dendron conjugates are not suitable for gene delivery as such; the concept should be further developed. 11.2 Lipid-coated DNA-complexes as stable gene delivery vectors Cationic lipoplexes and polyplexes are able to transfect cells in vitro, but in vivo they are susceptible to deactivation by several polyanionic proteins and polysaccharides, such as GAGs. Extracellular GAGs can bind to cationic complexes and are able to alter both cellular uptake and intracellular behavior thus decreasing the gene transfer (Ruponen et al. 2004). Optimal gene delivery vectors would be stable in extracellular space but are capable of releasing the cargo inside the cell. Coating of cationic DNA polyplexes with a stabilizing layer of anionic lipids results in envelope-type particles (Guo, Lee 2000, Mastrobattista et al. 2001, Nahde et al. 2001, Guo, Gosselin & Lee 2002, Khalil et al. 2007). The stability of such particles against GAGs has not been evaluated, however. 37 In this study, a method to coat the PEI/DNA complexes by anionic and fusogenic lipid mixture was developed. In this detergent removal coating procedure, the polyplexes are slowly coated by a mixed micellar solution of DOPE/CHEMS/octyl glucoside. The resulting mixture is subsequently diluted with buffer that leads to extraction of the surfactant from the mixed micelles and to the formation of a lipid bilayer. The lipid-coated DNA complexes (LCDC) showed a good stability against a model GAG, dextran sulphate, while uncoated polyplexes disintegrated rapidly in the presence of the GAGs. LCDCs prepared by the detergent removal coating method showed superior stability compared to the LCDCs prepared by ethanol injection procedure, in which the polyplexes were coated with preformed liposomes. The addition of mixed micellar solution to the polyplex surface apparently forms a more uniform coating compared to the addition of preformed liposomes. The coated complexes showed pH-sensitivity at low pH (5-6) by fusion and aggregation. This means that the complexes are able to fuse with endosomes and release their contents, which is important for efficient gene delivery. Because of the negative surface charge, the cellular uptake was lower than the uptake of non-coated cationic PEI/DNA complexes. This may explain why the transfection efficacy also remained lower compared to PEI/DNA polyplexes. Cellular uptake of LCDCs was improved by neutralizing the negative charges with PEGylation, but this modification reduced the pHsensitivity and lowered the level of transfection. PEG is ideal for preventing aggregation and thus prolonging the time in blood circulation, but it becomes unnecessary after particle internalization into the cells. To avoid the obstructing effects of PEG inside the cells, cleavable, acid labile PEG chains have been generated (Guo, Szoka 2001, Romberg, Hennink & Storm 2008). Local DNA delivery, for example in the eye or in some surgical situations, may not require PEGylation because the particles are not removed by the reticuloendothelial system in such cases. The anionic lipid coat or PEG coating may be useful however, in preventing the interactions with local anions, like hyaluronic acid in the vitreous body of the eye. Kurosaki et al. (2013) showed higher transfection activity in the retina of rabbits for PEI/DNA polyplexes coated with anionic polymer-coating than for uncoated cationic PEI/DNA polyplexes. Because of the aggregation, cationic polyplexes were immobilized in the vitreous and only a small amount could reach the target cells in the posterior eye. To improve the cellular uptake of LCDCs, cell penetrating agents could be employed. This kind of envelope-type structure, with a functionalized surface, could be one step towards a virus-mimicking gene delivery vector. In conclusion, LCDCs were stable against GAGs, but they were able to fuse with the endosomal membranes at low pH. The overall cellular uptake of the LCDCs remained low, however. By enhancing the cellular uptake with targeting peptides, the LCDCs might reach higher cellular uptake and become suitable for local gene delivery, to locations such as the eye. 38 11.3 Hindering effect of liposomal PEG on the targeting efficiency of a small hydrophobic peptide, AETP Steric stabilization of therapeutic nanoparticles with PEG is important for maintaining their stability and long blood circulation times, but, as discussed earlier, it can hamper complex internalization and endosomal membrane diffusion (Holland et al. 1996, Shi et al. 2002a). In addition, PEG shielding may also impair the interaction between the targeting moiety of the particle and the target antigen on the cell surface, as was hypothesized in the study of activated endothelium targeting peptide (AETP)-targeted liposomes. AETP was first discovered by phage display screening of activated human umbilical vein endothelial cells (HUVEC) and then of human Kaposi’s sarcoma xenograft in mice. It was thus expected to be an efficient targeting moiety towards neovascular endothelium. Despite successful phage display screening, the AETP was not able to act as a targeting moiety when conjugated to drugs (Bergman, M., personal communication). This was due to the very low water solubility of the peptide. Therefore, it was assumed that AETP could function as a targeting ligand on PEGylated liposomes. However, neither the cellular affinity nor the cytotoxic efficacy of AETP-targeted doxorubicin-containing liposomes was enhanced compared to the non-targeted ones. Pharmacokinetic investigation demonstrated that AETP-targeted liposomes had a similar or longer blood half-life compared to non-targeted, PEGylated liposomes. This was a positive result, since the peptide did not disturb the stealth effect of the PEG on the liposomes, but it could also mean that the peptide was covered with the PEG shield. AETP-targeted liposomes did not show any enhanced accumulation in the tumor tissue or enhanced specific binding to the endothelial cells in vivo when compared to non-targeted liposomes. The interaction between AETP moieties and liposomal PEG was demonstrated by molecular modeling. A well-known RGD-peptide that has been found to be an effective targeting ligand was used as a comparison in simulations. As a significantly more hydrophobic molecule, AETP was found to be covered by PEG while the surface of the more hydrophilic RGD was more exposed to water. In addition, AETP was also located deeper in the PEG shield than RGD peptide, rendering it unavailable to bind the receptors. An obscuring effect of PEG on targeting efficiency of PEG2000-liposomes targeted with PEG2000-folate has also been observed by Gabizon et al. (1999) and Shiokawa et al. (2005). When folate was coupled to the end of a clearly longer PEG arm, PEG3500 instead of PEG2000, clearly extruding from the PEG2000 shield, the cellular association of the liposomes increased. Recently, Stefanick et al. (2013) revealed the importance of linker length effect on cellular uptake of peptide-targeted PEGylated liposomes. However, using longer PEG-spacers may not work in the case of hydrophobic AETP because it tends to escape from the solvent and interact with the PEG molecules. PEG also has hydrophobic properties, even though it is described to form a hydrophilic sheath around the liposome. Hydrophobic interactions between AETP and PEG may explain the unfavorable orientation of AETP molecules for targeting purposes. Ligand docking studies showed a high binding affinity of human serum albumin (HSA) to both AETP and PEG. Although the effect of protein binding on AETP targetingability warrants further studies, molecular modeling shows very strong evidence that PEG coating hinders target binding of AETP. Protein binding to PEG has been previously 39 revealed by others experimentally (Price, Cornelius & Brash 2001, Dos Santos et al. 2007). Even though PEG presents many advantages and it is accepted for clinical use, it may not be suitable for all cases of drug delivery. In the case of having hydrophobic targeting ligands, PEG-coating on the liposomes could be replaced by more hydrophilic polymer to avoid the interactions between the ligand and the steric coating. More rigid polymer may also work, given that it can force the ligand out from the polymer cloud. But whether the stealth effect is then lost due to a firmer polymer structure should be assessed. Some possible alternatives for PEG have been suggested by Knop et al. (2010). When PEGylated nanosystems are used it might be beneficial to choose a hydrophilic targeting ligand. In a summary, AETP, a promising targeting candidate, failed to show any targeting efficiency in these studies. This was most likely due to the interference of liposomal PEG shield that may have prevented the interaction of the peptide with the target receptors. Nevertheless, cancer targeting is a complicated task, and many other factors may influence the efficacy of drug targeting. 11.4 Pre-targeting and local administration of liposomes as potential approaches in tumor targeting Tumor targeting with immunoliposomes has been studied in numerous institutes over the past 30 years. Targeting efficiency over the non-targeted liposomes was somewhat successful, but accelerated clearance from the blood circulation proved problematic, particularly when whole antibodies were used as targeting moieties (Aragnol, Leserman 1986). Shorter half-life usually compromises the benefit that has been achieved by targeting. When smaller fragments of antibodies have been used, half-lives comparable to non-targeted liposomes have been reached (Maruyama et al. 1997). Another option to prolong the residence time in blood circulation could be pre-targeting technology, since it has been noticed by Harding et al. (1997) that separate injections of cetuximab and PEGylated liposomes did not cause immune response, while immunogenicity was potentiated by antibody-coupled liposomes. In the current study, both direct targeting and pre-targeting (Figure 9) approaches were used to target PEGylated liposomes to ovarian adenocarcinoma cells (SKOV-3) in vitro and intraperitoneal xenografts in mice in vivo. Biotin-neutravidin technology was utilized in both cases to link the endothelial growth factor receptor (EGFR) antibody (cetuximab) to the liposomes. Direct targeting of the liposomes to SKOV-3 cells was receptor-specific and efficient in vitro, whilst the pre-targeting was not as efficient, possibly due to premature internalization of the cetuximab-receptor-complex. Antibody-neutravidincomplex should remain available for interaction with biotinylated liposomes. The other explanation could be the hindrance effect of PEG as discussed earlier. As a small molecule, biotin could be partly covered by the PEG shield and be hindered from the interaction with neutravidin that is bound to the receptor. 40 Figure 9 Schematic presention of pre-targeting and direct targeting approaches. In pretargeting method (A), antibody-linked neutravidin is administered first (step 1). Once antibody complex has found its target, biotinylated liposomes are administered (step 2). In direct targeting approach (B), antibodies are coupled on the surface of the liposomes and the formed immunoliposomes are administered as a single dose. In animal studies, accumulation of the targeted liposomes in the tumor was not higher than that of non-targeted liposomes with either targeting approach. However, intraperitoneally (i.p.) injected biotinylated liposomes, regardless of targeting, accumulated faster and reached a higher concentration in tumors compared to intravenously (i.v.) administered liposomes. This was comparable to the observation of Lin et al. (2009), who noticed rapid tumor accumulation of PEGylated liposomes after i.p. injection. I.p. administered liposomes can thus accomplish a fast local effect on intraperitoneal tumors, followed by systemic drug delivery after passing into the blood circulation. Because of the tendency of ovarian cancer to spread to the abdominal cavity, i.p. administration of cytotoxic drugs may be beneficial to reach both the primary tumor and the metastases. No significantly enhanced accumulation of targeted liposomes to the tumor was evident, when compared to non-targeted, PEGylated liposomes, even though higher uptake was seen in cell culture. This is somewhat expected, because due to the EPR effect, the uptake in the solid tumor tissue may not be further enhanced by targeting. Without higher accumulation at the tumor site, enhanced cancer cell-specific internalization (Kirpotin et al. 2006) and increased therapeutic efficacy (Mamot et al. 2005) for targeted liposomes over non-targeted ones have been observed. The pre-targeting method described here still requires optimization: the choice of the antibody should be reconsidered, but also the timing between the antibody administration and the liposome injections needs optimization. In principle, a pre-targeting approach has potential because of separate injections of targeting agents and drug formulation; different antigens can be targeted simultaneously with the same drug formulation. This flexibility enables the use of the same nanoformulation in different types of cancers. When antibody is not chemically attached to the liposome, the stability during storage could be improved, 41 and also the developmental costs may not rise as high as the costs for more complicated directly targeted formulation. Pre-targeting proved to be less efficient, compared to direct targeting, regarding uptake in cancer cells in vitro. Because of fast tumor accumulation in vivo, however, the concept of local tumor pre-targeting might be beneficial after further development. 42 12 Conclusions 1. Polymer architecture and composition had a clear effect on DNA complexation and gene transfer efficacy. Linear structure and rubbery poly-n-butyl acrylate block in the middle of PDMAEMA-chain improved DNA complexation and transfection efficiency, while star-shaped and glassy polystyrene core hindered DNA condensation as well as transfection. 2. Hydrophobin and BSA-conjugated polyamino dendrons bound to DNA with high efficacy and were biocompatible in vitro; but transfection was observed only for amphiphilic hydrophobin-dendron conjugates (second generation) with a low transgene expression. 3. Lipid-coated DNA complexes with a cationic PEI/DNA core covered with negatively charged DOPE/CHEMS mixture were successfully produced by detergent removal method. These complexes were resistant against extracellular glycosaminoglycans, and were able fuse with endosomal membrane at acidic pH and mediate transfection. 4. Molecular modeling revealed that hydrophobic AETP targeting moieties were located deep in the PEG layer of the liposomes, and thus might have been prevented from interacting with target receptors. In addition, peptide binding to serum proteins may further inhibit target binding. These findings may be useful in the development of targeted nanocarriers. 5. Direct targeting with EGFR-antibody liposomes was superior to non-targeted and pre-targeted liposomes in the cell studies, but in mice the accumulation in the tumor remained low. Tumoral accumulation after intraperitoneal administration of pre-targeted and non-targeted liposomes was faster and greater compared to intravenous administration. Local tumoral pre-targeting method warrants further studies as a potential approach in cancer therapy. 43 13 Future prospects Nanoparticles – drugs of the future? Nanoparticle technology in pharmaceutical research has provided great promise for more efficient and safe drug therapy. Targeted nanomedicines in particular have garnered growing enthusiasm among researchers reflected by a massively increasing number of publications. Despite decades of developmental work, there are no targeted nanoparticle formulations in clinical use and only a few are currently in clinical trials. When a drug formulation becomes more complicated, the risks in the development phase most definitely increase. Somewhat surprisingly, most of the nanoparticle systems are developed for the treatment of cancer. Development of systemically administered targeted nanoparticles faces many technical challenges as a risk of too short half-life, poor tumoral perfusion, and diffusion barriers at the binding site. The leakiness of the tumor vessels varies between the tumor types, in some cases the tumor core may be not well perfused (Chauhan et al. 2011). In addition, the expression levels of the target receptors can vary between different cancers (Perez-Soler 2004) and the receptor density may also decrease dramatically during the treatment (Jiang et al. 2008, Cheng et al. 2012). This brings challenges to nanoparticle delivery to tumors. Possibly, some other disease states in closed systems, such as in the eye, or the surgical situations, might be more beneficial delivery targets for nanomedicine development, because of the possibility of local administration. In this case, the nanomedicine would provide advantages, such as protection of the drug from enzymatic degradation, prolonged activity, cell specificity, and optimization of intracellular distribution. Non-malignant diseases also lack rapid mutation of the target cells which makes the targeting easier. The approval process for a nanoformulation is much more difficult than for parent drug. This likely reflects why big pharmaceutical companies do not want to invest money and time for a small increase in performance that might be achieved by reformulating a currently approved drug inside a nanocarrier (Venditto, Szoka Jr. 2013). For example, liposomal cancer therapeutics Caelyx and Ambisome are able to reduce the toxicity of the parent drug, but improving the efficacy is still modest. Even if reduced toxicity is very important for the patients, the minimal improvement in the efficacy may become an issue for nanotherapeutics (Juliano 2013). Although nanotherapeutics has not yet realised its promises, it will certainly find its place in the pharmaceutical field. Increasing knowledge of the strengths and the weaknesses of nanomedicines will be useful in the developmental phase. In addition, utilization of computational modeling may be helpful in screening the best nanocandidates before carrying out the costly preclinical and clinical studies. 44 References Adams, G.P., Schier, R., McCall, A.M., Simmons, H.H., Horak, E.M., Alpaugh, R.K., Marks, J.D. & Weiner, L.M. 2001, "High affinity restricts the localization and tumor penetration of single-chain fv antibody molecules", Cancer research, vol. 61, no. 12, pp. 4750-4755. Allen, C., Dos Santos, N., Gallagher, R., Chiu, G.N., Shu, Y., Li, W.M., Johnstone, S.A., Janoff, A.S., Mayer, L.D., Webb, M.S. & Bally, M.B. 2002, "Controlling the physical behavior and biological performance of liposome formulations through use of surface grafted poly(ethylene glycol)", Bioscience reports, vol. 22, no. 2, pp. 225-250. Allen, T.M., Hansen, C., Martin, F., Redemann, C. & Yau-Young, A. 1991, "Liposomes containing synthetic lipid derivatives of poly(ethylene glycol) show prolonged circulation half-lives in vivo", Biochimica et biophysica acta, vol. 1066, no. 1, pp. 2936. Allen, T.M., Brandeis, E., Hansen, C.B., Kao, G.Y. & Zalipsky, S. 1995, "A new strategy for attachment of antibodies to sterically stabilized liposomes resulting in efficient targeting to cancer cells", Biochimica et Biophysica Acta (BBA) - Biomembranes, vol. 1237, no. 2, pp. 99-108. Aragnol, D. & Leserman, L.D. 1986, "Immune clearance of liposomes inhibited by an anti-Fc receptor antibody in vivo", Proceedings of the National Academy of Sciences of the United States of America, vol. 83, no. 8, pp. 2699-2703. Boeckle, S., Fahrmeir, J., Roedl, W., Ogris, M. & Wagner, E. 2006, "Melittin analogs with high lytic activity at endosomal pH enhance transfection with purified targeted PEI polyplexes", Journal of Controlled Release, vol. 112, no. 2, pp. 240-248. Bolhassani, A. 2011, "Potential efficacy of cell-penetrating peptides for nucleic acid and drug delivery in cancer", Biochimica et biophysica acta, vol. 1816, no. 2, pp. 232246. Bolotin, E.M., Cohen, R., Bar, L.K., Emanuel, N., Ninio, S., Lasic, D.D. & Barenholz, Y. 1994, "Ammonium sulfate gradients for efficient and stable remote loading of amphiphilic weak bases into liposomes and ligandoliposomes", Journal of Liposome Research, vol. 4, no. 1, pp. 455-479. Boussif, O., Lezoualc'h, F., Zanta, M.A., Mergny, M.D., Scherman, D., Demeneix, B. & Behr, J.P. 1995, "A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine", Proceedings of the National Academy of Sciences of the United States of America, vol. 92, no. 16, pp. 7297-7301. Brinkhuis, R.P., Rutjes, F.P.J.T. & Van Hest, J.C.M. 2011, "Polymeric vesicles in biomedical applications", Polymer Chemistry, vol. 2, no. 7, pp. 1449-1462. 45 Chang, Y.J., Chang, C.H., Chang, T.J., Yu, C.Y., Chen, L.C., Jan, M.L., Luo, T.Y., Lee, T.W. & Ting, G. 2007, "Biodistribution, pharmacokinetics and microSPECT/CT imaging of 188Re-bMEDA-liposome in a C26 murine colon carcinoma solid tumor animal model", Anticancer Research, vol. 27, no. 4B, pp. 2217-2225. Chauhan, V.P., Stylianopoulos, T., Boucher, Y. & Jain, R.K. 2011, "Delivery of molecular and nanoscale medicine to tumors: transport barriers and strategies", Annual review of chemical and biomolecular engineering, vol. 2, pp. 281-298. Cheng, Z., Al Zaki, A., Hui, J.Z., Muzykantov, V.R. & Tsourkas, A. 2012, "Multifunctional nanoparticles: cost versus benefit of adding targeting and imaging capabilities", Science, vol. 338, no. 6109, pp. 903-910. Cho, K., Wang, X., Nie, S., Chen, Z.G. & Shin, D.M. 2008, "Therapeutic nanoparticles for drug delivery in cancer", Clinical cancer research, vol. 14, no. 5, pp. 1310-1316. Choosakoonkriang, S., Lobo, B.A., Koe, G.S., Koe, J.G. & Middaugh, C.R. 2003, "Biophysical characterization of PEI/DNA complexes", Journal of pharmaceutical sciences, vol. 92, no. 8, pp. 1710-1722. Chow, T.H., Lin, Y.Y., Hwang, J.J., Wang, H.E., Tseng, Y.L., Wang, S.J., Liu, R.S., Lin, W.J., Yang, C.S. & Ting, G. 2009, "Improvement of biodistribution and therapeutic index via increase of polyethylene glycol on drug-carrying liposomes in an HT-29/luc xenografted mouse model", Anticancer Research, vol. 29, no. 6, pp. 2111-2120. Chuang, K.H., Wang, H.E., Chen, F.M., Tzou, S.C., Cheng, C.M., Chang, Y.C., Tseng, W.L., Shiea, J., Lin, S.R., Wang, J.Y., Chen, B.M., Roffler, S.R. & Cheng, T.L. 2010, "Endocytosis of PEGylated agents enhances cancer imaging and anticancer efficacy", Molecular cancer therapeutics, vol. 9, no. 6, pp. 1903-1912. Conforti, G., Dominguez-Jimenez, C., Zanetti, A., Gimbrone, M.A.,Jr, Cremona, O., Marchisio, P.C. & Dejana, E. 1992, "Human endothelial cells express integrin receptors on the luminal aspect of their membrane", Blood, vol. 80, no. 2, pp. 437446. Danhier, F., Feron, O. & Preat, V. 2010, "To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery", Journal of controlled release, vol. 148, no. 2, pp. 135-146. Dash, P.R., Read, M.L., Barrett, L.B., Wolfert, M.A. & Seymour, L.W. 1999, "Factors affecting blood clearance and in vivo distribution of polyelectrolyte complexes for gene delivery", Gene therapy, vol. 6, no. 4, pp. 643-650. Dauty, E. & Verkman, A.S. 2005, "Actin cytoskeleton as the principal determinant of sizedependent DNA mobility in cytoplasm: a new barrier for non-viral gene delivery", The Journal of biological chemistry, vol. 280, no. 9, pp. 7823-7828. David, A., Kopečková, P., Minko, T., Rubinstein, A. & Kopeček, J. 2004, "Design of a multivalent galactoside ligand for selective targeting of HPMA copolymer– 46 doxorubicin conjugates to human colon cancer cells", European journal of cancer, vol. 40, no. 1, pp. 148-157. Dos Santos, N., Allen, C., Doppen, A.M., Anantha, M., Cox, K.A., Gallagher, R.C., Karlsson, G., Edwards, K., Kenner, G., Samuels, L., Webb, M.S. & Bally, M.B. 2007, "Influence of poly(ethylene glycol) grafting density and polymer length on liposomes: relating plasma circulation lifetimes to protein binding", Biochimica et biophysica acta, vol. 1768, no. 6, pp. 1367-1377. Dubey, P.K., Mishra, V., Jain, S., Mahor, S. & Vyas, S.P. 2004, "Liposomes modified with cyclic RGD peptide for tumor targeting", Journal of drug targeting, vol. 12, no. 5, pp. 257-264. Duncan, R. & Gaspar, R. 2011, "Nanomedicine(s) under the microscope", Molecular pharmaceutics, vol. 8, no. 6, pp. 2101-2141. Dunlap, D.D., Maggi, A., Soria, M.R. & Monaco, L. 1997, "Nanoscopic structure of DNA condensed for gene delivery", Nucleic acids research, vol. 25, no. 15, pp. 3095-3101. ElBayoumi, T.A. & Torchilin, V.P. 2009, "Tumor-targeted nanomedicines: enhanced antitumor efficacy in vivo of doxorubicin-loaded, long-circulating liposomes modified with cancer-specific monoclonal antibody", Clinical cancer research, vol. 15, no. 6, pp. 1973-1980. Erbacher, P., Bettinger, T., Belguise-Valladier, P., Zou, S., Coll, J., Behr, J. & Remy, J. 1999, "Transfection and physical properties of various saccharide, poly(ethylene glycol), and antibody-derivatized polyethylenimines (PEI)", The journal of gene medicine, vol. 1, no. 3, pp. 210-222. Fattal, E., Couvreur, P. & Dubernet, C. 2004, ""Smart" delivery of antisense oligonucleotides by anionic pH-sensitive liposomes", Advanced Drug Delivery Reviews, vol. 56, no. 7, pp. 931-946. Feron, O. 2004, "Targeting the tumor vascular compartment to improve conventional cancer therapy", Trends in pharmacological sciences, vol. 25, no. 10, pp. 536-542. Gabizon, A., Horowitz, A.T., Goren, D., Tzemach, D., Mandelbaum-Shavit, F., Qazen, M.M. & Zalipsky, S. 1999, "Targeting folate receptor with folate linked to extremities of poly(ethylene glycol)-grafted liposomes: in vitro studies", Bioconjugate chemistry, vol. 10, no. 2, pp. 289-298. Geng, Y., Dalhaimer, P., Cai, S., Tsai, R., Tewari, M., Minko, T. & Discher, D.E. 2007, "Shape effects of filaments versus spherical particles in flow and drug delivery", Nature nanotechnology, vol. 2, no. 4, pp. 249-255. Giacca, M. & Zacchigna, S. 2012, "Virus-mediated gene delivery for human gene therapy", Journal of Controlled Release, vol. 161, no. 2, pp. 377-388. 47 Godbey, W.T., Wu, K.K. & Mikos, A.G. 1999, "Size matters: molecular weight affects the efficiency of poly(ethylenimine) as a gene delivery vehicle", Journal of Biomedical Materials Research, vol. 45, no. 3, pp. 268-275. Görlich, D. & Mattaj, I.W. 1996, "Nucleoplasmic transport", Science, vol. 271, pp. 15131518. Guo, W., Gosselin, M.A. & Lee, R.J. 2002, "Characterization of a novel diolein-based LPDII vector for gene delivery", Journal of controlled release, vol. 83, no. 1, pp. 121-132. Guo, W. & Lee, R.J. 2000, "Efficient gene delivery using anionic liposome-complexed polyplexes (LPDII)", Bioscience reports, vol. 20, no. 5, pp. 419-432. Guo, X. & Szoka, F.C.,Jr 2001, "Steric stabilization of fusogenic liposomes by a low-pH sensitive PEG-diortho ester-lipid conjugate", Bioconjugate chemistry, vol. 12, no. 2, pp. 291-300. Haensler, J. & Szoka, F.C.,Jr 1993, "Polyamidoamine cascade polymers mediate efficient transfection of cells in culture", Bioconjugate chemistry, vol. 4, no. 5, pp. 372-379. Hafez, I.M. & Cullis, P.R. 2000, "Cholesteryl hemisuccinate exhibits pH sensitive polymorphic phase behavior", Biochimica et biophysica acta, vol. 1463, no. 1, pp. 107-114. Hafez, I.M., Maurer, N. & Cullis, P.R. 2001, "On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids", Gene therapy, vol. 8, no. 15, pp. 1188-1196. Hansen, C.B., Kao, G.Y., Moase, E.H., Zalipsky, S. & Allen, T.M. 1995, "Attachment of antibodies to sterically stabilized liposomes: evaluation, comparison and optimization of coupling procedures", Biochimica et biophysica acta, vol. 1239, no. 2, pp. 133144. Hanzlíková, M., Ruponen, M., Galli, E., Raasmaja, A., Aseyev, V., Tenhu, H., Urtti, A. & Yliperttula, M. 2011, "Mechanisms of polyethylenimine-mediated DNA delivery: Free carrier helps to overcome the barrier of cell-surface glycosaminoglycans", Journal of Gene Medicine, vol. 13, no. 7-8, pp. 402-409. Harasym, T.O., Bally, M.B. & Tardi, P. 1998, "Clearance properties of liposomes involving conjugated proteins for targeting", Advanced Drug Delivery Reviews, vol. 32, no. 1-2, pp. 99-118. Harding, J.A., Engbers, C.M., Newman, M.S., Goldstein, N.I. & Zalipsky, S. 1997, "Immunogenicity and pharmacokinetic attributes of poly(ethylene glycol)-grafted immunoliposomes", Biochimica et biophysica acta, vol. 1327, no. 2, pp. 181-192. 48 Hardy, J.G., Kostiainen, M.A., Smith, D.K., Gabrielson, N.P. & Pack, D.W. 2006, "Dendrons with spermine surface groups as potential building blocks for nonviral vectors in gene therapy", Bioconjugate chemistry, vol. 17, no. 1, pp. 172-178. Harrington, K.J., Rowlinson-Busza, G., Syrigos, K.N., Abra, R.M., Uster, P.S., Peters, A.M. & Stewart, J.S. 2000, "Influence of tumour size on uptake of(111)ln-DTPAlabelled pegylated liposomes in a human tumour xenograft model", British journal of cancer, vol. 83, no. 5, pp. 684-688. Hashizume, H., Baluk, P., Morikawa, S., McLean, J.W., Thurston, G., Roberge, S., Jain, R.K. & McDonald, D.M. 2000, "Openings between defective endothelial cells explain tumor vessel leakiness", The American journal of pathology, vol. 156, no. 4, pp. 1363-1380. Hillaireau, H. & Couvreur, P. 2009, "Nanocarriers' entry into the cell: relevance to drug delivery", Cellular and molecular life sciences, vol. 66, no. 17, pp. 2873-2896. Holland, J.W., Hui, C., Cullis, P.R. & Madden, T.D. 1996, "Poly(ethylene glycol)--lipid conjugates regulate the calcium-induced fusion of liposomes composed of phosphatidylethanolamine and phosphatidylserine", Biochemistry, vol. 35, no. 8, pp. 2618-2624. Hwang, S., Maitani, Y., Qi, X., Takayama, K. & Nagai, T. 1999, "Remote loading of diclofenac, insulin and fluorescein isothiocyanate labeled insulin into liposomes by pH and acetate gradient methods", International journal of pharmaceutics, vol. 179, no. 1, pp. 85-95. Iden, D.L. & Allen, T.M. 2001, "In vitro and in vivo comparison of immunoliposomes made by conventional coupling techniques with those made by a new post-insertion approach", Biochimica et biophysica acta, vol. 1513, no. 2, pp. 207-216. Iyer, A.K., Su, Y., Feng, J., Lan, X., Zhu, X., Liu, Y., Gao, D., Seo, Y., Vanbrocklin, H.F., Courtney Broaddus, V., Liu, B. & He, J. 2011, "The effect of internalizing human single chain antibody fragment on liposome targeting to epithelioid and sarcomatoid mesothelioma", Biomaterials, vol. 32, no. 10, pp. 2605-2613. Iyer, A.K., Khaled, G., Fang, J. & Maeda, H. 2006, "Exploiting the enhanced permeability and retention effect for tumor targeting", Drug discovery today, vol. 11, no. 17–18, pp. 812-818. Jain, R.K. 1990, "Physiological barriers to delivery of monoclonal antibodies and other macromolecules in tumors", Cancer research, vol. 50, no. 3 Suppl, pp. 814s-819s. Jiang, W., Kim, B.Y., Rutka, J.T. & Chan, W.C. 2008, "Nanoparticle-mediated cellular response is size-dependent", Nature nanotechnology, vol. 3, no. 3, pp. 145-150. Juliano, R. 2013, "Nanomedicine: is the wave cresting?", Nature reviews. Drug discovery, vol. 12, no. 3, pp. 171-172. 49 Kale, A.A. & Torchilin, V.P. 2007, ""Smart" drug carriers: PEGylated TATp-modified pH-sensitive liposomes", Journal of Liposome Research, vol. 17, no. 3-4, pp. 197203. Khalil, I.A., Kogure, K., Futaki, S., Hama, S., Akita, H., Ueno, M., Kishida, H., Kudoh, M., Mishina, Y., Kataoka, K., Yamada, M. & Harashima, H. 2007, "Octa-argininemodified multifunctional envelope-type nanoparticles for gene delivery", Gene therapy, vol. 14, no. 8, pp. 682-689. Kirchmeier, M.J., Ishida, T., Chevrette, J. & Allen, T.M. 2001, "Correlations between the rate of intracellular release of endocytosed liposomal Doxorubicin and cytotoxicity as determined by a new assay", Journal of Liposome Research, vol. 11, no. 1, pp. 15-29. Kirpotin, D., Park, J.W., Hong, K., Zalipsky, S., Li, W.L., Carter, P., Benz, C.C. & Papahadjopoulos, D. 1997, "Sterically stabilized anti-HER2 immunoliposomes: design and targeting to human breast cancer cells in vitro", Biochemistry, vol. 36, no. 1, pp. 66-75. Kirpotin, D.B., Drummond, D.C., Shao, Y., Shalaby, M.R., Hong, K., Nielsen, U.B., Marks, J.D., Benz, C.C. & Park, J.W. 2006, "Antibody targeting of long-circulating lipidic nanoparticles does not increase tumor localization but does increase internalization in animal models", Cancer research, vol. 66, no. 13, pp. 6732-6740. Knop, K., Hoogenboom, R., Fischer, D. & Schubert, U.S. 2010, "Poly(ethylene glycol) in drug delivery: pros and cons as well as potential alternatives", Angewandte Chemie, vol. 49, no. 36, pp. 6288-6308. Koltover, I., Salditt, T., Radler, J.O. & Safinya, C.R. 1998, "An inverted hexagonal phase of cationic liposome-DNA complexes related to DNA release and delivery", Science, vol. 281, no. 5373, pp. 78-81. Kreiss, P., Cameron, B., Rangara, R., Mailhe, P., Aguerre-Charriol, O., Airiau, M., Scherman, D., Crouzet, J. & Pitard, B. 1999, "Plasmid DNA size does not affect the physicochemical properties of lipoplexes but modulates gene transfer efficiency", Nucleic acids research, vol. 27, no. 19, pp. 3792-3798. Kullberg, M., Mann, K. & Owens, J.L. 2009, "A two-component drug delivery system using Her-2-targeting thermosensitive liposomes", Journal of drug targeting, vol. 17, no. 2, pp. 98-107. Kurosaki, T., Uematsu, M., Shimoda, K., Suzuma, K., Nakai, M., Nakamura, T., Kitahara, T., Kitaoka, T. & Sasaki, H. 2013, "Ocular gene delivery systems using ternary complexes of plasmid DNA, polyethylenimine, and anionic polymers", Biological & pharmaceutical bulletin, vol. 36, no. 1, pp. 96-101. Langer, K., Anhorn, M.G., Steinhauser, I., Dreis, S., Celebi, D., Schrickel, N., Faust, S. & Vogel, V. 2008, "Human serum albumin (HSA) nanoparticles: Reproducibility of preparation process and kinetics of enzymatic degradation", International journal of pharmaceutics, vol. 347, no. 1–2, pp. 109-117. 50 Lechardeur, D., Sohn, K.J., Haardt, M., Joshi, P.B., Monck, M., Graham, R.W., Beatty, B., Squire, J., O'Brodovich, H. & Lukacs, G.L. 1999, "Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer", Gene therapy, vol. 6, no. 4, pp. 482-497. Lechardeur, D., Verkman, A.S. & Lukacs, G.L. 2005, "Intracellular routing of plasmid DNA during non-viral gene transfer", Advanced Drug Delivery Reviews, vol. 57, no. 5, pp. 755-767. Lee, H., Hoang, B., Fonge, H., Reilly, R.M. & Allen, C. 2010, "In vivo distribution of polymeric nanoparticles at the whole-body, tumor, and cellular levels", Pharmaceutical research, vol. 27, no. 11, pp. 2343-2355. Lee, L.K., Mount, C.N. & Ayazi Shamlou, P. 2001, "Characterisation of the physical stability of colloidal polycation-DNA complexes for gene therapy and DNA vaccines", Chemical Engineering Science, vol. 56, no. 10, pp. 3163-3172. Lesch, H.P., Kaikkonen, M.U., Pikkarainen, J.T. & Ylä-Herttuala, S. 2010, "Avidin-biotin technology in targeted therapy", Expert opinion on drug delivery, vol. 7, no. 5, pp. 551-564. Lin, Y.Y., Li, J.J., Chang, C.H., Lu, Y.C., Hwang, J.J., Tseng, Y.L., Lin, W.J., Ting, G. & Wang, H.E. 2009, "Evaluation of pharmacokinetics of 111In-labeled VNBPEGylated liposomes after intraperitoneal and intravenous administration in a tumor/ascites mouse model", Cancer biotherapy & radiopharmaceuticals, vol. 24, no. 4, pp. 453-460. Loughrey, H., Bally, M.B. & Cullis, P.R. 1987, "A non-covalent method of attaching antibodies to liposomes", Biochimica et Biophysica Acta (BBA) - Biomembranes, vol. 901, no. 1, pp. 157-160. Lu, W.L., Qi, X.R., Zhang, Q., Li, R.Y., Wang, G.L., Zhang, R.J. & Wei, S.L. 2004, "A pegylated liposomal platform: pharmacokinetics, pharmacodynamics, and toxicity in mice using doxorubicin as a model drug", Journal of pharmacological sciences, vol. 95, no. 3, pp. 381-389. Lv, H., Zhang, S., Wang, B., Cui, S. & Yan, J. 2006, "Toxicity of cationic lipids and cationic polymers in gene delivery", Journal of Controlled Release, vol. 114, no. 1, pp. 100-109. Mamot, C., Drummond, D.C., Greiser, U., Hong, K., Kirpotin, D.B., Marks, J.D. & Park, J.W. 2003, "Epidermal growth factor receptor (EGFR)-targeted immunoliposomes mediate specific and efficient drug delivery to EGFR- and EGFRvIII-overexpressing tumor cells", Cancer research, vol. 63, no. 12, pp. 3154-3161. Mamot, C., Drummond, D.C., Noble, C.O., Kallab, V., Guo, Z., Hong, K., Kirpotin, D.B. & Park, J.W. 2005, "Epidermal growth factor receptor-targeted immunoliposomes significantly enhance the efficacy of multiple anticancer drugs in vivo", Cancer research, vol. 65, no. 24, pp. 11631-11638. 51 Marshall, E. 1999, "Gene therapy death prompts review of adenovirus vector", Science, vol. 286, no. 5448, pp. 2244-2245. Maruyama, K. 2011, "Intracellular targeting delivery of liposomal drugs to solid tumors based on EPR effects", Advanced Drug Delivery Reviews, vol. 63, no. 3, pp. 161-169. Maruyama, K., Takahashi, N., Tagawa, T., Nagaike, K. & Iwatsuru, M. 1997, "Immunoliposomes bearing polyethyleneglycol-coupled Fab′ fragment show prolonged circulation time and high extravasation into targeted solid tumors in vivo", FEBS letters, vol. 413, no. 1, pp. 177-180. Mastrobattista, E., Kapel, R.H.G., Eggenhuisen, M.H., Roholl, P.J.M., Crommelin, D.J.A., Hennink, W.E. & Storm, G. 2001, "Lipid-coated polyplexes for targeted gene delivery to ovarian carcinoma cells", Cancer gene therapy, vol. 8, no. 6, pp. 405-413. Mastrobattista, E., Koning, G.A. & Storm, G. 1999, "Immunoliposomes for the targeted delivery of antitumor drugs", Advanced Drug Delivery Reviews, vol. 40, no. 1-2, pp. 103-127. Mayer, L.D., Bally, M.B. & Cullis, P.R. 1986, "Uptake of adriamycin into large unilamellar vesicles in response to a pH gradient", Biochimica et Biophysica Acta Biomembranes, vol. 857, pp. 123-126. Mislick, K.A. & Baldeschwieler, J.D. 1996, "Evidence for the role of proteoglycans in cation-mediated gene transfer", Proceedings of the National Academy of Sciences of the United States of America, vol. 93, no. 22, pp. 12349-12354. Moghimi, S.M. & Szebeni, J. 2003, "Stealth liposomes and long circulating nanoparticles: Critical issues in pharmacokinetics, opsonization and protein-binding properties", Progress in lipid research, vol. 42, no. 6, pp. 463-478. Moreira, J.N., Hansen, C.B., Gaspar, R. & Allen, T.M. 2001, "A growth factor antagonist as a targeting agent for sterically stabilized liposomes in human small cell lung cancer", Biochimica et biophysica acta, vol. 1514, no. 2, pp. 303-317. Morikawa, S., Baluk, P., Kaidoh, T., Haskell, A., Jain, R.K. & McDonald, D.M. 2002, "Abnormalities in pericytes on blood vessels and endothelial sprouts in tumors", The American journal of pathology, vol. 160, no. 3, pp. 985-1000. Morille, M., Passirani, C., Vonarbourg, A., Clavreul, A. & Benoit, J. 2008, "Progress in developing cationic vectors for non-viral systemic gene therapy against cancer", Biomaterials, vol. 29, no. 24-25, pp. 3477-3496. Mounkes, L.C., Zhong, W., Cipres-Palacin, G., Heath, T.D. & Debs, R.J. 1998, "Proteoglycans mediate cationic liposome-DNA complex-based gene delivery in vitro and in vivo", The Journal of biological chemistry, vol. 273, no. 40, pp. 26164-26170. Muro, S., Garnacho, C., Champion, J.A., Leferovich, J., Gajewski, C., Schuchman, E.H., Mitragotri, S. & Muzykantov, V.R. 2008, "Control of endothelial targeting and 52 intracellular delivery of therapeutic enzymes by modulating the size and shape of ICAM-1-targeted carriers", Molecular therapy, vol. 16, no. 8, pp. 1450-1458. Müller-Eberhard, H.J. 1988, "Molecular Organization and Function of the Complement System", Annual Review of Biochemistry, vol. 57, pp. 321-347. Männistö, M., Reinisalo, M., Ruponen, M., Honkakoski, P., Tammi, M. & Urtti, A. 2007, "Polyplex-mediated gene transfer and cell cycle: effect of carrier on cellular uptake and intracellular kinetics, and significance of glycosaminoglycans", The journal of gene medicine, vol. 9, no. 6, pp. 479-487. Männistö, M., Vanderkerken, S., Toncheva, V., Elomaa, M., Ruponen, M., Schacht, E. & Urtti, A. 2002, "Structure-activity relationships of poly(L-lysines): effects of pegylation and molecular shape on physicochemical and biological properties in gene delivery", Journal of controlled release, vol. 83, no. 1, pp. 169-182. Mönkkönen, J. & Urtti, A. 1998, "Lipid fusion in oligonucleotide and gene delivery with cationic lipids", Advanced Drug Delivery Reviews, vol. 34, no. 1, pp. 37-49. Nahde, T., Müller, K., Fahr, A., Müller, R. & Brüsselbach, S. 2001, "Combined transductional and transcriptional targeting of melanoma cells by artificial virus-like particles", The journal of gene medicine, vol. 3, no. 4, pp. 353-361. Nobs, L., Buchegger, F., Gurny, R. & Allemann, E. 2006, "Biodegradable nanoparticles for direct or two-step tumor immunotargeting", Bioconjugate chemistry, vol. 17, no. 1, pp. 139-145. O'Brien, M.E., Wigler, N., Inbar, M., Rosso, R., Grischke, E., Santoro, A., Catane, R., Kieback, D.G., Tomczak, P., Ackland, S.P., Orlandi, F., Mellars, L., Alland, L., Tendler, C. & CAELYX Breast Cancer Study Group 2004, "Reduced cardiotoxicity and comparable efficacy in a phase III trial of pegylated liposomal doxorubicin HCl (CAELYX/Doxil) versus conventional doxorubicin for first-line treatment of metastatic breast cancer", Annals of Oncology, vol. 15, no. 3, pp. 440-449. Ogris, M., Brunner, S., Schüller, S., Kircheis, R. & Wagner, E. 1999, "PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery", Gene therapy, vol. 6, no. 4, pp. 595-605. Paasonen, L., Laaksonen, T., Johans, C., Yliperttula, M., Kontturi, K. & Urtti, A. 2007, "Gold nanoparticles enable selective light-induced contents release from liposomes", Journal of controlled release, vol. 122, no. 1, pp. 86-93. Pan, H., Han, L., Chen, W., Yao, M. & Lu, W. 2008, "Targeting to tumor necrotic regions with biotinylated antibody and streptavidin modified liposomes", Journal of controlled release, vol. 125, no. 3, pp. 228-235. 53 Park, J.W., Hong, K., Kirpotin, D.B., Meyer, O., Papahadjopoulos, D. & Benz, C.C. 1997, "Anti-HER2 immunoliposomes for targeted therapy of human tumors", Cancer letters, vol. 118, no. 2, pp. 153-160. Pasqualini, R., Koivunen, E. & Ruoslahti, E. 1997, "Alpha v integrins as receptors for tumor targeting by circulating ligands", Nature biotechnology, vol. 15, no. 6, pp. 542546. Pastorino, F., Brignole, C., Di Paolo, D., Nico, B., Pezzolo, A., Marimpietri, D., Pagnan, G., Piccardi, F., Cilli, M., Longhi, R., Ribatti, D., Corti, A., Allen, T.M. & Ponzoni, M. 2006, "Targeting liposomal chemotherapy via both tumor cell-specific and tumor vasculature-specific ligands potentiates therapeutic efficacy", Cancer research, vol. 66, no. 20, pp. 10073-10082. Pastorino, F., Brignole, C., Marimpietri, D., Cilli, M., Gambini, C., Ribatti, D., Longhi, R., Allen, T.M., Corti, A. & Ponzoni, M. 2003a, "Vascular damage and antiangiogenic effects of tumor vessel-targeted liposomal chemotherapy", Cancer research, vol. 63, no. 21, pp. 7400-7409. Pastorino, F., Brignole, C., Marimpietri, D., Sapra, P., Moase, E.H., Allen, T.M. & Ponzoni, M. 2003b, "Doxorubicin-loaded Fab' fragments of anti-disialoganglioside immunoliposomes selectively inhibit the growth and dissemination of human neuroblastoma in nude mice", Cancer research, vol. 63, no. 1, pp. 86-92. Perez-Soler, R. 2004, "HER1/EGFR targeting: refining the strategy", The oncologist, vol. 9, no. 1, pp. 58-67. Pollard, H., Remy, J.S., Loussouarn, G., Demolombe, S., Behr, J.P. & Escande, D. 1998, "Polyethylenimine but not cationic lipids promotes transgene delivery to the nucleus in mammalian cells", The Journal of biological chemistry, vol. 273, no. 13, pp. 75077511. Price, M.E,, Cornelius, R.M. & Brash, J.L. 2001, "Protein adsorption to polyethylene glycol modified liposomes from fibrinogen solution and from plasma", Biochimica et Biophysica Acta, vol. 1512, no. 2, pp. 191-205. Pulkkinen, M., Pikkarainen, J., Wirth, T., Tarvainen, T., Haapa-aho, V., Korhonen, H., Seppälä, J. & Järvinen, K. 2008, "Three-step tumor targeting of paclitaxel using biotinylated PLA-PEG nanoparticles and avidin-biotin technology: Formulation development and in vitro anticancer activity", European journal of pharmaceutics and biopharmaceutics, vol. 70, no. 1, pp. 66-74. Radler, J.O., Koltover, I., Salditt, T. & Safinya, C.R. 1997, "Structure of DNA-cationic liposome complexes: DNA intercalation in multilamellar membranes in distinct interhelical packing regimes", Science, vol. 275, no. 5301, pp. 810-814. Rawat, M., Singh, D., Saraf, S. & Saraf, S. 2006, "Nanocarriers: promising vehicle for bioactive drugs", Biological & pharmaceutical bulletin, vol. 29, no. 9, pp. 1790-1798. 54 Riviere, K., Huang, Z., Jerger, K., Macaraeg, N. & Szoka, F.C.,Jr 2011, "Antitumor effect of folate-targeted liposomal doxorubicin in KB tumor-bearing mice after intravenous administration", Journal of drug targeting, vol. 19, no. 1, pp. 14-24. Romberg, B., Hennink, W.E. & Storm, G. 2008, "Sheddable coatings for long-circulating nanoparticles", Pharmaceutical research, vol. 25, no. 1, pp. 55-71. Roth, P., Hammer, C., Piguet, A., Ledermann, M., Dufour, J. & Waelti, E. 2007, "Effects on hepatocellular carcinoma of doxorubicin- loaded immunoliposomes designed to target the VEGFR-2", Journal of drug targeting, vol. 15, no. 9, pp. 623-631. Ruoslahti, E. 2002, "Specialization of tumour vasculature", Nature reviews. Cancer, vol. 2, no. 2, pp. 83-90. Ruponen, M., Honkakoski, P., Tammi, M. & Urtti, A. 2004, "Cell-surface glycosaminoglycans inhibit cation-mediated gene transfer", Journal of Gene Medicine, vol. 6, no. 4, pp. 405-414. Ruponen, M., Ylä-Herttuala, S. & Urtti, A. 1999, "Interactions of polymeric and liposomal gene delivery systems with extracellular glycosaminoglycans: physicochemical and transfection studies", Biochimica et biophysica acta, vol. 1415, no. 2, pp. 331-341. Sapra, P. & Allen, T.M. 2003, "Ligand-targeted liposomal anticancer drugs", Progress in lipid research, vol. 42, no. 5, pp. 439-462. Seftor, R.E., Seftor, E.A. & Hendrix, M.J. 1999, "Molecular role(s) for integrins in human melanoma invasion", Cancer metastasis reviews, vol. 18, no. 3, pp. 359-375. Sharkey, R.M., McBride, W.J., Karacay, H., Chang, K., Griffiths, G.L., Hansen, H.J. & Goldenberg, D.M. 2003, "A universal pretargeting system for cancer detection and therapy using bispecific antibody", Cancer research, vol. 63, no. 2, pp. 354-363. Shew, R.L. & Deamer, D.W. 1985, "A novel method for encapsulation of macromolecules in liposomes", BBA - Biomembranes, vol. 816, no. 1, pp. 1-8. Shi, J., Votruba, A.R., Farokhzad, O.C. & Langer, R. 2010, "Nanotechnology in Drug Delivery and Tissue Engineering: From Discovery to Applications", Nano Letters, vol. 10, pp. 3223-3230. Shi, F., Wasungu, L., Nomden, A., Stuart, M.C., Polushkin, E., Engberts, J.B. & Hoekstra, D. 2002a, "Interference of poly(ethylene glycol)-lipid analogues with cationic-lipidmediated delivery of oligonucleotides; role of lipid exchangeability and non-lamellar transitions", The Biochemical journal, vol. 366, no. Pt 1, pp. 333-341. Shi, G., Guo, W., Stephenson, S.M. & Lee, R.J. 2002b, "Efficient intracellular drug and gene delivery using folate receptor-targeted pH-sensitive liposomes composed of cationic/anionic lipid combinations", Journal of controlled release, vol. 80, no. 1-3, pp. 309-319. 55 Shiokawa, T., Hattori, Y., Kawano, K., Ohguchi, Y., Kawakami, H., Toma, K. & Maitani, Y. 2005, "Effect of polyethylene glycol linker chain length of folate-linked microemulsions loading aclacinomycin A on targeting ability and antitumor effect in vitro and in vivo", Clinical cancer research, vol. 11, no. 5, pp. 2018-2025. Simões, S., Moreira, J.N., Fonseca, C., Düzgünes, N. & Pedroso de Lima, M.C. 2004, "On the formulation of pH-sensitive liposomes with long circulation times", Advanced Drug Delivery Reviews, vol. 56, no. 7, pp. 947-965. Simões, S., Slepushkin, V., Düzgünes, N. & Pedroso de Lima, M.C. 2001, "On the mechanisms of internalization and intracellular delivery mediated by pH-sensitive liposomes", Biochimica et.biophysica acta, vol. 1515, no. 1, pp. 23-37. Sonawane, N.D., Szoka, F.C.,Jr & Verkman, A.S. 2003, "Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes", The Journal of biological chemistry, vol. 278, no. 45, pp. 44826-44831. Stefanick, J.F., Ashley, J.D., Kiziltepe, T. & Bilgicer, B. 2013, "A Systematic Analysis of Peptide Linker Lenght and Liposomal Polyethylene Glycol Coating on Cellular Uptake of Peptide-Targeted Liposomes", ACS Nano, vol 7, no. 4, pp. 2935-2947. Subrizi, A., Tuominen, E., Bunker, A., Róg, T., Antopolsky, M. & Urtti, A. 2012, "Tat(4860) peptide amino acid sequence is not unique in its cell penetrating properties and cell-surface glycosaminoglycans inhibit its cellular uptake", Journal of Controlled Release, vol. 158, no. 2, pp. 277-285. Sugahara, K.N., Teesalu, T., Karmali, P.P., Kotamraju, V.R., Agemy, L., Greenwald, D.R. & Ruoslahti, E. 2010, "Coadministration of a tumor-penetrating peptide enhances the efficacy of cancer drugs", Science, vol. 328, no. 5981, pp. 1031-1035. Szoka Jr., F. & Papahadjopoulos, D. 1978, "Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation", Proceedings of the National Academy of Sciences of the United States of America, vol. 75, no. 9, pp. 4194-4198. Tao, L., Hu, W., Liu, Y., Huang, G., Sumer, B.D. & Gao, J. 2011, "Shape-specific polymeric nanomedicine: emerging opportunities and challenges", Experimental biology and medicine, vol. 236, no. 1, pp. 20-29. Temming, K., Schiffelers, R.M., Molema, G. & Kok, R.J. 2005, "RGD-based strategies for selective delivery of therapeutics and imaging agents to the tumour vasculature", Drug resistance updates, vol. 8, no. 6, pp. 381-402. Tirosh, O., Barenholz, Y., Katzhendler, J. & Priev, A. 1998, "Hydration of polyethylene glycol-grafted liposomes", Biophysical journal, vol. 74, no. 3, pp. 1371-1379. Tong, R., Yala, L., Fan, T.M. & Cheng, J. 2010, "The formulation of aptamer-coated paclitaxel–polylactide nanoconjugates and their targeting to cancer cells", Biomaterials, vol. 31, no. 11, pp. 3043-3053. 56 Torchilin, V.P. 2005, "Recent advances with liposomes as pharmaceutical carriers", Nature reviews. Drug discovery, vol. 4, no. 2, pp. 145-160. Torchilin, V.P., Rammohan, R., Weissig, V. & Levchenko, T.S. 2001, "TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors", Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 15, pp. 8786-8791. Toropainen, E., Hornof, M., Kaarniranta, K., Johansson, P. & Urtti, A. 2007, "Corneal epithelium as a platform for secretion of transgene products after transfection with liposomal gene eyedrops", The journal of gene medicine, vol. 9, no. 3, pp. 208-216. Tros de Ilarduya, C., Sun, Y. & Düzgünes, N. 2010, "Gene delivery by lipoplexes and polyplexes", European journal of pharmaceutical sciences, vol. 40, no. 3, pp. 159170. Ulrich, A.S. 2002, "Biophysical aspects of using liposomes as delivery vehicles", Bioscience reports, vol. 22, no. 2, pp. 129-150. Vauthier, C., Persson, B., Lindner, P. & Cabane, B. 2011, "Protein adsorption and complement activation for di-block copolymer nanoparticles", Biomaterials, vol. 32, no. 6, pp. 1646-1656. Venditto, V.J. & Szoka Jr., F.C. 2013, "Cancer nanomedicines: So many papers and so few drugs!", Advanced Drug Delivery Reviews, vol. 65, no. 1, pp. 80-88. Voinea, M., Dragomir, E., Manduteanu, I. & Simionescu, M. 2002, "Binding and uptake of transferrin-bound liposomes targeted to transferrin receptors of endothelial cells", Vascular Pharmacology, vol. 39, no. 1–2, pp. 13-20. Wang, T., Upponi, J.R. & Torchilin, V.P. 2011, "Design of multifunctional non-viral gene vectors to overcome physiological barriers: Dilemmas and strategies", International journal of pharmaceutics, vol. 427, no. 1, pp. 3-20. Weber, P.C., Ohlendorf, D.H., Wendoloski, J.J. & Salemme, F.R. 1989, "Structural origins of high-affinity biotin binding to streptavidin", Science, vol. 243, pp. 85-88. Wightman, L., Kircheis, R., Rossler, V., Carotta, S., Ruzicka, R., Kursa, M. & Wagner, E. 2001, "Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo", The journal of gene medicine, vol. 3, no. 4, pp. 362-372. Wiseman, J.W., Goddard, C.A., McLelland, D. & Colledge, W.H. 2003, "A comparison of linear and branched polyethylenimine (PEI) with DCChol/DOPE liposomes for gene delivery to epithelial cells in vitro and in vivo", Gene therapy, vol. 10, no. 19, pp. 1654-1662. Xiang, S., Tong, H., Shi, Q., Fernandes, J.C., Jin, T., Dai, K. & Zhang, X. 2012, "Uptake mechanisms of non-viral gene delivery", Journal of Controlled Release, vol. 158, no. 3, pp. 371-378. 57 Xiao, Z., McQuarrie, S.A., Suresh, M.R., Mercer, J.R., Gupta, S. & Miller, G.G. 2002, "A three-step strategy for targeting drug carriers to human ovarian carcinoma cells in vitro", Journal of Biotechnology, vol. 94, no. 2, pp. 171-184. Xiong, X.B., Huang, Y., Lu, W.L., Zhang, H., Zhang, X. & Zhang, Q. 2005, "Enhanced intracellular uptake of sterically stabilized liposomal Doxorubicin in vitro resulting in improved antitumor activity in vivo", Pharmaceutical research, vol. 22, no. 6, pp. 933-939. Xu, F.J., Zhang, Z.X., Ping, Y., Li, J., Kang, E.T. & Neoh, K.G. 2009, "Star-shaped cationic polymers by atom transfer radical polymerization from beta-cyclodextrin cores for nonviral gene delivery", Biomacromolecules, vol. 10, no. 2, pp. 285-293. Xu, Y. & Szoka, F.C.,Jr 1996, "Mechanism of DNA release from cationic liposome/DNA complexes used in cell transfection", Biochemistry, vol. 35, no. 18, pp. 5616-5623. Ye, G., Gupta, A., DeLuca, R., Parang, K. & Bothun, G.D. 2010, "Bilayer disruption and liposome restructuring by a homologous series of small Arg-rich synthetic peptides", Colloids and surfaces.B, Biointerfaces, vol. 76, no. 1, pp. 76-81. Yuan, F., Dellian, M., Fukumura, D., Leunig, M., Berk, D.A., Torchilin, V.P. & Jain, R.K. 1995, "Vascular permeability in a human tumor xenograft: molecular size dependence and cutoff size", Cancer research, vol. 55, no. 17, pp. 3752-3756. Zalipsky, S., Puntambekar, B., Boulikas, P., Engbers, C.M. & Woodle, M.C. 1995, "Peptide attachment to extremities of liposomal surface grafted PEG chains: preparation of the long-circulating form of laminin pentapeptide, YIGSR", Bioconjugate chemistry, vol. 6, no. 6, pp. 705-708. Zelphati, O. & Szoka, F.C.,Jr 1996, "Mechanism of oligonucleotide release from cationic liposomes", Proceedings of the National Academy of Sciences of the United States of America, vol. 93, no. 21, pp. 11493-11498. Zelphati, O., Uyechi, L.S., Barron, L.G. & Szoka Jr., F.C. 1998, "Effect of serum components on the physico-chemical properties of cationic lipid/oligonucleotide complexes and on their interactions with cells", Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, vol. 1390, no. 2, pp. 119-133. Zhang, L., Gu, F.X., Chan, J.M., Wang, A.Z., Langer, R.S. & Farokhzad, O.C. 2008, "Nanoparticles in Medicines: Therapeutic Applications and Developments", Clinical Pharmacology & Therapeutics, vol. 83, no. 5, pp. 761-769. Zhang, Z.Y. & Smith, B.D. 2000, "High-generation polycationic dendrimers are unusually effective at disrupting anionic vesicles: membrane bending model", Bioconjugate chemistry, vol. 11, no. 6, pp. 805-814. 58