expression of human genes to cells of the nervous systems, selective destruction of ... mediated gene transfer is a promising tool for the delivery of the therapeutic gene .... Cell penetrating peptides (CPPs) have proven to be an efficient intracellular delivery system overcoming the lipophilic barrier of cell membranes [54].
7.7 DELIVERY SYSTEMS FOR PEPTIDES/ OLIGONUCLEOTIDES AND LIPOPHILIC NUCLEOSIDE ANALOGS R.A. Schwendener1 and Herbert Schott2 1 2
Institute of Molecular Cancer Research, University of Zurich, Zurich, Switzerland Institute of Organic Chemistry, University of Tübingen, Tübingen, Germany
Chapter Contents 7.7.1 State of the Art of Nanosized Delivery Systems 7.7.2 Comparison of Viral vs. NonViral Gene Delivery Systems 7.7.3 Viral Systems 7.7.3.1 Adenoviruses 7.7.3.2 Poxviruses 7.7.3.3 Herpex Simplex Virus 7.7.3.4 Lentiviruses 7.7.3.5 Adeno-Associated Virus 7.7.4 NonViral Systems 7.7.4.1 Cationic Polymers, Lipoplexes 7.7.4.2 NonLipidic Polycation Gene Delivery Systems 7.7.4.3 Other Methods 7.7.4.4 Virosomes 7.7.5 Liposomes and Cationic Liposome–DNA Complexes (Lipoplexes) 7.7.6 Peptide and Oligonucleotide Liposome Vaccine Formulations 7.7.7 Liposomes as Carriers of Lipophilic and Amphiphilic Nucleoside Analogs 7.7.8 Outlook and Future Directions References
1150 1150 1152 1152 1152 1153 1153 1153 1154 1155 1155 1156 1157 1158 1162 1163 1167 1167
Handbook of Pharmaceutical Biotechnology, Edited by Shayne Cox Gad. Copyright © 2007 John Wiley & Sons, Inc.
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7.7.1 STATE OF THE ART OF NANOSIZED DELIVERY SYSTEMS The earliest developments of drug delivery systems (DDS) date back to the 1950s where the fi rst microencapsulated drugs were introduced. In the 1960s polymerbased slow release systems appeared, and as the first nanosized DDS with spherical shape properties, the liposomes were recognized as models to study membranes and as carriers of both hydrophilic and lipophilic molecules. Liposomes, small unilamellar phospholipid bilayer vesicles, undoubtedly represent the most extensively and advanced drug delivery vehicles. After a long period of research and development efforts, liposome-formulated drugs have now entered the clinics to treat cancer and systemic fungal infections, mainly because they are biologically inert and biocompatible and do not cause unwanted toxic or antigenic reactions [1–3]. For the delivery of genetic material (DNA, ribozymes, DNAzymes, aptamers, (antisense-) oligonucleotides, small interfering RNAs), the liposomes, in particular lipid–DNA complexes termed lipoplexes, compete with viral gene transfection systems, as will be outlined in the following sections. Nanoparticles, nanospheres, polymersomes, nanogels, micelles, dendrimers, and virosomes are other main types of nanocarrier systems used for drug delivery [4–7]. As shown in Figure 7.7-1, these drug delivery systems vary in their compositions, shapes, sizes, drug loading capacity, as well as their pharmacokinetic and organ or tissue targeting properties [8]. DDS are developed for drugs with nonideal properties that include (1) poor solubility, where a conventional pharmaceutic formulation is difficult to prepare as poorly water soluble drugs may precipitate in aqueous media. (2) Tissue damage caused by inadvertent extravasation of drugs, e.g., tissue necrosis caused by cytotoxic drugs. (3) Loss of drug activity after administration, e.g., enzymatic and fast metabolic degradation. (4) Unfavorable pharmacokinetic properties and poor biodistribution. (5) Lack of selectivity for target organs or tissues. Systemic drug distribution may cause toxic side effects, and low concentrations in target tissues may cause suboptimal therapeutic effects. The formulation of pharmacologically active drug molecules in DDS can improve or abolish these unfavorable properties. However, there are also drawbacks in DDS development, such as system complexity, unwanted biologic effects, stability, costs of development and scale-up, as well as intellectual property issues. In the limited format of this review, it is not possible to cover all methods and references in the field. Hence, we concentrate this review on DDS for the delivery of peptides, DNA, plasmids, oligodeoxynucleotides, siRNA, and lipophilic nucleoside derivatives. Vaccine delivery systems will also be mentioned, and examples will be provided to demonstrate the general development trends.
7.7.2 COMPARISON OF VIRAL VS. NONVIRAL GENE DELIVERY SYSTEMS Viral gene delivery has become an important therapeutic strategy for the development of novel treatment approaches. A deeper understanding of vector biology and the molecular mechanisms of disease together with remarkable advances in molecular biology and vector technology have considerably advanced the field of human
COMPARISON OF VIRAL VS. NONVIRAL GENE DELIVERY SYSTEMS
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Phospholipid
dendrimer
worm micelle
Hydrophobic chain Protein drug PEG
500 nm
Antibody
nanoparticle nanogel
polymer micelle liposome
polymersome 500 nm
1000 nm
Figure 7.7-1. Nanocarriers for vascular drug delivery. Schematic representation of several main types of nanocarriers depicted in relative size scale with limited and oversimplified structural features. All nanocarriers can be surface-conjugated with targeting antibodies (or alternative affi nity moieties) and PEG polymer providing stealth features. Dendrimers are the smallest of nanocarriers, in the maximum range of tens of nanometers. They possess multiple end groups suitable for a high extent of coupling targeting or active agents. Liposomes, composed of biologically derived phospholipids, are the most common form of nanocarriers with large aqueous loading potential; yet they are also the least stable of carriers. Polymersomes, one of three self-assembled polymeric nanocarriers, are the synthetic polymer analog of liposomes, possessing enhanced stability, dense PEG coating, and therefore prolonged circulation in vivo. Worm micelles are long, flexible cylindrical polymer micelles that possess one of the longest circulation times recorded in vivo with a yet unknown potential for therapeutic applications. Polymeric micelles are the smallest of the self-assembling polymer aggregate carriers and are the least stable self-assembler. Nanogels are composed of cross-linked polymers with drug entrapped into the ensuing matrix. Their circulation and potential therapeutic uses remain to be studied. Nanoparticles are solid polymer structures formed through processing rather than through self-assembly methods. They represent the largest of the carriers, have the greatest active protein loading capacity measured to date, and can protect encapsulated therapeutic enzymes from external proteolysis. (With permission from Ref. 8.)
gene therapy development. However, most viral gene delivery systems used to date have demonstrated limitations in practicality and safety, mainly due to low levels and short duration of recombinant transgene expression, induction of host immunogenicity to vector constituents, and suboptimal transgene expression to tissues or cells. A recent, additional cause for concern over using viral vectors is the phenomenon known as insertional mutagenesis, in which the chromosomal integration of viral gene material either interrupts the expression of a tumor suppressor gene or activates an oncogene, leading to the malignant transformation of cells. Thus, safer, nonviral delivery approaches are needed and the latest advances indicate that efficient, long-term gene expression can be achieved by nonviral
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means. In particular, integration of DNA can be targeted to specific genomic sites without harmful consequences, and it is possible to maintain transgenes as small episomal plasmids or artificial chromosomes. The application of these approaches to human gene therapy is progressively becoming a reality. Here, we briefly compare use, properties, advantages, and disadvantages of viral and nonviral gene delivery, the two main types of DDS that are used in gene therapy and vaccine approaches.
7.7.3
VIRAL SYSTEMS
The lack of efficient nontoxic gene delivery systems is still the major impediment to the successful application of gene therapy. Having evolved to deliver their genes to target cells, viruses are effective means of gene delivery and they can be manipulated to express therapeutic genes or to replicate specifically in certain cells. The fi rst viral vector systems were developed more than 25 years ago [9], and since then viral gene therapy strategies has been progressively developed [10]. A variety of virus vectors has been employed and modified to deliver genes to cells to provide either transient, such as adenovirus [11–13], poxviruses (vaccinia) [14], or herpes virus [15], or permanent, such as retroviruses (lentivirus) [16–18] and adeno-associated virus [19], transgene expression; each approach has its characteristic advantages and disadvantages. 7.7.3.1
Adenoviruses
Replication-defective adenoviruses are vectors of choice for delivering corrective genes into human cells. Major efforts are directed to design new generations of adenoviral vectors that feature reduced immunogenicity and improved targeting ability. Various targeting strategies have been attempted aiming at obtaining optimized and specific cellular transduction, including that of genetic manipulation of the viral capsid. Modification of the tropism-determining fiber protein and other capsid proteins has yielded vectors that are superior to the first-generation adenoviruses employed for gene therapy [11]. Adenoviral-based vectors are susceptible both to cytotoxic T-lymphocyte and humoral immune responses. In addition, leaky adenoviral genes also render transduced cells susceptible to host immune responses. These are the main reasons why adenoviral-based vectors are not suitable to correct genetic disorders, which require long-term expression of the transgene [20]. The production of adenoviral vectors for gene therapy applications still faces several challenges that limit the availability of high-quality material for clinical applications [21, 22]. 7.7.3.2
Poxviruses
Poxviruses represent a heterogenous group of DNA viruses that have been used to express a multitude of foreign genes. Vaccinia virus is the prototypical recombinant poxvirus that can generate potent antibody and T-cell responses. Recombinant vaccinia viruses (rVVs) as nonreplicating viral vectors have been demonstrated for their great potential as vaccines, diminished cytopathic effects, high levels of protein expression, and strong immunogenicity, and they are relatively safe in
VIRAL SYSTEMS
1153
animals and in human patients. These properties have led to the use of rVVs as vaccines against HIV and cancer [14, 23]. 7.7.3.3
Herpes Simplex Virus
Attenuated genetically engineered herpes simplex virus (HSV) vectors are potential vectors for several human therapy applications. These include delivery and expression of human genes to cells of the nervous systems, selective destruction of cancer cells, prophylaxis against infection with HSV or other infectious diseases, and targeted infection to specific tissues or organs [15]. 7.7.3.4
Lentiviruses
Lentiviruses, members of the retroviral family, have the ability to infect cells at both mitotic and postmitotic stages of the cell cycle, thus opening the possibility to target nondividing cells and tissues. Human Immunodeficiency Virus (HIV)-based vectors have been used in vitro and in vivo in several situations; however, safety concerns still exist. Therefore, the development of vector systems based on primate as well as nonprimate lentiviruses is ongoing. Recent developments in the modification of the virus coat allow more targeted approaches and open new possibilities for the systemic delivery of therapeutic viruses [24]. However, the specific mechanisms used by different retroviruses to efficiently deliver their genes into cell nuclei remain largely unclear. Understanding these molecular mechanisms may reveal features to improve the efficacy of current retroviral vectors [25]. 7.7.3.5
Adeno-Associated Virus
Vectors based on the adeno-associated virus (AAV) have attracted much attention as potent gene-delivery vehicles, mainly because of the persistence of this nonpathogenic virus in the host cell and its sustainable therapeutic gene expression. The principal historical limitation of this vector system, efficiency of recombinant AAV-mediated (rAAV) transduction, has recently observed a dramatic increase as the titer, purity, and production capacity of rAAV preparations have improved [26]. AAV vectors have been used in phase I clinical trials for the treatment of neurological disorders, such as Parkinson’s and Canavan’s diseases. Indeed, AAVmediated gene transfer is a promising tool for the delivery of the therapeutic gene into the central and peripheral nervous systems. AAV-mediated gene transfer was also applied in phase I and II clinical trials for the treatment of cystic fibrosis and in phase I trials for the treatment of hemophilia B. In the context of cancer, the ability of attenuated viruses to replicate specifically in tumor cells has already yielded some impressive results in clinical trials, allowing the design of new therapeutic approaches, particularly when combined with other approved anticancer therapies. Despite the remark-able progress that has been reported, the design of further optimized vectors is still required. As it stands, AAV-mediated gene transfer has a limited capacity in accommodating foreign genes. In addition, some preclinical studies have shown that AAV-derived vectors can cause tumors in animals due to mutagenic random vector integration into the genome [27]. To circumvent this problem, a novel approach to AAV-mediated gene therapy based on gene targeting through homologous recombination has been introduced that allows efficient, high-fidelity, nonmutagenic gene repair in a host cell [28].
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7.7.4
DELIVERY SYSTEMS
NONVIRAL SYSTEMS
Currently, the most severe limitations of the nonviral gene therapy systems are low transfection efficiency of gene material into the target cells, physico-chemical instability, and cytotoxicity. The major obstacles encountered in the transfer of foreign genetic material into the body include interactions with blood components and vascular endothelial cells and uptake by the mononuclear phagocyte system (MPS). The degradation of DNA by serum nucleases is another major obstruction for functional delivery to the target. In addition to targeting a specific cell type, an ideal nonviral vector (liposomes, lipoplexes, virosomes), once taken up by a target cell, must manifest an efficient endosomal escape, provide sufficient protection of DNA in the cytosol and help facilitate an easy passage of cytosolic DNA to the nucleus (Figure 7.7-2).
Liposome
Drug
A
B
Lysosome
F
G
Endosome
Nucleus
C
H
E
D
Figure 7.7-2. Liposome–cell interactions. Drug-loaded liposomes can specifically (A) or nonspecifically (B) adsorb onto the cell surface. Liposomes can also fuse with the cell membrane (C) and release their contents into the cell cytoplasm, or they can be destabilized by certain cell membrane components when adsorbed on the surface (D) so that the released drug can enter the cell via micro-pinocytosis. Liposome can undergo the direct or transfer-protein mediated exchange of lipid components with the cell membrane (E) or be taken up by specific or nonspecific endocytosis (F). In the case of endocytosis, a liposome can be delivered by the endosome into the lysosome (G), or en route to the lysosome, the liposome can provoke endosome destabilization (H), which results in drug liberation into the cytoplasm. (With permission from Ref. 29.)
NONVIRAL SYSTEMS
7.7.4.1
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Cationic Polymers, Lipoplexes
Cationic polymers have a great potential for DNA complexation and have shown to be useful as nonviral vectors for gene therapy applications. For the past 15–20 years liposomes composed of cationic lipids, termed lipoplexes, have routinely been used for the delivery of nucleic acids such as plasmids, oligodeoxynucleotides, and siRNA to cells in culture and in vivo. Many of these reagents are commercially available or can be formulated in the laboratory [30–32]. Most cationic lipid–DNA complexes form a multilayered structure with DNA sandwiched between the cationic lipids. Much more rarely, an inverted hexagonal structure with single DNA strands encapsulated in lipid tubules is observed [33]. Among other advantages, lipoplexes have the ability to transfer very large genes into cells. However, as the understanding of their mechanisms of action is still incomplete, their transfection efficiencies remain low compared with those of viruses. Particularly in cultured cells, toxicity remains a significant problem. In addition, these complexes are immunostimulatory, a fact that may either be harmful or beneficial. The development of cationic lipids that are safe to use, especially for in vivo applications, and possess enhanced transfection capabilities is an ongoing process. The lipoplexes are described in detail in Section 7.7.5.
7.7.4.2
NonLipidic Polycation Gene Delivery Systems
Of the many nonlipidic polycation gene delivery systems developed in the past decades, poly(L-lysine) (PLL) was the fi rst polycation used for nonviral gene delivery [34]. Among a vast number of other positively charged polymers, polyethylenimine (PEI) has been widely used for nonviral transfection in vitro and in vivo and has an advantage over other polycations in that it combines strong DNA condensation capacity with an intrinsic endosomolytic activity [35–38]. Other synthetic and natural polycations developed as nonviral vectors are polyamidoamine dendrimers, the synthetic cationic polymer poly(2-dimethylamino)ethyl methacrylate (PDMAEMA) [39], and chitosan. Polyamidoamine (PAMAM) dendrimers represent a novel class of polycationic synthetic polymers that can be used for gene transfer [40, 41]. The three-dimensional spherical structure of dendrimers offers synthesis control of the molecule in terms of degree and generation of branching. The control of branching of the dendrimers during synthesis allows the production of polymer particles with a very high degree of monodispersity, which is a significant advantage over other polymers such as polylysine that generate highly polydisperse particles (see Figure 7.7-1). Low polydispersity can lead to reproducible gene delivery and a clinically reliable formulation. The cationic amino acid residues in the polymeric structure of PAMAM dendrimers can help in DNA condensation and endosome release. Dendrimers that protect oligonucleotides from serum nucleases have been used to enhance oligonucleotide delivery [42]. Chitosan, a natural-based polymer obtained by alkaline deacetylation of chitin, is nontoxic, biocompatible, and biodegradable. These properties make chitosan a promising candidate for conventional and novel drug delivery systems. Because of the high affi nity of chitosan for cell membranes, it has been used as a coating agent for liposome formulations [43–45].
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7.7.4.3
Other Methods
Various other methods, such as DNA electrotransfer, electroporation-based gene transfer, calcium phosphate nanoparticles, peptide nucleic acids, and cell penetrating peptides, round off the currently used methods in the field of nonviral gene delivery techniques. DNA electrotransfer, the use of electric pulses to transfect various types of cells, is well known and regularly used in vitro for bacteria and eukaryotic cell transformation. Electric pulses can also be delivered in vivo either through the skin or with electrodes in direct contact with the target tissues. After injection of naked DNA in a tissue, appropriate local electric pulses can result in a very high expression of the transferred genes [46]. Electroporation has been applied in preclinical autoimmune and/or inflammatory diseases to deliver either cytokines and anti-inflammatory agents or immunoregulatory molecules. The method is also effective for the intratumoral delivery of therapeutic vectors, and it strongly boosts DNA vaccination against infectious agents or tumor antigens. Electroporation gene therapy has become a widely used method for nonviral gene delivery, including applications for intramuscular and intratumoral electro-gene transfer and for the transfection of dendritic and stem cells [47, 48]. The current challenges faced by both in vitro and in vivo applications comprise the enhancement of transfection efficiency, extention of the duration of gene expression, and increase of the survival rate for in vitro cell transfections. Virus- or liposome-like-sized calcium phosphate nanoparticles of 20–200 nm mean diameter have been found to overcome many of the known limitations in delivering genes to the nucleus of specific cells. It has been demonstrated that calcium ions play an important role in endosomal escape, cytosolic stability, and enhanced nuclear uptake of DNA through nuclear pore complexes. The role of exogenous calcium ions to overcome the major obstacles encountered in the practical accomplishment of gene delivery suggests that calcium phosphate nanoparticles can be designated as a new generation of nonviral vectors [49]. Peptide nucleic acid (PNA) is a powerful new biomolecular tool with a wide range of important applications. PNA mimics the behavior of DNA and binds complementary nucleic acid strands and RNA sequences with high affinity and selectivity. The unique chemical, physical, and biological properties of PNA are exploited to produce powerful biomolecular tools, antisense and antigene agents, molecular probes, and biosensors [50–53]. Cell penetrating peptides (CPPs) have proven to be an efficient intracellular delivery system overcoming the lipophilic barrier of cell membranes [54]. CPPs can deliver a wide range of large cargo molecules such as proteins, peptides, oligonucleotides, and even small particles as liposomes to a variety of cell types and to different cellular compartments. The CPPs are basic, lysine-, or arginine- rich amphipathic peptides. The peptides originate from different sources, either as naturally occurring peptide sequences, virally derived (TAT, VP22), from transcription factors (pAntp), as chimeric (transportan) or as synthetic peptides (polyarginines) and others [55]. As depicted in Figure 7.7-3, CPPs can either form complexes with peptides, proteins, plasmids, oligonucleotides, or siRNA molecules or they can be covalently linked to these cargo molecules [54]. Larger structures such as liposomes have also
NONVIRAL SYSTEMS
1157
CPP
Peptide
Antisense ON
Plasmid
Protein
siRNA
A B
C
D
E
Inverted micelle
Direct translocation Endocytotic uptake
Figure 7.7-3. Cell penetrating peptides, CPPs. Suggested uptake mechanisms for CPPs and examples of delivered cargoes. (A) CPP and peptide in single amino acid chain. (B) Oligodeoxynucleotides either in complex or covalently linked. (C) Plasmid in complex by electrostatic interaction. (D) Protein either as fusion protein or in complex with CPP. (E) siRNA, covalently linked or as complex. (With permission from Ref. 54.)
been decorated with the TAT [56] or pAntp CPPs [57], demonstrating higher cell uptake rates in vitro. Unfortunately, their usefulness as drug delivery systems is hampered by their ability to penetrate virtually any cell type both in vitro and in vivo. This feature makes CPP applications as target specific drug delivery systems complicated and their application as therapeutic drug carrier systems seems unlikely, unless their target cell specificity can be significantly improved. 7.7.4.4
Virosomes
Virosomes were developed from liposomes by combining liposomes with fusogenic viral envelope proteins. Almeida et al. [58] were the fi rst to report on the generation of lipid vesicles containing viral spike proteins derived from influenza virus. Using preformed liposomes and hemagglutinin (HA) and neuraminidase (NA), purified from influenza virus, they succeeded to generate membrane vesicles with spike proteins protruding from the vesicle surface. Visualization of these vesicles by electron microscopy revealed that they very much resembled native influenza virus. Consequently, they were named virosomes. Reconstituted viral envelopes (virosomes, artificial viral envelopes) appear to be ideally suited as vaccine formulations for the delivery of protein antigens to the cytosol of antigen presenting cells (APCs), and thus for the introduction of antigenic peptides into the MHC class I presentation pathway. Cytotoxic T-cell activity can be induced by immunization of mice with an antigenic peptide or an entire protein encapsulated in virosomes. As shown schematically in Figure 7.7-4, the action of influenza virosomes is likely to involve both delivery of the enclosed antigen to the cytosol of antigen presenting
1158
DELIVERY SYSTEMS Liposome Viral components
A Drug
B
Endosome
C Nucleus
D
Drug efflux
Figure 7.7-4. Virosome–cell interactions. Liposome modified with specific viral components (A) and loaded with a drug can specifically interact with cells (B), provoke endocytosis, and via the interaction of viral components with the inner membrane of the endosome (C), allow for drug efflux into the cell cytoplasm (D). (With permission from Ref. 29.)
cells and the powerful helper activity of the virosomal hemagglutinin. Although the immune responses elicited by DNA-virosomes are moderate, they are promising and warrant further research to ultimately develop effective DNA-based virosomal vaccines [59–61]. 7.7.5 LIPOSOMES AND CATIONIC LIPOSOME-DNA COMPLEXES (LIPOPLEXES) Liposomes have become known as one of the most versatile tools for the delivery of DNA, DNA-related, and many other therapeutic molecules [6, 7, 29]. Liposomes are spherical vesicles that consist of an aqueous compartment enclosed in a phospholipid bilayer. If multiple bilayers of lipids are formed around the primary core, the structures that are generated are known as multilamellar vesicles (MLVs). MLVs are formed spontaneously by reconstitution of lipid fi lms in aqueous media. Small unilamellar vesicles (SUVs) of specific size (100–500 nm) are produced by high-pressure extrusion of MLVs through polycarbonate membranes. SUVs (25– 90 nm) are also obtained by sonication of MLVs or larger SUVs, by detergent dialysis [62] and by many other, less important methods. Both hydrophilic and
LIPOSOMES AND CATIONIC LIPOSOME-DNA COMPLEXES (LIPOPLEXES)
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hydrophobic drugs can be entrapped in the liposomes, and the choice of the lipid composition as well as the surface modification of the liposomes provides them with a high versatility such as long circulation half-life and sustained and targeted delivery (Figure 7.7-5) [29, 62]. Liposomes can be used as DNA drug delivery systems either by entrapping the DNA-based therapeutics inside the aqueous core or by complexing them to positively charged lipids (lipoplexes, see below). Liposomes offer significant advantages over viral delivery systems; for example, liposomes are generally nonimmunogenic because of the absence of proteinaceous components. As the phospholipid composition in the liposome bilayers can be varied, liposomal delivery systems are of high versatility and customized formulations can be easily engineered to obtain desired sizes, surface charge, composition, and morphology. Liposome encapsulated DNA molecules are protected from nuclease activity and thus improve their biological stability. Long circulating (“stealth”) liposomes are sterically stabilized liposomal formulations that include poly(ethylene glycol) (PEG)-conjugated lipids or other hydrophilic coating molecules. PEGylation prevents the opsonization and recognition of the liposomal vesicles by the MPS. PEGlyation has also been used in conjunction with other polymeric delivery systems such as poly(L-lysine) to achieve longer circulation half-lives. Immunoliposomes are complex drug or gene delivery systems that can be used for cell targeting by the incorporation of functionalized antibodies attached to lipid bilayers. The “state-of-the-art” immunoliposomes are long circulating PEGliposomes to which receptor specific molecules are attached, preferably at the distal tips of the PEG chains [62–65]. Immunoliposomes target specific receptors and facilitate receptor-mediated endocytosis for cell uptake (see Fig. 2). Immunoliposomes decorated with single-chain antibody fragments against the ED-B isoform of fibronectin were successfully used in targeted delivery of cytotoxic drugs into tumors in vivo [66]. Tissue-specific gene delivery using immunoliposomes has also been achieved by antitransferrin receptor immunoliposomes [67]. To release encapsulated material into the cytoplasm, pH-sensitive liposomes can be generated by the inclusion of dioleyl-phosphatidylethanolamine (DOPE) into liposomes composed of acidic lipids such as cholesterylhemisuccinate or oleic acid. At the neutral cellular pH 7, these lipids have the typical bilayer structure; however, upon endosomal compartmentalization, they undergo protonation and collapse into nonbilayer structures, thereby leading to the disruption and destabilization of the endosomal bilayer, which in turn helps in the rapid release of encapsulated molecules into the cytoplasm [7]. There is an important difference between liposome vesicles and cationic lipid– DNA complexes, the lipoplexes. Lipoplexes are cationic lipid–DNA complexes that are formed spontaneously in aqueous media upon mixing DNA and preformed liposome vesicles composed of cationic and neutral lipids [68]. From a physical point of view, lipoplexes are ordered, self-assembled, composite aggregates whose spatial geometry and phase behavior are controlled by the electrostatic interactions between the positively charged lipids and the negatively charged DNA molecules. Numerous studies have demonstrated the use of cationic liposome formulations for the delivery of different plasmid constructs in a wide range of cells, both in vivo and in vitro. Despite their cytotoxicity, their nonimmunogenic nature and the
1160
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A
C f d
e c
b a
D
E h
j i q
g
k
s r
p
l o
n
m
Figure 7.7-5. Evolution of liposomes. (A) Early traditional “plain” liposomes with watersoluble drug (a) entrapped into the aqueous liposome interior, and lipophilic drug (b) incorporated into the liposomal membrane. (B) Antibody-targeted immunoliposome with antibody covalently coupled (c) to the reactive phospholipids in the membrane, or hydrophobically anchored (d) into the liposomal membrane after preliminary modification with a hydrophobic moiety. (C) Long circulating liposome grafted with a protective polymer (e) such as PEG, which shields the liposome surface from the interaction with opsonizing proteins (f). (D) Long circulating immunoliposome simultaneously bearing both protective polymer and antibody, which can be attached to the liposome surface (g) or, preferably, to the distal end of the grafted polymeric chain (h). (E) New-generation liposome, the surface of which can be modified (separately or simultaneously) by different ways. Among these modifications are the attachment of protective polymer (i) or protective polymer and targeting ligand, such as antibody (j); the attachment/incorporation of a diagnostic label (k); the incorporation of positively charged lipids (l) allowing for the complexation with DNA yielding lipoplex structures (m); the incorporation of stimuli-sensitive lipids (n); the attachment of a stimuli-sensitive polymer (o); the attachment of a cell-penetrating peptide (p); and the incorporation of viral components (q). In addition to a drug, liposomes can be loaded with magnetic particles (r) for magnetic targeting and/or with colloidal gold, silver particles, or fluorescent molecules (s) for microscopic analysis. (Adapted with permission from Ref. 29.)
LIPOSOMES AND CATIONIC LIPOSOME-DNA COMPLEXES (LIPOPLEXES)
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simplicity of production of these systems make them attractive tools for gene transfer. Currently, many gene therapy trials in progress employ nonviral liposomal vectors for transgene delivery. Lipoplex formulations generally consist of mixtures of cationic and neutral (zwitterionic) lipids. Commonly used cationic lipids are 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 2,3-dioleoyloxy-N-[2-(sperminecarboxamido) ethyl]N,N-dimethyl-1-propanaminium (DOSPA), dioctadecyl amido glycil spermine (DOGS), and 3,[N-(N1,N-dimethylethylenediamine)-carbamoyl] cholesterol (DC-chol) [69–71]. Commonly used neutral molecules, also known as helper or colipids, are DOPE and cholesterol. The cationic lipids in the liposomal formulation serve as a DNA complexation and DNA condensation agent during the formation of the lipoplex. The positive charge also helps in cellular association. The colipids facilitate membrane perturbation and fusion. Many proprietary reagents of cationic lipids such as Lipofectamine (Invitrogen, Carlsbad, CA), Effectene (Qiagen, Valencia, CA), and Tranfectam (Promega, Madison, WI) are commercially available. However, most of these transfection reagents can only be used for in vitro gene transfection applications. Despite the appreciable success of cationic lipids in gene transfer, toxicity is a main issue for both in vitro and in vivo applications. Inflammatory toxicity represents a typical toxicity associated with systemic administration of lipoplexes. Results obtained from in vivo studies indicate that lipoplex gene delivery systems mediate uptake of plasmid DNA by the liver, mainly by Kupffer cells, in which a large amount of cytokines is produced [72, 73]. Upon administration via the airways, cytokinemediated pulmonary toxicity and TNF-α induction by cationic lipids in lung tissue have been reported [74]. Reduction in toxicity was observed by a modification of DOPE polyplexes with cetylated PEI, resulting in remarkable transgene efficiency with low cytotoxicity [72]. In another study, a sterically stabilized immunolipoplex composed of a p53 DNA–lipid complex to which PEG molecules and an antitransferrin receptor single-chain antibody fragment were attached resulted in improved delivery of the complex to tumor cells in vivo [75]. The negative factors of lipoplex-mediated gene transfer are low transfection efficiencies, which have been attributed to the heterogeneity and instability of the lipoplex formulations. Lipoplex size heterogeneity also adversely affects their quality control, scale-up, and long-term shelf stability, which are important issues for their pharmaceutical development. Compared with viral vectors, the transfection efficiencies of cationic liposomal vectors are significantly lower. Another drawback in the use of cationic lipids is the rapid inactivation of their cargo in the presence of serum [76]. As an alternative to cationic lipids, the potential of anionic lipids for DNA delivery has been investigated. However, in recent years, only a few studies using anionic liposomal DNA delivery vectors have been reported [77, 78]. These vectors have limited applications, mainly because of inefficient entrapment of DNA molecules within anionic liposomes and lack of toxicity data. Lacking progress of these systems may be attributed to the poor association between DNA molecules and anionic lipids, which is caused by electrostatic repulsion between these negatively charged species.
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7.7.6 PEPTIDE AND OLIGONUCLEOTIDE LIPOSOME VACCINE FORMULATIONS With the increasing availability of defi ned antigens such as highly purified proteins or synthetic peptides, more effective and safer vaccines are developed. As many antigens are often poorly immunogenic when administered alone, the development of suitable adjuvants, possessing the ability to potentiate the immunogenicity of a given antigen, preferably with little or no side effects, is required. Based on their principal mechanisms of action, adjuvants can be divided into two groups: (1) vaccine delivery systems and (2) immunostimulatory adjuvants. As described in this chapter, vaccine delivery systems are generally particulate delivery systems of size dimensions comparable with pathogens as bacteria and viruses (e.g., liposomes, microemulsions, immunostimulatory complexes, and other nano- or microparticle systems in the size range of 20–500 nm). Their function is mainly to target antigens to antigen-presenting cells (APCs; dendritic cells, macrophages) and to act as adjuvants. The rationale for the development of an optimal vaccine is to ensure that both antigen and adjuvant are delivered into the same population of APCs, thereby reducing systemic distribution and minimizing the potential to induce adverse reactions. Small unilamellar liposomes have an important potential as delivery systems for the coadministration of antigens and of immunostimulatory adjuvants, including synthetic oligodeoxynucleotides containing immunostimulatory deoxycytidylyl-deoxyguanosine dinucleotides (CpGs) or DNA encoding antigens [79–81]. Additionally, the efficiency of the liposomes can be improved by targeting them more effectively and specifically to the APCs by exploiting various scavengers and other receptors as their targets or by enhancing their cell uptake by modification with cell penetrating peptides as recently shown by us [57]. Using the lymphocytic choriomeningitis virus (LCMV) model system, we successfully prepared efficient peptide vaccines with liposomes as the carrier [82]. Liposome-encapsulated antigenic peptides were highly immunogenic when administered intradermally, and they elicited protective antiviral immunity. An optimized formulation contained immunostimulatory oligonucleotides leading to activation of dendritic cells and antitumor immunity in LCMV peptide transfected EL4 thymomas. In a follow-up study we could confi rm the efficacy of such peptide– liposome vaccines in a hepatitis C virus model in HLA-A2.1 transgenic mice [83]. These fi ndings clearly indicate that liposomal antigen delivery in vivo is a promising approach to induce efficient antiviral and antitumor immune responses with relevance for human applications. A cautionary note to the potential dangers of all viral gene products, transgenes, viral proteins and peptides, and CpG DNA sequences in siRNA or plasmids formulated in DDS has to be given. Immune responses induced by these molecules may lead to problems such as transient gene expression, nonefficient readministration of the same vectors and to severe side effects in clinical trials [84]. Due to their particulate nature, the DDS are recognized as foreign in an organism that reacts with an immune response. However, the immunomodulating activities of the DDS depend largely on their composition, size, and homogeneity. Synthetic polymers can exhibit significant immunomodulating activity, whereas liposomes prepared with natural phospholipids and cholesterol are known to be less immunogenic [85].
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7.7.7 LIPOSOMES AS CARRIERS OF LIPOPHILIC AND AMPHIPHILIC NUCLEOSIDE ANALOGS Most applications of liposomes as therapeutic drug carrier systems are based on the encapsulation of water-soluble molecules within the trapped aqueous volume of the liposomes. Long circulating PEG-modified liposomes with cytotoxic antitumor drugs doxorubicine, paclitaxel, vincristine, and methotrexate are examples of clinically applied chemotherapeutic liposome formulations [86–88]. In contrast to the extensive exploitation of the trapped aqueous volume of the liposomes that serves as a nanocontainer for the water-soluble molecules, the phospholipid bilayer has not been given the same attention for its use as the carrier matrix for lipophilic drugs. Hence, the development of liposomal drug formulations with lipophilic drugs is less popular. This difference may have several reasons, the main probably consisting in the chemistry required to transform water-soluble molecules into lipophilic compounds that allow incorporation into the lipid bilayer core. The most favorable chemical modifications to obtain a molecule that intercalates in a stable fashion into the lipophilic moiety of a lipid bilayer consist in the attachment of long-chain fatty acyl or alkyl residues, for example saturated or unsaturated fatty acids, preferably palmitic or stearic acid and alkylamines, preferably hexadecyl- or octadecylamine to a suitable functional group of the hydrophilic part of the molecule. Some recent examples of lipophilic modifications of antitumor drugs and their formulation in liposomes are gemcitabine, 5-iodo-2′-deoxyuridine, methotrexate, paclitaxel, cytosine arabinoside, and a lipophilic topoisomerase inhibitor, DB67 [89–96]. Drugs that are highly lipophilic by their own nature, e.g., taxanes, epothilones, and cyclosporins, can only be used therapeutically by the addition of possibly toxic solubilizing agents (e.g., Cremophor EL) in complex pharmaceutical formulations [97–99]. One of several feasible means of obtaining nontoxic parenterally applicable formulations of such drugs is their incorporation into the bilayer matrix of phospholipid liposomes. Nucleoside analogs are a major class of chemotherapeutic agents for the treatment of cancer and viral diseases. Natural endogenous nucleosides must be phosphorylated to corresponding 5′-triphosphates to be incorporated into the DNA or RNA synthesized within the cell. The fi rst phosphorylation step, leading to the formation of nucleoside-5′-monophosphate, is commonly performed by a nucleoside kinase encoded by either the host cell or the virus infecting the host cell. Hence, cellular and virally encoded kinases play a vital role in the metabolism and replication of cells and viruses. Nucleoside analogs used for chemotherapy of cancer and viral infections are in essence prodrugs because they must be phosphorylated in the cytoplasm like the natural nucleosides to triphosphates before they can exert their activities. Thus, administration of phosphorylated nucleoside analogs would circumvent the enzymatic phosphorylation step, which transforms inactive nucleosides into active drugs. Phosphate groups possess an anionic charge at nearly all physiological pH values, making them very polar. This high polarity can be responsible for many deficiencies in terms of efficient drug delivery. Nucleotides are too hydrophilic to penetrate the lipid-rich cell membrane. In addition, blood and cell-surface phosphohydrolases rapidly metabolize the nucleotides to the corresponding nucleosides. Due to their polarity, nucleotides often exhibit a low
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DELIVERY SYSTEMS
volume of distribution and are therefore efficiently cleared by renal elimination. In an attempt to overcome these shortcomings, various prodrugs or “pronucleotide” approaches have been devised and investigated for the in vivo delivery of pharmacologically active nucleotides. The general aim of these approaches has been to promote passive diffusion through cell membranes and to increase the bioavailability of phosphorylated nucleoside analogs [100, 101]. A simple solution for the delivery of nucleoside monophosphate analogs into cells consists in the chemical neutralization of the ionizable phosphate group via chemical derivatization for example by esterification, resulting in nucleotide phosphodi- and phosphotriester derivatives or by the synthesis of nucleotide phophorodiamidates, cyclic phosphoramidates, and phosphoramidate mono- and diesters. These compounds have a neutral charge and should be capable of entering cells via passive diffusion. To retain the advantage of nucleoside phosphotriesters with regard to their improved cellular uptake, a variety of biolabile moieties have been evaluated as potential protecting groups for nucleoside phosphotriesters. We chose the approach of the chemical transformation of water-soluble nucleosides of known cytotoxic and antiviral properties into lipophilic drugs or prodrugs. Thus, we reversed the paradigm of transforming lipophilic molecules into hydrophilic derivatives [102]. The fi rst cytotoxic nucleoside that we transformed into lipophilic derivatives was 1-β-D-arabino-furanosyl cytosine (ara-C) because its major clinical disadvantages are a very short plasma half-life and rapid degradation by deamination to the inactive metabolite 1-β-D-arabino-furanosyluracil (ara-U), a shortcoming that also impedes the oral application of ara-C. To reduce these limitations, a large number of 5′- and N4 -substituted ara-C derivatives have been synthesized and characterized in the past (reviewed in Ref. 103). We synthesized a new class of N4 -alkyl-ara-C derivatives with alkyl chain lengths ranging between 6 and 22 C-atoms, demonstrating a typical structure-activity correlation between the length of the alkyl side chains and their antitumor activity profi le [104]. The most effective derivative, N4 -octadecyl-ara-C (NOAC), which is highly lipophilic and extremely resistant toward deamination exerted excellent antitumor activity after oral and parenteral therapy in several mouse tumor models and showed to have distinct pharmacological properties compared with ara-C [105]. Consequently, we further modified NOAC by the synthesis of a new generation of lipophilic/amphiphilic heterodinucleoside phosphate derivatives, termed “duplex drugs” that combine the clinically used antitumor drugs ara-C and 5-fluorodeoxyuridine (5-FdU) with NOAC yielding the heterodinucleoside phosphates arabinocytidylyl-N4 -octadecyl-1-β-D-arabino-furanosylcytosine (ara-C-NOAC) and 2′-deoxy-5-fluorouridylyl-N4 -octadecyl-1-β-D-arabinofuranosy-lcytosine (5-FdUNOAC) (Figure 7.7-6) [106, 107]. Ethynylcytidine (1-(3-C-ethynyl-β-D-ribopentafuranosyl)-cytosine, ETC) is a novel nucleoside that was found to be highly cytotoxic [108–110]. Thus, by combination of ETC with NOAC, we obtained the lipophilic duplex drug ETC-NOAC (3′-C-ethynylcytidylyl-(5′ → 5′)-N4 -octadecyl-1-β-Darabinofuranosylcytosine). Due to the combination of the effects of both active molecules that can be released into the cytoplasm as monomers or as the corresponding monophosphates (MPs), the cytotoxic activity of the duplex drugs is expected to be more pronounced as compared with the monomeric drugs. Furthermore, it can be anticipated that the monophosphorylated nucleosides ara-CMP, 5-FdU-MP, and ETC-MP, respec-
LIPOPHILIC AND AMPHIPHILIC NUCLEOSIDE ANALOGS
1165
tively, are directly released into the cell after enzymatic cleavage of the duplex drugs. Thus, monophosphorylated molecules would not have to pass the first phosphorylation step, which is known to be rate limiting [100, 101]. The lipophilic side chains allow a stable incorporation of these duplex drugs into liposomes, allowing the exploitation of the advantages liposome formulations are offering. Due to their high polarity, the nonderivatized heterodinucleoside phosphodiesters are less suited for liposomal formulations. In comparison with the nonpolar heterodinucleoside phosphotriesters that have good properties to be taken up by cells, but whose capacity to be cleaved enzymatically is limited, the cleavage of the natural phosphodiester bond of the duplex drugs is not constrained. A delayed intracellular release of nucleoside and nucleotide analogs from the duplex molecules provides a depot effect that may be therapeutically of advantage. A structure-related disadvantage of the duplex drugs is that upon enzymatic cleavage of the phosphodiester bonds, a 1-to-1 ratio of nucleoside to nucleotide is obtained (see Figure 7.7-6). Thus, the desired 5′-phosphorylated nucleotide is only formed at maximally 50%. Additionally, one of the two nucleosides has to be transformed into a lipophilic derivative without loss of antitumor or antiviral activity. On the other hand, it was shown that the lipophilic derivatization of nucleosides can result in enhanced activity and modulation of cell specificity [106]. The disadvantage of obtaining only a 50% yield of 5′-phosphorylated nucleoside analogs, which results from the enzymatic cleavage of the duplex drugs, can be avoided by the synthesis of glycerol–lipid–heteronucleotides, the so-called “multiplex drugs.” Figure 7.7-7 shows an example of such a compound where the two cytotoxic nucleosides ara-C and 5-FdU are linked at their 5′-hydroxyl groups via a phosphodiester to the 1,3-hydroxy groups of glycerol. To introduce amphiphilic/
NH N O N O O HO
HO P O
NOAC
OH O N O
O O
H N
F 5-FdU
OH
Figure 7.7-6. Duplex drugs. Structure of the anticancer amphiphilic heterodinucleosidyl phosphodiester (Duplex-drug) combining the hydrophilic 5-fluoro-deoxyuridine (5-FdU) with the lipophilic N4 -octadecylarabino-furanosylcytosine (NOAC). Upon enzymatic hydrolysis of the duplex drugs, different anticancer nucleotides and nucleosides are formed with additive or synergistic activities.
1166
DELIVERY SYSTEMS O F NH O O
P O
N
O
5-FdU
O
OH OH O
NH2
C18H37 N O
O P O OH
N O O HO
Ara-C
OH
Figure 7.7-7. Multiplex drugs. Example of an amphiphilic glyceryllipid-heterodinucleotide (Multiplex drug). The two hydrophilic nucleoside-5′-monophosphates p5-FdU and paraC are esterified with the terminal hydroxyl residues of the lipophilic 2-octadecylglycerol. By the enzymatic cleavage, several different anticancer metabolites with additive or synergistic activities are obtained.
lipophilic properties, an octadecyl chain is coupled to the 2-hydroxy position of glycerol. After metabolic degradation by phosphodiesterase, both nucleosides are released as 5′-nucleotides. In case of different hydrolysis kinetics, other active intermediate products may be formed. The structure of the glycerol–lipid–heteronucleotides provides a programmed release of differently active drugs that can penetrate a cell membrane when the cleavage takes place outside of a cell. These compounds may also be formulated in DDS, such as liposomes or micellar systems. In vitro tests of the glycerol–lipid–heteronucleotides shown in Figure 7.7-7 revealed that these multiplex drugs inhibited colony formation of 5-FU sensitive and resistant human colon tumor cell lines and induced dose-dependent apoptosis in colon tumor cells as well as in mouse leukemia cells. No significant difference in the cytotoxicity could be observed between 5-FU sensitive and resistant cells, indicating that the multiplex drugs might be useful for the treatment of 5-FU resistant tumors [111, 112]. The effectiveness of the postulated mechanisms and advantages of the multiplex drugs will have to be elucidated in in vivo experiments. We conclude that the chemical modification of water-soluble molecules by attachment of long alkyl chains and their stable incorporation into the bilayer membranes of small unilamellar liposomes represent a very promising example of taking advantage of the high loading capacity lipid bilayers offer for lipophilic drugs. The combination of chemical modifications of water-soluble drugs with their pharmaceutical formulation in liposomes is a valuable method for the development of novel pharmaceutical preparations not only for the treatment of tumors or infectious diseases, but also for many other disorders.
REFERENCES
7.7.8
1167
OUTLOOK AND FUTURE DIRECTIONS
The development of DDS is a challenging venture that combines research efforts of experts in various areas, including bioengineering, nanotechnology, biomaterials, pharmaceutics, biochemistry, and cell and molecular biology. Specific characteristics of pathological processes and cell or tissue types that are the subject of therapeutic interventions govern the path from target selection to the development of specific DDS formulations. The identification of novel cellular targets, for example, easily accessible vascular endothelial cells, in contrast to tumor cells or other less reachable tissues, will lead to optimized pharmaceutical drug delivery formulations and preparation technologies. Refi nement of DDS to overcome unwanted properties such as toxicity, unspecific tissue distribution, and uncontrolled release of entrapped active molecules will be the major challenges in the field. Future DDS will mostly be based on DNA therapeutics such as DNA, ribozymes, DNAzymes, aptamers, (antisense-) oligonucleotides, small interfering RNAs, and their next-generation analogs and derivatives. The use of highthroughput systems for lead identification will yield many new therapeutic targets. Validation and optimization of these disease targets will provide a tremendous stimulus in developing newer potent molecules and their corresponding delivery systems. Further advances in the study of gene function and identification of singlenucleotide polymorphisms will not only help in fi ne-tuning DNA-based therapeutics but will also fulfi ll the ultimate goal of providing individualized therapies and medicines.
REFERENCES 1. Bangham A (1992). Liposomes: Realizing their promise. Hospital Prac. 27:51–62. 2. Kim S (1993). Liposomes as carriers of cancer chemotherapy. Drugs. 46:618–638. 3. Drummond D C, Meyer O, Hong K, et al. (1999). Optimizing liposomes for delivery of chemotherapeutic agents to solid tumors. Pharmacol. Rev. 51:691–743. 4. Haag R, Kratz F (2006). Polymer therapeutics: Concepts and applications. Angewandte Chemie Internat. Edi. Eng. 45:1198–1215. 5. Tiera M J, Winnik F O, Fernandes J C (2006). Synthetic and natural polycations for gene therapy: state of the art and new perspectives. Curr. Gene Ther. 6:59–71. 6. Patil S D, Rhodes D G, Burgess D J (2005). DNA-based therapeutics and DNA delivery systems: A comprehensive review. AAPS J. 7:E61–E77. 7. Torchilin V P (2006). Recent approaches to intracellular delivery of drugs and DNA and organelle targeting. Annual Rev. Biomedi. Engin. 8:1.1–1.31. 8. Ding B-S, Dziubla T, Shuvaev V V, et al. (2006). Advanced drug delivery systems that target the vascular endothelium. Molec. Intervent. 6:98–112. 9. Wei C M, Gibson M, Spear P G, et al. (1981). Construction and isolation of a transmissible retrovirus containing the src gene of Harvey murine sarcoma virus and the thymidine kinase gene of herpes simplex virus type 1. J. Virol. 39:935–944. 10. Young L S, Searle P F, Onion D, et al. (2006). Viral gene therapy strategies: from basic science to clinical application. J. Pathol. 208:299–318. 11. Noureddini S C, Curiel D T (2005). Genetic targeting strategies for adenovirus. Molec. Pharmaceuti. 2:341–347.
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12. Barouch D H (2006). Rational design of gene-based vaccines. J. Pathol. 208:283– 289. 13. Nadeau I, Kamen A (2003). Production of adenovirus vector for gene therapy. Biotechnol. Adv. 20:475–489. 14. Moroziewicz D, Kaufman H L (2005). Gene therapy with poxvirus vectors. Curr. Opin. Molec. Therapeuti. 7:317–325. 15. Argnani R, Lufi no M, Manservigi M, et al. (2005). Replication-competent herpes simplex vectors: design and applications. Gene Ther. 12:S1, S170–S177. 16. Wiznerowicz M, Trono D (2005). Harnessing HIV for therapy, basic research and biotechnology. TRENDS Biotechnol. 23:42–47. 17. Sinn P L, Sauter S L, McCray Jr P B (2005). Gene therapy progress and prospects: Development of improved lentiviral and retroviral vectors—design, biosafety, and production. Gene Ther. 12:1089–1098. 18. Trono D (2000). Lentiviral vectors: turning a deadly foe into a therapeutic agent. Gene Ther. 7:20–23. 19. Vasileva A, Jessberger R (2005) Precise hit: adeno-associated virus in gene targeting. Nat. Rev. Microbiol. 3:837–847. 20. Romano G (2006). The controversial role of adenoviral-derived vectors in gene therapy programs: Where do we stand? Drug News Perspecti. 19:99–106. 21. Kaplan J M (2005). Adenovirus-based cancer gene therapy. Curr. Gene Ther. 5:595– 605. 22. Kanerva A, Hemminki A (2005). Adenoviruses for treatment of cancer. Ann. Med. 37:33–43. 23. Guo Z S, Bartlett D L (2004). Vaccinia as a vector for gene delivery. Expert Opin. Biolog. Ther. 4:901–917. 24. Bartosch B, Cosset F L (2004). Strategies for retargeted gene delivery using vectors derived from lentiviruses. Curr. Gene Ther. 4:427–443. 25. Anderson J L, Hope T J (2005). Intracellular trafficking of retroviral vectors: Obstacles and advances. Gene Ther. 12:1667–1678. 26. Kapturczak M H, Flotte T, Atkinson M A (2001). Adeno-associated virus (AAV) as a vehicle for therapeutic gene delivery: improvements in vector design and viral production enhance potential to prolong graft survival in pancreatic islet cell transplantation for the reversal of type 1 diabetes. Curr. Molec. Med. 1:245–258. 27. Romano G (2005). Current development of adeno-associated viral vectors. Drug News Perspecti. 18:311–316. 28. Vasileva A, Jessberger R (2005). Precise hit: Adeno-associated virus in gene targeting. Nat. Rev. Microbiol. 3:837–847. 29. Torchilin V P (2005). Recent advances with liposomes as pharmaceutical carriers. Nat. Rev. Drug Disc. 4:145–160. 30. Ewert K, Evans H M, Ahmad A, et al. (2005). Lipoplex structures and their distinct cellular pathways. Adv. Genet. 53:119–155. 31. Spagnou S, Miller A D, Keller M (2004). Lipidic carriers of siRNA: differences in the formulation, cellular uptake, and delivery with plasmid DNA. Biochem. 43:13348– 13356. 32. Shuey D J, McCallus D E, Giordano T (2002). RNAi: gene-silencing in therapeutic intervention. Drug Disc. Today. 7:1040–1046. 33. Safi nya C R (2001). Structures of lipid-DNA complexes: supramolecular assembly and gene delivery. Curr. Opin. Struct. Biol. 11:440–448.
REFERENCES
1169
34. Wu G Y, Wu C H (1988). Evidence for targeted gene delivery to Hep G2 hepatoma cells in vitro. Biochem. 27:887–892. 35. Lungwitz U, Breunig M, Blunk T, et al. (2005). Polyethylenimine-based non-viral gene delivery systems. Euro. J. Pharm. Biopharm. 60:247–266. 36. Neu M, Fischer D, Kissel T (2005). Recent advances in rational gene transfer vector design based on poly(ethylene imine) and its derivatives. J. Gene Med. 7: 992–1009. 37. Cho Y W, Kim J D, Park K (2003). Polycation gene delivery systems: escape from endosomes to cytosol. J. Pharm. Pharmacol. 55:721–734. 38. Demeneix B, Behr J P (2005). Polyethylenimine (PEI). Adv. Genet. 53:217–230. 39. Haensler J, Szoka F C Jr (1993). Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconj. Chem. 4:372–379. 40. Eichman J D, Bielinska A U, Kukowska-Latallo J F, et al. (2000). The use of PAMAM dendrimers in the efficient transfer of genetic material into cells. Pharmaceutical Sci. Technol. Today. 3:232–245. 41. Zinselmeyer B H, Mackay S P, Schatzlein A G, et al. (2002). The lower-generation polypropylenimine dendrimers are effective gene-transfer agents. Pharmaceut. Res. 19:960–967. 42. Yoo H, Juliano R L (2000). Enhanced delivery of antisense oligonucleotides with fluorophore-conjugated PAMAM dendrimers. Nucl. Acids Res. 28:4225–4231. 43. Lee K Y, Kwon I C, Kim Y H, et al. (1998). Preparation of chitosan self-aggregates as a gene delivery system. J. Control. Rel. 51:213–220. 44. Prabaharan M, Mano J F (2005). Chitosan-based particles as controlled drug delivery systems. Drug Del. 2:41–57. 45. Shi C, Zhu Y, Ran X, et al. (2006). Therapeutic potential of chitosan and its derivatives in regenerative medicine. J. Surg. Res. 133:185–192. 46. Andre F, Mir L M (2004). DNA electrotransfer: Its principles and an updated review of its therapeutic applications. Gene Ther. S1, S33–42. 47. Li S (2004). Electroporation gene therapy: New developments in vivo and in vitro. Curr. Gene Ther. 4:309–316. 48. Prud’homme G J, Glinka Y, Khan A S, et al. (2006). Electroporation-enhanced nonviral gene transfer for the prevention or treatment of immunological, endocrine and neoplastic diseases. Curr. Gene Ther. 6:243–273. 49. Maitra A (2005). Calcium phosphate nanoparticles: Second-generation nonviral vectors in gene therapy. Exp. Rev. Molec. Diagnos. 5:893–905. 50. Nielsen P E, Egholm M (1999). An introduction to peptide nucleic acid. Curr. Iss. Molec. Biol. 1:89–104. 51. Marin V L, Roy S, Armitage B A (2004). Recent advances in the development of peptide nucleic acid as a gene-targeted drug. Exp. Opin. Biolog. Ther. 4:337–348. 52. Simonson O E, Svahn M G, Tornquist E, et al. (2005). Bioplex technology: Novel synthetic gene delivery pharmaceutical based on peptides anchored to nucleic acids. Curr. Pharmac. Design. 11:3671–3680. 53. Nielsen P E, Egholm M, Buchardt O (1994). Peptide nucleic acid (PNA). A DNA mimic with a peptide backbone. Bioconj. Chem. 5:3–7. 54. Järver P, Langel U (2006). Cell-penerating peptides—A brief introduction. Biochim. Biophys. Acta. 1758:260–263. 55. Wagstaff K M, Jans D A (2006). Protein transduction: cell penetrating peptides and their therapeutic applications. Curr. Med. Chem. 13:1371–1387.
1170
DELIVERY SYSTEMS
56. Torchilin V P, Rammohan R, Weissig V, et al. (2001). TAT peptide on the surface of liposomes affords their efficient intracellular delivery even at low temperature and in the presence of metabolic inhibitors. Proc. Nati. Acad. Sci. USA. 98:8786–8791. 57. Marty C, Meylan C, Schott H, et al. (2004). Enhanced heparan sulfate proteoglycanmediated uptake of cell-penetrating peptide-modified liposomes. Cell. Molec. Life Sci. 61:1785–1794. 58. Almeida J D, Edwards D C, Brand C M, et al. (1975). Formation of virosomes from influenza subunits and liposomes. Lancet. 2:899–901. 59. Kaneda Y (2000). Virosomes: Evolution of the liposome as a targeted drug delivery system. Adv. Drug Del. Rev. 43:197–205. 60. Felnerova D, Viret J F, Gluck R, et al. (2004). Liposomes and virosomes as delivery systems for antigens, nucleic acids and drugs. Curr. Opin. Biotechnol. 15:518–529. 61. Daemen T, de Mare A, Bungener L, et al. (2005). Virosomes for antigen and DNA delivery. Adv. Drug Del. Rev. 57:451–463. 62. Allen T M (2002). Ligand-targeted therapeutics in anticancer therapy. Nat. Rev. Cancer. 2:750–763. 63. Fenske D B, Cullis P R (2005). Entrapment of small molecules and nucleic acid-based drugs in liposomes. Meth. Enzymol. 391:7–40. 64. Sapra P, Tyagi P, Allen T M (2005). Ligand-targeted liposomes for cancer treatment. Curr. Drug Del. 2:369–381. 65. Park J W, Benz C C, Martin F J (2004). Future directions of liposome- and immunoliposome-based cancer therapeutics. Seminars Oncol. 6(suppl 13):196–205. 66. Marty C, Odermatt B, Schott H, et al. (2002). Cytotoxic targeting of F9 teratocarcinoma tumours with anti-ED-B fibronectin scFv antibody modified liposomes. British J. Cancer. 87:106–112. 67. Xu L, Huang C C, Huang W, et al. (2002). Systemic tumor-targeted gene delivery by anti-transferrin receptor scFv-immunoliposomes. Molec. Cancer Ther. 1:337–346. 68. May S, Ben-Shaul A (2004). Modeling of cationic lipid-DNA complexes. Curr. Med. Chem. 11:151–167. 69. Ferrari M E, Rusalov D, Enas J, et al. (2002). Synergy between cationic lipid and colipid determines the macroscopic structure and transfection activity of lipoplexes. Nucleic Acids Res. 30:1808–1816. 70. Tranchant I, Thompson B, Nicolazzi C, et al. (2004). Physicochemical optimisation of plasmid delivery by cationic lipids. J. Gene Med. 6:S24–S35. 71. Zhdanov R I, Podobed O V, Vlassov V V (2002). Cationic lipid-DNA complexeslipoplexes-for gene transfer and therapy. Bioelectrochem. 58:53–64. 72. Matsuura M, Yamazaki Y, Sugiyama M, et al. (2003). Polycation liposome-mediated gene transfer in vivo. Biochim. Biophys. Acta. 1612:136–143. 73. Zhang J S, Liu F, Huang L (2005). Implications of pharmacokinetic behavior of lipoplex for its inflammatory toxicity. Adv. Drug Del. Rev. 57:689–698. 74. Freimark B D, Blezinger H P, Florack V J, et al. (1998) Cationic lipids enhance cytokine and cell influx levels in the lung following administration of plasmid: cationic lipid complexes. J. Immunol. 160:4580–4586. 75. Yu W, Pirollo K F, Rait A, et al. (2004). A sterically stabilized immunolipoplex for systemic administration of a therapeutic gene. Gene Ther. 11:1434–1440. 76. Audouy S, Molema G, De Leij L, et al. (2000). Serum as a modulator of lipoplexmediated gene transfection: dependence of amphiphile, cell type and complex stability. J. Gene Med. 2:465–476.
REFERENCES
1171
77. Son K K, Tkach D, Hall K J (2000). Efficient in vivo gene delivery by the negatively charged complexes of cationic liposomes and plasmid DNA. Biochim. Biophys. Acta. 1468:6–10. 78. Lakkaraju A, Dubinsky J M, Low W C, et al. (2001). Neurons are protected from excitotoxic death by p53 antisense oligonucleotides delivered in anionic liposomes. J. Biol. Chem. 276:32000–32007. 79. Rothenfusser S, Tuma E, Wagner M, et al. (2003). Recent advances in immunostimulatory CpG oligonucleotides. Curr. Opin. Molec. Ther. 5:98–106. 80. Wang H, Rayburn E, Zhang R (2005). Synthetic oligodeoxynucleotides containing deoxycytidyl-deoxyguanosine dinucleotides (CpG ODNs) and modified analogs as novel anticancer therapeutics. Curr. Pharmaceu. Des. 11:2889–2907. 81. Chen W C, Huang L (2005). Non-viral vector as vaccine carrier. Adv. Gene. 54: 315–337. 82. Ludewig B, Barchiesi F, Pericin M, et al. (2001). In vivo antigen loading and activation of dendritic cells via a liposomal peptide vaccine mediates protective antiviral and anti-tumour immunity. Vaccine. 19:23–32. 83. Engler O B, Schwendener R A, Dai Wen J, et al. (2004). A liposomal peptide vaccine inducing CD8+ T cells in HLA-A2.1 transgenic mice, which recognise human cells encoding hepatitis C virus (HCV) proteins. Vaccine. 23:58–68. 84. Zhou H, Liu D, Liang C (2004). Challenges and strategies: The immune responses in gene therapy. Med. Res. Rev. 24:748–761. 85. Storni T, Kündig T M, Senti G, et al. (2005). Immunity in response to particulate antigen-delivery systems. Adv. Drug Del. Rev. 57:333–355. 86. Rose P G (2005). Pegylated liposomal doxorubicin: optimizing the dosing schedule in ovarian cancer. Oncol. 10:205–214. 87. Cattel L, Ceruti M, Dosio F (2004). From conventional to stealth liposomes: A new Frontier in cancer chemotherapy. J. Chemother. S4:94–97. 88. Straubinger R M, Arnold R D, Zhou R, et al. (2004). Antivascular and antitumor activities of liposome-associated drugs. Anticancer Res. 24(2A):397–404.R 89. Immordino M L, Brusa P, Rocco F, et al. (2004). Preparation, characterization, cytotoxicity and pharmacokinetics of liposomes containing lipophilic gemcitabine prodrugs. J. Control. Rel. 100:331–346. 90. Bergman A M, Kuiper C M, Noordhuis P, et al. (2004). Antiproliferative activity and mechanism of action of fatty acid derivatives of gemcitabine in leukemia and solid tumor cell lines and in human xenografts. Nucleosides Nucleot. Nucleic Acids. 23:1329–1333. 91. Harrington K J, Syrigos K N, Uster P S, et al. (2004). Targeted radiosensiti-sation by pegylated liposome-encapsulated 3′, 5′-O-dipalmitoyl 5-iodo-2′-deoxyuridine in a head and neck cancer xenograft model. British J. Cancer. 91:366–373. 92. Pignatello R, Puleo A, Puglisi G, et al. (2003). Effect of liposomal delivery on in vitro antitumor activity of lipophilic conjugates of methotrexate with lipoamino acids. Drug Del. 10:95–100. 93. Stevens P J, Sekido M, Lee R J (2004). A folate receptor-targeted lipid nanoparticle formulation for a lipophilic paclitaxel prodrug. Pharmaceu. Res. 21:2153–2157. 94. Lundberg B B, Risovic V, Ramaswamy M, et al. (2003). A lipophilic paclitaxel derivative incorporated in a lipid emulsion for parenteral administration. J. Control. Rel. 86:93–100. 95. Bergman A M, Kuiper C M, Myhren F, et al. (2004). Antiproliferative activity and mechanism of action of fatty acid derivatives of arabinosylcytosine (ara-C) in leukemia and solid tumor cell lines. Nucleosides Nucleot. Nucleic Acids. 23:1523–1526.
1172
DELIVERY SYSTEMS
96. Lopez-Barcons L A, Zhang J, Siriwitayawan G, et al. (2004). The novel highly lipophilic topoisomerase I inhibitor DB67 is effective in the treatment of liver metastases of murine CT-26 colon carcinoma. Neoplasia. 6:457–467. 97. Fahr A, van Hoogevest P, May S, et al. (2005). Transfer of lipophilic drugs between liposomal membranes and biological interfaces: consequences for drug delivery. Euro. J. Pharmaceu. Sci. 26:251–265. 98. Strickley R G (2004). Solubilizing excipients in oral and injectable formulations. Pharmaceu. Res. 21:201–230. 99. ten Tije A J, Verweij J, Loos W J, et al. (2003). Pharmacological effects of formulation vehicles: Implications for cancer chemotherapy. Clin. Pharmacokin. 42:665–685. 100. Krise J P, Stella V J (1996) Prodrugs of phosphates, phosphonates, and phosphinates. Adv. Drug Del. Rev. 19:287–310. 101. Wagner C R, Iyer V V, McIntee E J (2000). Pronucleotides: Toward the in vivo delivery of antiviral and anticancer nucleotides. Med. Res. Rev. 20:417–451. 102. Schwendener R A, Schott H (2005). Lipophilic arabinofuranosyl cytosine derivatives in liposomes. Meth. Enzymol. 391:58–70. 103. Hamada A, Kawaguchi T, Nakano M (2002). Clinical pharmacokinetics of cytarabine formulations. Clinical Pharmakinet. 41:705–718. 104. Schwendener R A, Schott H (1996). Lipophilic 1-β-D-arabino-furanosylcytosine derivatives in liposomal formulations for oral and parenteral antileukemic therapy in the murine L1210 leukemia model. J. Cancer Res. Clini. Oncol. 122:723–726. 105. Schwendener R A, Friedl K, Depenbrock H, et al. (2001). In vitro activity of liposomal N4octadecyl-1-β-D-arabino-furanosylcytosine (NOAC), a new lipophilic derivative of 1-β-D-arabino-furanocylcytosine on biopsized clonogenic human tumor cells and hematopoietic precursor cells. Investigat. New Drugs. 19:203–210. 106. Cattaneo-Pangrazzi R M C, Schott H, Wunderli-Allenspach H, et al. (2000). The novel heterodinucleoside dimer 5-FdU-NOAC is a potent cytotoxic drug and a p53independent inducer of apoptosis in the androgen-independent human prostate cancer cell lines PC-3 and DU-145. Prostate. 45:8–18. 107. Cattaneo-Pangrazzi R M C, Schott H, Wunderli-Allenspach H, et al. (2000). New amphiphilic heterodinucleoside phosphate dimers of 5-fluorodeoxyuridine (5FdUrd): Cell cycle dependent cytotoxicity and induction of apoptosis in PC-3 prostate tumor cells. Biochem. Pharmacol. 60:1887–1896. 108. Takatori S, Kanda H, Takenaka K, et al. (1999). Antitumor mechanisms and metabolism of the novel antitumor nucleoside analogues, 1-(3-C-ethynyl-β-D-ribopentofuranosyl)cytosine and 1-(3-C-ethynyl-β-D-ribopento-furanosyl)-uracil. Cancer Chemother. Pharmacol. 44:97–104. 109. Tabata S, Tanaka M, Endo Y, et al. (1997). Anti-tumor mechanisms of 3′-ethynyluridine and 3′-ethynylcytidine as RNA synthesis inhibitors: Development and characterization of 3′-etyhynyluridine-resistant cells. Cancer Lett. 116:225–231. 110. Yokogawa T, Naito T, Kanda H, et al. (2005). Inhibitory mechanisms of 1-(3C-ethynyl-β-D-ribopentofuranosyl)uracil (EUrd) on RNA synthesis. Nucleosides Nucleot. Nucleic Acids. 24:227–232. 111. Ludwig P S, Schwendener R A, Schott H (2005). Synthesis and anticancer activities of amphiphilic 5-fluoro-2′-deoxyuridylic acid prodrugs. Euro. J. Med. Chem. 40:494– 504. 112. Saiko P, Horvarth Z, Bauer W, et al. (2004). In vitro and in vivo antitumor activity of novel amphiphilic dimers consiting 5-fluorodeoxyuridine and arabinofuranosylcytosine. Internat. J. Oncol. 25:357–364.
