**2. Non viral gene delivery systems**

#### **2.1. Naked DNA**

Naked DNA such as plasmids remain popular as vectors for gene therapy today for their low immunogenicity and low risk of causing insertional mutagenesis. However, their episomal feature resulting in transient gene expression makes them unsuitable as gene therapy vectors when long-term gene expression is needed for treatment. DNA transposons have the proper‐ ties of naked DNA and plasmids as well as the ability to insert transgenes into host chromo‐ somes for long-term expression. DNA transposons are natural genetic elements residing in the genome as repetitive sequences that move through a direct cut-and-paste mechanism.

A simple transposon is characterized by terminal inverted repeats flanking a gene encoding transposase, an enzyme required for its translocation (Meir et al., 2011). The cut-and-paste process, called transposition, makes DNA transposons particularly attractive as gene delivery tools. To turn DNA transposons into a gene delivery tool, a two-plasmid system, consisting of a helper plasmid expressing the transposase and a donor plasmid with the terminal repeat sequences flanking genes of interest, has been developed. Using this system, transposons have been utilized extensively as genetic tools in invertebrates and in plants for transgenesis and insertional mutagenesis (Spradling et al., 1982; Hayes et al., 2003).

Plasmid DNA is an attractive alternative due to its inherent simplicity and because it can easily be produced in bacteria and manipulated using standard recombinant DNA techniques. It shows very little dissemination and transfection at distant sites following delivery and can be re-administered multiple times into mammals without inducing an antibody response against itself (Jiao et al., 1992). Also, considerable long term foreign gene expression from naked plasmid DNA (pDNA) is possible even without chromosome integration if the target cell is post-mitotic or has low mitotic rate and if an immune reaction against the foreign protein is not generated (Herweijer *et al*., 2001; Wolff *et al*., 1992). The poor expression levels represent a major constraint in the use of these vectors for gene transfer/therapy. However, the low efficient expression by direct injection of naked plasmids was improved by ballistic technol‐ ogy, cationic lipids and neutral polymers (Prud'homme et al., 2001; Gao et al., 1995; Lemieux et al., 2000) and most efficiently by electroporation (Sandri et al, 2003), an established technol‐ ogy that transiently permeabilizes cell membrane by short voltage pulse, allowing the uptake of a wide spectrum of biological molecules.

The stability of DNA vectors with high-molecular-weight is a central point for the improve‐ ment of gene delivery. All high-molecular-weight DNA vectors are susceptible to damage. The self-compacting option (self-entangling) can be defined as the folding of single DNA molecules into a configuration with mutual restriction by the individual segments of bent DNA. A negatively charged phosphate backbone makes DNA self-repulsive, so it is reasonable to assume that a certain number of 'sticky points' dispersed within DNA could facilitate the entangling. Tolmachov, proposes that the spontaneous entanglement of vector DNA can be enhanced by the interlacing of the DNA with sites capable of mutual transient attachment through the formation of non-B-DNA forms, such as interacting cruciform structures, intersegment triplexes, slipped-strand DNA, left-handed duplexes (Z-forms) or G-quadruplexes. (Tolmachov, 2012).

#### **2.2. Liposomes**

**•** Novel systems were the viral and non-viral methods merge will be also addressed.

166 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

Naked DNA such as plasmids remain popular as vectors for gene therapy today for their low immunogenicity and low risk of causing insertional mutagenesis. However, their episomal feature resulting in transient gene expression makes them unsuitable as gene therapy vectors when long-term gene expression is needed for treatment. DNA transposons have the proper‐ ties of naked DNA and plasmids as well as the ability to insert transgenes into host chromo‐ somes for long-term expression. DNA transposons are natural genetic elements residing in the genome as repetitive sequences that move through a direct cut-and-paste mechanism.

A simple transposon is characterized by terminal inverted repeats flanking a gene encoding transposase, an enzyme required for its translocation (Meir et al., 2011). The cut-and-paste process, called transposition, makes DNA transposons particularly attractive as gene delivery tools. To turn DNA transposons into a gene delivery tool, a two-plasmid system, consisting of a helper plasmid expressing the transposase and a donor plasmid with the terminal repeat sequences flanking genes of interest, has been developed. Using this system, transposons have been utilized extensively as genetic tools in invertebrates and in plants for transgenesis and

Plasmid DNA is an attractive alternative due to its inherent simplicity and because it can easily be produced in bacteria and manipulated using standard recombinant DNA techniques. It shows very little dissemination and transfection at distant sites following delivery and can be re-administered multiple times into mammals without inducing an antibody response against itself (Jiao et al., 1992). Also, considerable long term foreign gene expression from naked plasmid DNA (pDNA) is possible even without chromosome integration if the target cell is post-mitotic or has low mitotic rate and if an immune reaction against the foreign protein is not generated (Herweijer *et al*., 2001; Wolff *et al*., 1992). The poor expression levels represent a major constraint in the use of these vectors for gene transfer/therapy. However, the low efficient expression by direct injection of naked plasmids was improved by ballistic technol‐ ogy, cationic lipids and neutral polymers (Prud'homme et al., 2001; Gao et al., 1995; Lemieux et al., 2000) and most efficiently by electroporation (Sandri et al, 2003), an established technol‐ ogy that transiently permeabilizes cell membrane by short voltage pulse, allowing the uptake

The stability of DNA vectors with high-molecular-weight is a central point for the improve‐ ment of gene delivery. All high-molecular-weight DNA vectors are susceptible to damage. The self-compacting option (self-entangling) can be defined as the folding of single DNA molecules into a configuration with mutual restriction by the individual segments of bent DNA. A negatively charged phosphate backbone makes DNA self-repulsive, so it is reasonable to assume that a certain number of 'sticky points' dispersed within DNA could facilitate the

insertional mutagenesis (Spradling et al., 1982; Hayes et al., 2003).

of a wide spectrum of biological molecules.

**2. Non viral gene delivery systems**

**2.1. Naked DNA**

The liposomes, lipids arranged in lamellar structures, are concentric bilayered vesicles surrounded by a phospholipid membrane. They are related to micelles which are generally composed of a monolayer of lipids. The amphiphilic nature of liposomes, their ease of surface modification, and a good biocompatibility profile make them an appealing solution for increasing the circulating half-life of peptides, proteins, cDNAs and siRNAs (Bhavsar et al., 2012). They may contain hydrophilic compounds, which remain encapsulated in the aqueous interior, or hydrophobic compounds, which may escape encapsulation through diffusion out of the phospholipid membrane (Figure 1).

**Figure 1.** Non-viral gene delivery using lipoplexes. DNA is complexed with cationic liposomes and is internalized through receptor mediated endocytosis. After their internalization large amounts of complexes are degraded in the endolysosomal compartments. Only a small fraction enters into the nucleus and elicits desired gene expression. (From: Pankajakshan Divya and Devendra K. Agrawal, 2013).

Liposomes can be designed to adhere to cellular membranes to deliver drugs or cDNAs after endocytosis (Bangham et al., 1995). The first formulation was prepared in 1986 by the Christian Dior laboratories in collaboration with the Pasteur Institute (Bangham et al., 1995). Liposomes have been used as delivery systems due to their versatility, charge and surface functionalities that improve their effectiveness *in vivo*. Presumably, the lack of widespread medical impact is due to their limited biological stability. Hydrophobic nanoparticles such as unmodified liposomes are rapidly cleared via the reticuloendothelial system. Liposome formulations with prolonged circulation time have been obtained upon functionalization with PEG (polyethyle‐ neglycol, a biologically inert polymer). The longer circulation half-life of these PEG-coated liposomes may allow a better control of therapeutic drug delivery (Gabizon A et al, 1988).

complexes (Rockenberger et al., 1999), and by thermal decomposition of iron pentacarbonyl followed by oxidation. When optimized, these methods may afford monodisperse nanopar‐ ticles with sizes ranging from 3 to 20 nm for magnetite and 4 to 16 nm for maghemite (Sun et al., 2002). The circulation times of these particles can be greatly increased simply by hydrophilic surface modification with PEG. Furthermore, iron oxide nanoparticles also display fairly easy surface modification capabilities that presents an attractive prospect for direct drug or

Gene Delivery Systems

169

http://dx.doi.org/10.5772/56869

Nanoparticle-based delivery (NBD) has emerged as a promising approach to improve the

Quantum dots are luminescent nanoparticles typically used for imaging in biological systems. Their primary components —core (cadmium with selenium or tellurium), shell, and coating— give the photochemical properties. Qdots have small size and versatile surface chemistry and offer superb optical properties for real-time monitoring as transport vehicles at both cellular and systemic levels. Qdots offer great potential providing mechanisms for monitoring intracellular and systemic nanocarrier distribution, degradation, drug release, and clearance. They can be manufactured with diameters from a few nanometers to micrometers and a narrow size distribution using techniques requiring high annealing temperatures (Yum et al., 2009). Capping of quantum dots with ZnS has been shown to augment stability and enhance luminescence (Park et al., 2009). However, ZnS capping alone is not sufficient to fully stabilize the core, especially in biological systems. PEGyla‐ tion plays a dual role in increasing biocompatibility and improving the core stability in

Though the direct use of QDots for drug delivery remains questionable due to their potential long-term toxicity, the QDot core can be easily replaced with other organic drug carriers or more biocompatible inorganic contrast agents (such as gold and magnetic nanoparti‐ cles) based on their similar size and surface properties, facilitating translation of well characterized NBD vehicles to the clinic, maintaining NBD imaging capabilities, and potentially providing additional therapeutic functionalities such as photothermal therapy

Magnetofection is a methodology developed in the early 2000's (Scherer et al., 2002). It is based on the association of MNPs with non-viral or viral vectors in order to optimize gene delivery in the presence of a magnetic field, and to concentrate therapeutic complexes in target areas. The association of viral vector-based gene delivery with nanotechnology now offers the possibility to develop more efficient and less invasive gene therapy strategies for a number of

major pathologies and diseases *(See details in Section 3.5 of this chapter).*

biomolecule payload attachment (Longmuir et al., 2006).

*2.3.1. Quantum dots (Qdots)*

biological systems (He et al., 2010).

and magneto-transfection.

*2.3.2. Magnetic nanoparticles (MNPs)*

efficacy and the development of new therapies (Probst et al., 2012).

There are additional unique mesophasic structures of lipids formed as a result of lipid structure polymorphisms, which include cubic-, hexagonal- or sponge-phases. These structures have been utilized for gene, vaccine and drug delivery and provide the advantages of stability and production feasibility compared with liposomes (Shanmugam et al., 2011).

#### **2.3. Nanoparticles and nanotechnology**

Nanotechnology is a rapidly expanding field, encompassing the development of man-made materials in the 5–200 nanometer size range. This dimension vastly exceeds that of standard organic molecules, but its lower range approaches that of many proteins and biological macromolecules. The origin of nanotechnology can be traced back to 1959 when physicist Richard Feynman (1960) recognized the potential of manipulating individual atoms and molecules at the nanometer scale and suggested that materials at this scale possess unique physical properties. The nanotechnology field encompasses concepts and approaches deeply rooted in physics, polymer and colloidal chemistry, pharmaceutics, biomaterials, as well as cell and molecular biology and biophysics. The main theme of nanotechnology is the devel‐ opment and use of nanometer-scale materials that display unique functional properties not shown by bulk materials. Nanomaterials can interact with biological systems at a molecular and supra-molecular level, they can be coated to respond to specific cell environments and even to induce desired physiological responses in cells, while minimizing unwanted side effects. The first practical applications of nanotechnology can be traced to advances in communications, engineering, physics, chemistry, biology, robotics, and medicine. Nanotech‐ nology has been utilized in medicine for therapeutic drug delivery and the development of treatments for a variety of diseases and disorders. An intracellular nanoparticle, consequently, may act as a drug depot within the cell and provide an intracellular sanctuary to protect therapeutic compounds from efflux or degradation.

Several synthetic strategies exist to prepare ferromagnetic iron oxide nanoparticles (Gobe et al., 1983). For maghemite (c-Fe2O3) and magnetite (Fe3O4) nanoparticles, this precipitation technique requires alkalization of a solution of metal salt with subsequent hydrolysis in microemulsions. Additionally, biosynthetic routes exist utilizing "magnetic bacteria"; the resulting nanoparticles typically range from 50 to 100 nm in diameter (Matsunaga et al., 1998). The synthesis of iron oxide nanoparticles has also been achieved by sonochemical decomposition of iron pentacarbonyl (Faraji et al., 2009), thermal decomposition of other iron complexes (Rockenberger et al., 1999), and by thermal decomposition of iron pentacarbonyl followed by oxidation. When optimized, these methods may afford monodisperse nanopar‐ ticles with sizes ranging from 3 to 20 nm for magnetite and 4 to 16 nm for maghemite (Sun et al., 2002). The circulation times of these particles can be greatly increased simply by hydrophilic surface modification with PEG. Furthermore, iron oxide nanoparticles also display fairly easy surface modification capabilities that presents an attractive prospect for direct drug or biomolecule payload attachment (Longmuir et al., 2006).

Nanoparticle-based delivery (NBD) has emerged as a promising approach to improve the efficacy and the development of new therapies (Probst et al., 2012).

#### *2.3.1. Quantum dots (Qdots)*

Liposomes can be designed to adhere to cellular membranes to deliver drugs or cDNAs after endocytosis (Bangham et al., 1995). The first formulation was prepared in 1986 by the Christian Dior laboratories in collaboration with the Pasteur Institute (Bangham et al., 1995). Liposomes have been used as delivery systems due to their versatility, charge and surface functionalities that improve their effectiveness *in vivo*. Presumably, the lack of widespread medical impact is due to their limited biological stability. Hydrophobic nanoparticles such as unmodified liposomes are rapidly cleared via the reticuloendothelial system. Liposome formulations with prolonged circulation time have been obtained upon functionalization with PEG (polyethyle‐ neglycol, a biologically inert polymer). The longer circulation half-life of these PEG-coated liposomes may allow a better control of therapeutic drug delivery (Gabizon A et al, 1988).

There are additional unique mesophasic structures of lipids formed as a result of lipid structure polymorphisms, which include cubic-, hexagonal- or sponge-phases. These structures have been utilized for gene, vaccine and drug delivery and provide the advantages of stability and

Nanotechnology is a rapidly expanding field, encompassing the development of man-made materials in the 5–200 nanometer size range. This dimension vastly exceeds that of standard organic molecules, but its lower range approaches that of many proteins and biological macromolecules. The origin of nanotechnology can be traced back to 1959 when physicist Richard Feynman (1960) recognized the potential of manipulating individual atoms and molecules at the nanometer scale and suggested that materials at this scale possess unique physical properties. The nanotechnology field encompasses concepts and approaches deeply rooted in physics, polymer and colloidal chemistry, pharmaceutics, biomaterials, as well as cell and molecular biology and biophysics. The main theme of nanotechnology is the devel‐ opment and use of nanometer-scale materials that display unique functional properties not shown by bulk materials. Nanomaterials can interact with biological systems at a molecular and supra-molecular level, they can be coated to respond to specific cell environments and even to induce desired physiological responses in cells, while minimizing unwanted side effects. The first practical applications of nanotechnology can be traced to advances in communications, engineering, physics, chemistry, biology, robotics, and medicine. Nanotech‐ nology has been utilized in medicine for therapeutic drug delivery and the development of treatments for a variety of diseases and disorders. An intracellular nanoparticle, consequently, may act as a drug depot within the cell and provide an intracellular sanctuary to protect

Several synthetic strategies exist to prepare ferromagnetic iron oxide nanoparticles (Gobe et al., 1983). For maghemite (c-Fe2O3) and magnetite (Fe3O4) nanoparticles, this precipitation technique requires alkalization of a solution of metal salt with subsequent hydrolysis in microemulsions. Additionally, biosynthetic routes exist utilizing "magnetic bacteria"; the resulting nanoparticles typically range from 50 to 100 nm in diameter (Matsunaga et al., 1998). The synthesis of iron oxide nanoparticles has also been achieved by sonochemical decomposition of iron pentacarbonyl (Faraji et al., 2009), thermal decomposition of other iron

production feasibility compared with liposomes (Shanmugam et al., 2011).

168 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

**2.3. Nanoparticles and nanotechnology**

therapeutic compounds from efflux or degradation.

Quantum dots are luminescent nanoparticles typically used for imaging in biological systems. Their primary components —core (cadmium with selenium or tellurium), shell, and coating— give the photochemical properties. Qdots have small size and versatile surface chemistry and offer superb optical properties for real-time monitoring as transport vehicles at both cellular and systemic levels. Qdots offer great potential providing mechanisms for monitoring intracellular and systemic nanocarrier distribution, degradation, drug release, and clearance. They can be manufactured with diameters from a few nanometers to micrometers and a narrow size distribution using techniques requiring high annealing temperatures (Yum et al., 2009). Capping of quantum dots with ZnS has been shown to augment stability and enhance luminescence (Park et al., 2009). However, ZnS capping alone is not sufficient to fully stabilize the core, especially in biological systems. PEGyla‐ tion plays a dual role in increasing biocompatibility and improving the core stability in biological systems (He et al., 2010).

Though the direct use of QDots for drug delivery remains questionable due to their potential long-term toxicity, the QDot core can be easily replaced with other organic drug carriers or more biocompatible inorganic contrast agents (such as gold and magnetic nanoparti‐ cles) based on their similar size and surface properties, facilitating translation of well characterized NBD vehicles to the clinic, maintaining NBD imaging capabilities, and potentially providing additional therapeutic functionalities such as photothermal therapy and magneto-transfection.

#### *2.3.2. Magnetic nanoparticles (MNPs)*

Magnetofection is a methodology developed in the early 2000's (Scherer et al., 2002). It is based on the association of MNPs with non-viral or viral vectors in order to optimize gene delivery in the presence of a magnetic field, and to concentrate therapeutic complexes in target areas. The association of viral vector-based gene delivery with nanotechnology now offers the possibility to develop more efficient and less invasive gene therapy strategies for a number of major pathologies and diseases *(See details in Section 3.5 of this chapter).*
