**3.1.2 Cationic liposomes**

526 Non-Viral Gene Therapy

chromosome should theoretically result in the disruption of the chromosome, killing the

Fig. 1. Plasmid Integration Events: A miniplasmid that undergoes a single recombination event with the host chromosome should be rare due to the removal of all elements except the gene of interest. Any integration events by: A. A lcc miniplasmid should result in chromosomal disruption that is likely lethal and cannot be replicated or segregated; B. A ccc

Non-viral gene delivery is emerging as a realistic alternative to the use of viral vectors with the potential to have a significant impact on clinical therapies. Synthetic vectors provide flexibility in formulation design and can be tailored to the size and topology of the DNA cargo and the specific route of vector administration, and can be delivered selectively to a specific tissue type through the incorporation of a targeting ligand. Compared to viral vectors, synthetic vectors are potentially less immunogenic, relatively easy to produce in clinically relevant quantities, and are associated with fewer safety concerns (Liu and Huang 2002). As a result of these advantages, their use has rapidly moved from transfection of cell

Synthetic vectors designed for parenteral administration encompass a wide range of formulations. These include unmodified (naked) DNA, which is designed for direct intratissue injection, cationic polymer–DNA complexes, cationic lipid–DNA complexes, and cationic polymer–lipid–DNA ternary complexes (lipopolyplexes), which are mostly aimed at

The simplest employed method of transgene delivery is the injection of naked DNA. It shows very little dissemination and transfection at distant sites following delivery and can be re-administered multiple times into mammals (including primates) without inducing an antibody response against itself (i.e., no anti-DNA antibodies generated) (Wolff and Budker, 2005). The simplicity of this approach is offset by serious limitations such as inefficient uptake of the therapeutic gene into the target cells and rapid clearance of the DNA from the circulation. Direct *in vivo* gene transfer with naked DNA was first demonstrated by efficient transfection of myofibers following injection of mRNA or pDNA into skeletal muscle (Wolff JA, 1990). This novel approach was heralded as a superior method of *in vivo* transfection and its application was later advanced to confer high level expression in hepatocytes in mice by the rapid injection of naked DNA in large volumes into the tail vein (Zhang et al., 1999). This hydrodynamic tail vein (HTV) procedure has proven very useful not only to gene expression studies, but also more recently for the delivery of siRNA (Lewis et al., 2002; McCaffrey et al., 2002). Ultrasoundmediated eruption of polyethyleneglycol (PEG)-modified liposomes loaded with naked plasmid-DNA is also a feasible and efficient technique for gene delivery (Negishi et

miniplasmid can integrate without disrupting the host chromosome.

cultures to clinical cancer gene therapy applications.

systemic administration.

**3.1.1 Naked DNA** 

al. 2010).

cell (see Figure 1).

Cationic liposomes are one of the most efficient, and among the most widely used non-viral vector systems. They are composed of positively charged lipid bilayers that can be complexed to DNA through electrostatic interactions resulting in complexes, termed lipoplexes. The application of cationic liposomes to gene therapy was first described in 1987 by Felgner (Felgner et al. 1987). The lipoplexes are generally composed of a positively charged lipidic component (see Figure 2), such as dioleylpropyltrimethylammonium chloride, dioleoyl triethylammonium propane (DOTAP), or dimethylaminoethane carbamoyl cholesterol (DC-Chol), that is capable of complexing and condensing the DNA (Giatrellis et al. 2009). Most lipoplex formulations include a ''helper'' lipid, such as dioleoylphosphatidyl-ethanolamine (DOPE, as seen in Figure 2), or cholesterol that provides added stability to the lipoplexes and enhances DNA release from endosomal compartments. While transfection activity of lipoplexes is shown to vary depending on their DNA/cationic lipid ratio, it is necessary for them to be positively charged to interact with the negatively charge cell membranes and exhibit transfection activity. Lipoplex uptake occurs through endosome formation (via a number of mechnisms including caveosomes, clathrin dependent, etc.), followed by disruption of the endosomal membrane by fusion with, or incorporation of the lipoplex lipids resulting in DNA release.

The advantages of liposomes in delivery system designs include their simplicity in preparation, ability to complex relatively large amounts of DNA, versatility for use with any size or type of DNA/RNA, ability to transfect non-dividing cells and overall stability (Dutta et al. 2010). In addition, lipids are non-immunogenic allowing for repeated administration without adverse immunologic reaction(Barron et al. 1999). The primary disadvantages of lipoplex delivery vectors include low tumor transfection efficiency and lack of tumor specificity.

In order to enhance gene transfection, based upon structure-activity relationship, various gemini surfactants (Gemini surfactants consist of two hydrophobic chains and two polar headgroups linked chemically by a spacer group) have been designed. In order to enhance gene transfection based upon structure-activity relationship, various gemini surfactants have been designed, synthesized and tested for gene delivery in our laboratory (Donkuru et al. 2010;Wang and Wettig 2011;Wettig et al. 2007a;Wettig et al. 2007b;Wettig and Verrall 2001). Recent reports have shown that obstacles can be overcome by exploiting receptormediated endocytosis for highly efficient internalization of ligands naturally employed by eukaryotic cells. Advances along this line include the conjugation of mAbs ("immunoliposomes"), ligands such as growth factors, or hormones to liposomes to confer targeting capability. Nanoparticle based anti-cancer gene/drug delivery first reached clinical trial in mid 1980s and the first nanomedicine Doxil® (liposomal encapsulated doxorubicin) was marketed in 1995. Other example of liposome-mediated drug delivery include daunorubicin (Daunoxome), which is also currently being marketed as liposome delivery systems. Numerous new nanoparticle based cancer gene therapy systems are under development.

### **3.1.3 Polyplexes**

Polyplexes differ from lipoplexes in that they are comprised of charged complexes of plasmid DNA and a cationic polymer, such as poly-L-lysine (PLL), polyethylenimine, polyamidoamine (starburst) dendrimers, and chitosan with a net positive charge (See Figure 2). Cationic polymers differ from cationic lipids primarily in that they do not contain

Nanomedicine Based Approaches to Cancer Diagonsis and Therapy 529

outer surface of the complexes, reducing hydrophobic interactions with serum proteins and components of the reticulo-endothelial system (RES). In addition to decreasing toxicity of the polyplexes, this strategy also confers increased biocirculation times and therefore, further increases transfection efficiencies. [Please see van Vlerken et al. 2007 for a recent review of PEG modification of nanocarriers]. The presence of the terminal alcohol groups in this PEG outer shell also provides sites for further modification that have been conjugated to various types of targeting ligands, including glycoprotein, transferring, carbohydrates, folate and epidermal growth factors, facilitating tissuespecific gene delivery (Guo and Lee 1999;Han et al. 1999;Lai et al. 2009). Such complexes have been found to mediate efficient gene transfer into tumor cell lines in a receptordependent and cell-cycle-dependent manner. While the modifications described here have resulted in significant improvements in both the transfection efficiencies and toxicity profiles of polycation-based transfection vectors, a number of questions relating to mechanisms of endosome escape (Tros, I et al. 2010), structure-activitiy relationships, pharmacokinetics, and *in-vitro* vs *in-vivo* application remain (Jere et al. 2009). Overall, while proving to be a very promising strategy toward gene therapy design, polyplex systems still require much further testing and improvement prior to entering clinical trials

Lipopolyplexes (lipid-polymer-DNA complexes or LPDs) combine plasmid DNA with both a cationic polymer and liposomes via electrostatic interactions. In general, these vectors are compact particles that exhibit superior colloidal stability, reduced cytotoxicity, and provide elevated transfection efficiency compared to either polyplexes or lipoplexes alone. The cationic polymer may be covalently linked to the liposomes (e.g. lipopolylysine) or be noncovalently incorporated into a ternary lipid–polymer–DNA complex by a charge-mediated self-assembly process. The polycation component facilitates the optimal condensation of plasmid DNA, whereas lipidic components, to which targeting ligands can be attached, further stabilize the vector formulation and mediate the efficient endosomal escape of the vector following cellular internalization. LPD particles prepared using protamine as the cationic polymer and DOTAP/Chol cationic liposomes have been reported to inhibit tumor growth following i.v. administration in mice (Whitmore et al. 1999). Both *in vitro* and *in vivo* studies have demonstrated improved outcomes of (liposomes/protamine/DNA) LPDmediated gene transfer over conventional liposomes (El-Aneed 2004) . It is believed that the small size of LPD (100 to 250 nm, which is almost three to five times less than conventional

lipoplexes) will facilitate endocytosis and increase the *in vivo* circulating half life.

To facilitate efficient gene expression, delivered plasmid DNA must initially circumnavigate various barriers to cellular and nuclear entry as seen in Figure 3. The lipoplex/polyplex must first be internalized by the cell membrane,for which there are many different possible routes including receptor mediated endocytosis, pinocytosis and phagocytosis (Godbey and Mikos 2001). Receptor-mediated endocytosis or clathrindependent internalization is the most common of these and can be exploited to engineer polyplexes to express attached ligands to facilitate this process (Morille et al. 2008).

**4. Challenges in nanoparticle based gene therapy** 

(Midoux et al., 2008).

**3.1.4 Lipopolyplexes** 

a hydrophobic moiety and are completely soluble in water. A wide variety of cationic polymers that transfect cells *in vitro* have been characterized (Midoux et al., 2008). A key determinant of polyplex gene transfer efficiency is the positive (on amine nitrogen atoms in the polymer) to negative charge ratio or the related negative to positive (N/P) ratio. Given their polymeric nature, cationic polymers can be synthesized in different lengths, with different geometry (linear versus branched), and with substitutions or additions of functional groups with relative ease and flexibility, which opens the way to extensive structure/function relationship studies.

Fig. 2. Examples of commonly used lipids (DOPE and DOTAP) and polymers (PEI and poly-L-lysine) in gene therapy.

Polymer-based nanoparticles are now widely used for gene and drug delivery and targeted therapy. One of the most widely applied cationic polymers used for DNA transfections is polyethyleneimine (Choosakoonkriang et al. 2003). DNA complexation with PEI, has not been found to result in an alteration of DNA conformation, remaining essentially in the B form, and the utility of PEI as a gene delivery vector has been demonstrated in numerous studies (Jere et al. 2009;Moore et al. 2009). High molecular weight PEI has been shown to be one of the most successful polymeric vectors due to the large number of protonatable amine groups that result in an enhanced ability to escape from the endosome following uptake by the cell via the so-called "proton sponge" effect. This benefit is contrasted by the high level of cellular toxicity also imparted by the number of amine groups within the polymer. Attempts to overcome this increased level of toxicity have involved using low molecular weight PEI; however, transfection efficiencies are directly correlated with decreases in molecular weight while the tendency to aggregate can increase with decreasing polymer molecular weight. Another successful strategy has involved the shielding of the polyethylenimine/DNA core with a shell of polyethylene glycol (PEG). This approach results in the formation of a dense hydrophilic outer surface of the complexes, reducing hydrophobic interactions with serum proteins and components of the reticulo-endothelial system (RES). In addition to decreasing toxicity of the polyplexes, this strategy also confers increased biocirculation times and therefore, further increases transfection efficiencies. [Please see van Vlerken et al. 2007 for a recent review of PEG modification of nanocarriers]. The presence of the terminal alcohol groups in this PEG outer shell also provides sites for further modification that have been conjugated to various types of targeting ligands, including glycoprotein, transferring, carbohydrates, folate and epidermal growth factors, facilitating tissuespecific gene delivery (Guo and Lee 1999;Han et al. 1999;Lai et al. 2009). Such complexes have been found to mediate efficient gene transfer into tumor cell lines in a receptordependent and cell-cycle-dependent manner. While the modifications described here have resulted in significant improvements in both the transfection efficiencies and toxicity profiles of polycation-based transfection vectors, a number of questions relating to mechanisms of endosome escape (Tros, I et al. 2010), structure-activitiy relationships, pharmacokinetics, and *in-vitro* vs *in-vivo* application remain (Jere et al. 2009). Overall, while proving to be a very promising strategy toward gene therapy design, polyplex systems still require much further testing and improvement prior to entering clinical trials (Midoux et al., 2008).
