**5. Micro-/nano-particles**

36 Non-Viral Gene Therapy

• **PEI-PEG:** PEI-grafted PEGs (PEI-g-PEG) with different PEG grafting ratios were synthesized to address the cytotoxicity and aggregation problems of PEI. Cell cytotoxicity of PEI-g-PEG was greatly reduced, while the transfection efficiency of PEIg-PEG was still comparable to that of PEI. Cytotoxicity was independent of molecular

weight of PEG but affected by the degree of PEG substitution [Park et al., 2006]. • **PEI conjugates with targeting moieties:** a) PEI-g-PEG-RGD: An angiogenic endothelial cell targeted gene delivery system (PEI-g-PEG-RGD) was developed by incorporating the ανβ3/ανβ5 integrin binding RGD peptide. b) Antibody-conjugated PEIs: A monoclonal antibody against human epidermal growth factor receptor-2 (HER-2) was conjugated to linear PEI for targeted gene transfer to cancer cells. The HER-2 antibody-PEI conjugate showed enhanced transfection efficiency in HER-2 over-expressing human breast adenocarcinoma cells (Sk-Br-3) compared to unmodified PEI [Park et al.,

• **Folate-conjugated PEIs:** Folate-polyethylene glycol-folate-grafted-polyethylenimine (FPF-g-PEI) was synthesized by grafting folate-PEG-folate to PEI. A PEI-PEG-folate (PEI-PEG-FOL) conjugate was used as a carrier for a plasmid encoding small interfering RNA (siRNA) targeting green fluorescence protein (GFP). The complexes between the PEI-PEG-FOL and the siRNA-expressing plasmid showed an efficient suppression of GFP expression compared to unmodified PEI complexes in folate receptor over-

Water-soluble lipopolymer (WSLP): The water-soluble lipopolymer (WSLP) was synthesized by conjugating chlosteryl chloroformate to a low molecular weight PEI (1.8 kDa). WSLP interacts with DNA to form stable colloidal particles (70 nm). The PEI moiety of WSLP confers a buffering effect, which could facilitate endosomal escape of the WSLP/DNA complex. It was also reported that the dodecylation of PEI enhanced the cellular uptake and transfection efficiency. In a similar way, the hydrophobic cholesterol moiety of WSLP would give a chance to form small and stable complexes, resulting in enhanced cellular uptake and transfection efficiency. WSLP showed higher transfection efficiency and much lower cytotoxicity than 25 kDa PEI, suggesting that WSLP has the advantages from PEI as well as from chlosterol. Intratumoral injection of WSLP/p2CMVmIL-12 complexes to tumor-bearing mice showed a significant improvement

The backbone linkages of most polymeric gene carriers consist of a –C-C- bond or amide bond, which are not degraded in physiological solutions. The non-degradable non-viral carriers are not easily removed by physiological clearance systems and therefore, can possibly accumulate within cells or tissues to elicit further cytotoxicity [Park et al., 2006]. To solve the problems, several biodegradable polycations have been synthesized and evaluated as potential gene carriers. Generally, the biodegradable polycations showed much less cytotoxicity and higher transfection efficiency compared to an unmodified polycations, such

expressing cells (KB), which stably expressed GFP [Park et al., 2006].

in the retardation of tumor growth and survival rate [Park et al., 2006].

different spectra utilizing PEI as described in following sections:

2006].

**Biodegradable polycations** 

during the maturation of the endosome to lysosome, facilitating intracellular trafficking of DNA. High cation density of PEI also contributes to the formation of highly condensed particles by interacting with DNA. However, the property may confer significant cytotoxicity. Studies with linear PEIs showed even higher transfection efficiency and lower cytotoxicity compared to branched PEI [Park et al., 2006]. There are

> Another approach to DNA-vaccine delivery involves microparticle-based technologies to target APCs [Ulmer et al., 2006]. Microencapsulation of DNA, or association of DNA with microcapsules, has led to enhancement of CTL responses to encoded proteins [Doria-Rose and Haigwood, 2003]. Biodegradable, non-antigenic poly-lactide polyglycolide (PLGA or PLG) microspheres offer many advantages as a vaccine delivery system. Both cellular and humoral immune responses can be elicited to antigens encapsulated in, or conjugated onto PLG microspheres. Particles used typically range in size from 1 to 10 μm in diameter, a size that is readily phagocytosed by dendritic cells and other antigen-presenting cells (APCs). Microspheres elicit both CD8+ and CD4+ T cell responses by releasing antigen intracellularly [Doria-Rose and Haigwood, 2003]. Biodegradable PLGA nanoparticles (NPs) have been investigated for sustained and targeted/localized delivery of different agents, including drugs, proteins and peptides and recently, plasmid DNA owing to their ability to protect DNA from degradation in endolysosomes. PLGA-based nanotechnology has been widely used in diagnosis and treatment of cancer. These NPs have been shown to stimulate the immune response as measured by an increase in IL-2 and IFN-γ in spleen homogenates [Lu et al., 2009]. The majority of the existing literature involving PLGA polymers has tended to be focused on PLGA microspheres. In the last 10 years, microspheres have been used extensively for the injectable delivery of vaccine antigens, both for viral and bacterial antigens. Similar to microspheres, PLGA NPs have been shown to effectively enhance immune responses. The major obstacle is providing delivery vehicles with the adequate surface molecules for recognition by the immune system and for more effective targeting. It is likely, therefore, that future studies of PLGA NPs as vaccine candidates will focus on improving these features, as recently tested by grafting RGD peptides (arginine–glycine– aspartic acid-containing synthetic peptides) covalently onto PEG moieties on the surface of PLGA NPs [Lu et al., 2009].

Non-Viral Delivery Systems in Gene Therapy and Vaccine Development 39

nodes in a tumor vaccine model. Mice were immunized with a tumor cell–based vaccine that was labeled with SPIO. APCs that had captured SPIO were imaged over time as they accumulated in lymph nodes. It was indicated that MRI is capable of monitoring, *in vivo*, the trafficking of magnetically labeled APCs inducing a tumor-specific immune response, and that these cells can be magnetically recovered *ex vivo*. Excellent correlation was observed between *in vivo* and *ex vivo* quantification of APCs, with resolution sufficient to detect

The studies have shown that a number of peptides and proteins are able to penetrate the cell membrane and enter the cell. It has been observed that many cargo molecules that are covalently attached to these peptides will be translocated into the cell. Recently, various natural and/ or synthetic cell-penetrating peptides (CPP) have known as efficient tools in vaccine design as they are capable of delivering therapeutic targets into cellular compartments. In fact, the cell membrane is impermeable to hydrophilic substances and delivery into cells could be facilitated by linking to CPP. Different cargos such as drugs, peptide/ protein, oligonucleotide/ DNA/ RNA, nanoparticles, liposomes, bacteriophages, fluorescent dyes and quantum dots have been linked to CPPs for intracellular delivery with possible use in future vaccine design [Brooks et al., 2010]. Two applications of CPP already validated in vaccine studies are delivery of tumor-associated antigens into antigenpresenting cells (APCs) and use as a non-viral gene delivery vehicle in DNA vaccines. There are two methods for designing CPP incorporating immunogenic antigens: A) chemical linking via covalent bonds B) coupling via recombinant fusion constructs produced by bacterial expression vectors. The orientation of the peptide and cargo and the type of linkage are likely important [Brooks et al., 2010]. In addition, the utilized CPP, attached cargo, concentration and cell type, all significantly affect the mechanism of internalization. The mechanism of cellular uptake and subsequent processing still remains controversial. It is now apparent that CPP mediate intracellular delivery via both endocytic and non-endocytic pathways [Brooks et al., 2010; Jarver and Langel, 2004; Wagstaff and Jans, 2006]. An attractive feature of using polypeptides as gene delivery vectors is incorporating multiple functional domains into one polypeptide chain, such as a DNA-binding domain linked with a receptor-targeting domain. This kind of polypeptides will recognize and bind to cell surface receptors that are unique to target cells and deliver the bound DNA into the cells through receptor-mediated endocytosis. Therefore, this process may ensure the therapeutic effect in desired cells and limit the potential side effects caused by transgene expression into

Several studies have shown that oligo-deoxynucleotides (ODN) with immune-stimulating sequences (ISS) containing CpG motifs facilitate the priming of MHC class I- restricted CD8+ T cell responses to proteins or peptides. Therefore, ODN/cationic peptide complexes are potent tools for priming CD8+ T cell immunity [Schirmbeck et al., 2003]. The complex formation required electrostatic linkage of the positively charged peptide to the negatively charged ODN. Conjugation of immunostimulatory DNA or ODN to protein antigens facilitates the rapid, long-lasting, and potent induction of cell-mediated immunity. It was shown that ODN (with or without CpG-containing sequences) are potent Th1-promoting adjuvants when bound to cationic peptides covalently linked to antigenic epitopes, a mode of antigen delivery existing in many viral nucleocapsids [Schirmbeck et al., 2003]. Table 3

increased APC trafficking elicited by an adjuvant [Long et al., 2009].

the non-targeted cells [Zeng and Wang, 2005].

**6. Cationic peptides/cell-penetrating peptides (CPP)/trojan peptides** 

These polymers have been designated as feasible candidates for drug delivery systems, anticancer agents and vaccine immunotherapy. For example, DNA vaccine delivery to APCs has been facilitated by microencapsulation of plasmid DNA, which encodes HPV E6/E7 antigenic proteins. The capsule is formed from polymeric PGLA microparticles. These resulting microparticles have a greater propensity toward APC uptake compared to naked DNA. This technique allows HPV DNA plasmid to be condensed inside the microparticle. The physical and chemical properties of the PGLA scaffold make DNA inaccessible to nuclease and preventing degradation, allowing for a sustained release of DNA and enhancing transfection efficiency *in vitro* [Lin et al., 2010]. In mice, microspheres containing HPV plasmid encoding HPV E6/E7 antigens have been shown to elicit a strong antigenspecific cytotoxic T cell response. Using this technology, microencapsulated DNA vaccine termed ZYC-101 encoding multiple HLA-A2 restricted HPV E7 epitopes has undergone Phase I trials in patients with CIN2/3 lesions and high-grade anal intraepithelial neoplasia. In both trials, intramuscularly administered vaccine was well tolerated, and in some patients had resulted in histological regression of the lesions as well as generation of E7-specific IFNγ expressing T cells. A newer version of the DNA vaccine, ZYC-101a, which encodes HPV16 and HPV18 E6- and E7-derived epitopes has been used in phase II clinical trial in patients with CIN 2/3 lesions [Lin et al., 2010].

The multi-functional nano-devices based on the dendritic polymer or dendrimers are also being applied to a variety of cancer therapies to improve their safety and efficacy. Technical advances have been focused on the development of a linking strategy that allows the dendrimer molecules to be linked via complementary oligonucleotides. At present, further applications of dendrimers in photodynamic therapy, boron neutron capture therapy, and gene therapy for cancer are being examined [Baker, 2009].

Recently, the modified fluorescent nanoparticles have been synthesized as a targeting and delivery system, by conjugating both tumor targeting agent and chemokines to the nanoparticles, in order to attract immune cells toward tumor cells. Biodegradable chitosan nanoparticles encapsulating quantum dots were prepared, with suitable surface modification to immobilize both tumor targeting agent and chemokine on their surfaces [Chatterjee and Zhang, 2007]. Fluorescent chitosan coated quantum dots (QDs) were used to act as bi-functional bridging units between cancer and immune cells. This nanoparticulate form of delivery promises the advantages of enhanced tumor selectivity and longer halflives, thereby enhancing effectiveness of the immune response and reduction in systemic toxicity [Chatterjee and Zhang, 2007]. Furthermore, the rapid development of Quantum Dots (QDs) technology has already fulfilled some of the hopes of developing new, more effective cancer-imaging probes. First, stable encapsulation of QDs with amphiphilic polymers has prevented the quenching of QD fluorescence in the aqueous *in vivo* environment. Second, QDs are relatively inert and stable. Finally, successful conjugation of QDs with biomolecules has probably made active targeting them to tumors. Despite their success so far in cancer imaging, there are challenges in enhancing sensitivity, maximizing specificity and minimizing toxicity of QDs, which must be undertaken before clinical applications can proceed [Zhang et al., 2008]. A major parameter limiting immune responses to vaccination is the number of activated APCs that capture antigen and migrate to draining lymph nodes. The use of cellular magnetic resonance imaging (MRI) is a promising approach for this purpose [Long et al., 2009]. In a study, an *in vivo* labeling method was described, which relies upon cell-to-cell transfer of super-paramagnetic iron oxide (SPIO) from tumor cells to endogenous APCs, *in situ*, for quantification of APC delivery to lymph

These polymers have been designated as feasible candidates for drug delivery systems, anticancer agents and vaccine immunotherapy. For example, DNA vaccine delivery to APCs has been facilitated by microencapsulation of plasmid DNA, which encodes HPV E6/E7 antigenic proteins. The capsule is formed from polymeric PGLA microparticles. These resulting microparticles have a greater propensity toward APC uptake compared to naked DNA. This technique allows HPV DNA plasmid to be condensed inside the microparticle. The physical and chemical properties of the PGLA scaffold make DNA inaccessible to nuclease and preventing degradation, allowing for a sustained release of DNA and enhancing transfection efficiency *in vitro* [Lin et al., 2010]. In mice, microspheres containing HPV plasmid encoding HPV E6/E7 antigens have been shown to elicit a strong antigenspecific cytotoxic T cell response. Using this technology, microencapsulated DNA vaccine termed ZYC-101 encoding multiple HLA-A2 restricted HPV E7 epitopes has undergone Phase I trials in patients with CIN2/3 lesions and high-grade anal intraepithelial neoplasia. In both trials, intramuscularly administered vaccine was well tolerated, and in some patients had resulted in histological regression of the lesions as well as generation of E7-specific IFNγ expressing T cells. A newer version of the DNA vaccine, ZYC-101a, which encodes HPV16 and HPV18 E6- and E7-derived epitopes has been used in phase II clinical trial in patients

The multi-functional nano-devices based on the dendritic polymer or dendrimers are also being applied to a variety of cancer therapies to improve their safety and efficacy. Technical advances have been focused on the development of a linking strategy that allows the dendrimer molecules to be linked via complementary oligonucleotides. At present, further applications of dendrimers in photodynamic therapy, boron neutron capture therapy, and

Recently, the modified fluorescent nanoparticles have been synthesized as a targeting and delivery system, by conjugating both tumor targeting agent and chemokines to the nanoparticles, in order to attract immune cells toward tumor cells. Biodegradable chitosan nanoparticles encapsulating quantum dots were prepared, with suitable surface modification to immobilize both tumor targeting agent and chemokine on their surfaces [Chatterjee and Zhang, 2007]. Fluorescent chitosan coated quantum dots (QDs) were used to act as bi-functional bridging units between cancer and immune cells. This nanoparticulate form of delivery promises the advantages of enhanced tumor selectivity and longer halflives, thereby enhancing effectiveness of the immune response and reduction in systemic toxicity [Chatterjee and Zhang, 2007]. Furthermore, the rapid development of Quantum Dots (QDs) technology has already fulfilled some of the hopes of developing new, more effective cancer-imaging probes. First, stable encapsulation of QDs with amphiphilic polymers has prevented the quenching of QD fluorescence in the aqueous *in vivo* environment. Second, QDs are relatively inert and stable. Finally, successful conjugation of QDs with biomolecules has probably made active targeting them to tumors. Despite their success so far in cancer imaging, there are challenges in enhancing sensitivity, maximizing specificity and minimizing toxicity of QDs, which must be undertaken before clinical applications can proceed [Zhang et al., 2008]. A major parameter limiting immune responses to vaccination is the number of activated APCs that capture antigen and migrate to draining lymph nodes. The use of cellular magnetic resonance imaging (MRI) is a promising approach for this purpose [Long et al., 2009]. In a study, an *in vivo* labeling method was described, which relies upon cell-to-cell transfer of super-paramagnetic iron oxide (SPIO) from tumor cells to endogenous APCs, *in situ*, for quantification of APC delivery to lymph

with CIN 2/3 lesions [Lin et al., 2010].

gene therapy for cancer are being examined [Baker, 2009].

nodes in a tumor vaccine model. Mice were immunized with a tumor cell–based vaccine that was labeled with SPIO. APCs that had captured SPIO were imaged over time as they accumulated in lymph nodes. It was indicated that MRI is capable of monitoring, *in vivo*, the trafficking of magnetically labeled APCs inducing a tumor-specific immune response, and that these cells can be magnetically recovered *ex vivo*. Excellent correlation was observed between *in vivo* and *ex vivo* quantification of APCs, with resolution sufficient to detect increased APC trafficking elicited by an adjuvant [Long et al., 2009].
