**Polyethylenimine (PEI)-based gene carriers**

• PEI has been one of the most popularly employed cationic gene carriers due to its superior transfection efficiency in many different types of cells. The buffering property of PEI leads to protect the DNA from degradation in the endosomal compartment

Non-Viral Delivery Systems in Gene Therapy and Vaccine Development 37

as PLL or PEI [Park et al., 2006]. Some examples include: Poly (α-[4-aminobutyl]-L-glycolic acid) (PAGA), a biodegradable PLL analogue; Poly(β-amino ester)s; Poly(2-aminoethyl propylene phosphate) (PPE-EA); Degradable PEIs; The biodegradable PEIs were synthesized by crosslinking low molecular weight PEI (0.8 kDa) with either PEG-bissuccinimidyl succinate or disulfide-containing cross-linkers, such as dithiobis (succinimidylpropionate) (DSP) and dimethyl 3,3'-dithiobispropionimidate (DTBP) [Park et

Although, cationic polymers, which electrostatically interact with DNA to neutralize its negative charge and condense DNA into nanosized particles, are generally considered as gene carriers, neutral polymers such as polyvinyl alcohol (PVA) and polyvinyl-pyrolidone (PVP) can also be used for gene transfer. They can protect DNA from enzymatic degradation and facilitate cellular uptake of DNA. These polymers may interact with DNA via hydrogen bonding and/or van der Waals interactions; van der Waals interaction of the hydrophobic vinyl backbones may cover around DNA to make its surface more hydrophobic. In a study, intramuscularly administered PVP/plasmid DNA formulation resulted in a significant increase in the number and distribution of the reporter-gene

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

**Neutral and non-condensing polymer-based gene delivery systems** 

expressing cells in rat tibialis, compared to naked plasmid [Park et al., 2006].

al., 2006].

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

PLGA NPs [Lu et al., 2009].

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 different spectra utilizing PEI as described in following sections:


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

#### **Biodegradable polycations**

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 as PLL or PEI [Park et al., 2006]. Some examples include: Poly (α-[4-aminobutyl]-L-glycolic acid) (PAGA), a biodegradable PLL analogue; Poly(β-amino ester)s; Poly(2-aminoethyl propylene phosphate) (PPE-EA); Degradable PEIs; The biodegradable PEIs were synthesized by crosslinking low molecular weight PEI (0.8 kDa) with either PEG-bissuccinimidyl succinate or disulfide-containing cross-linkers, such as dithiobis (succinimidylpropionate) (DSP) and dimethyl 3,3'-dithiobispropionimidate (DTBP) [Park et al., 2006].
