**9. Concluding remarks**

568 Non-Viral Gene Therapy

(a mixture of Pluronic L61 and F127; also called as SP1017) has been reported to deliver plasmid DNA in skeletal and cardiac muscle, as well as in solid tumors. Unlike other polycations, Pluronic® does not bind and condense the nucleic acids, it does not protect DNA from degradation or facilitate transport of the DNA into the cell and its effects involve transcriptional activation of gene expression (Kabanov, 2006). The effect of Pluronic® was reported to be related to the activation of gene expression by activating the NF-κB and p53 signaling pathways, in which pro-apoptotic AP-l gene that is frequently regulated by the NF-κB system, was not responsive. This, perhaps, indicates that Pluronic-mediated influence on transcription is selective and it is not a result of a general nonspecific activation of immune defense system such as NO-mediated burst (Kabanov, 2006). Nonetheless, to ensure about this supposition, it is essential to recruit global gene expression screening methods such as microarray technology as we have witnessed dramatic alterations in gene expression *in vitro* and *in vivo* upon treatment with different polymers using microarray technology. Kabanov's group has reported that Pluronic block copolymers interact with biomembranes and induce gene expressions through mechanisms that differ from the delivery of the DNA into the cell. They also questioned whether upregulation of expression of genes delivered into cells can also take place by other nonviral polymer-based gene delivery systems? We have observed that various polymers, in particular polycations, are able to alter gene expressions related to immune response and cell defense (Barar et al.,

It appears that the cytotoxicity of nonviral vectors is largely dependent upon the cationic nature of the vector, which attains different level to different structural architecture. For cationic lipid, the cytotoxic effects are mainly determined by the structure of its hydrophilic group (Prokop & Davidson, 2008), e.g. the quaternary ammonium amphiphiles are more toxic than their tertiary amine counterparts. Such toxicity (due to positive charge of the head group) can be reduced by importing a heterocyclic ring such

The biodegradability potential of the advanced nanobiomaterials are also determined their toxicity. For example, poly(lactic-co-glycolic acid) nanoparticles elicit very low level of cytotoxicity and toxicogenomic compared to cationic polymers, but not the modified PLGA-

Surprisingly, the effect of hydrophobic chain on toxicity has not been adequately addressed to date even though it is deemed that the hydrophobic moieties may disrupt the integrity of lipid bilayer. Like cationic lipid, cationic polymers with acid-labile linkage can be rapidly degraded and less toxic. It has been reported that the toxicity of polymers (e.g., PEI, PLL or dendrimers) increases with high molecular weight (Bieber & Elsasser, 2001). Polymers synthesized by linking low molecular weight with acid-labile show low toxicity (Li et al., 2004). The creation of amphiphilic cationic polymer based on PEI or PLL, by linking PEG or other groups, reduces toxicity without compromising the gene delivery efficiency (Zhang et

Upon our observations the biodegradable cationic polymers (e.g., polysaccharides) which display high degree of biodegradability possess low toxicity, thus we speculate that they may be extensively used for *in vivo* transfection in the future. Further, high transfection efficiency and low toxicity can be obtained by the addition of co-lipids or co-polymers (PEGylation). Water soluble lipopolymer, to combine the advantages of both cationic polymer and liposome, seems to be our next approach for optimized gene transfer. Besides, adding cell-specific biomolecules (e.g. aptamer, peptide ligands, antibodies or nanobodies)

2009; Hollins et al., 2007; Omidi et al., 2008).

grafted poly(L-lysine) nanosystems (Omidi & Davaran, 2011).

as imidazolium or pyridinium.

al., 2008).

Synthetic lipids or polymers used for gene delivery may impose selective "phenotypic effects" in cells by affecting cell signaling involved in various biological functions such as cell defense, inflammation, differentiation, proliferation and apoptosis. It is believed that these effects result basically from their interactions with cell membranes, intracellular organelles and subcellular biomolecules, as a result the target cells can respond to these effects phenotypically or genotypically. In some cases, these effects can be relatively benign as they do not induce sever cytotoxic effects, while in the case of nonviral cationic vectors it is not the case since the interaction of the polycationic gene delivery nanosystems with target cells is significantly greater than non-cationic polymers. It is now deemed that one unifying property of polycationic gene delivery nanosystems is their potential to interact with cellular/subcellular biomolecules, upon which profound changes in various cell processes may occur. From this standpoint, it becomes clear that these polycations are able to penetrate into cells and reach different critical subcellular targets and induce inventible biological functions, for which the nanoscaled range of sizes is an important factor. Different cell types as biological targets may response differently, and even modify the activities of such nanomaterials. While the genome-based therapeutics (e.g., oligonucleotides and gene silencing siRNAs) have already been lined up for clinical trials (up to 1700 trials), our knowledge is lacking upon genomic signature of such gene based medicines. As concluding statement, it is suggested that the inadvertent intrinsic genomic signature of nonviral delivery systems should be assessed and taken into consideration for a gene therapy trial since gene silencing/stimulation experiments are to target a specific gene while the gene delivery system may potentially mask or interfere with the desired genotype and/or phenotype end-point of gene therapy. The upregulation or downregulation of genes induced by gene delivery systems or any other drug carriers and excipients appears to instigate a new directionality such as "functional excipients". But, this approach simply represents the gene expression changes which are solely based on intensities of expressed genes for various signaling pathways, while we should look for ways to correlate such gene expression intensities with functional genomics.
