**8. Genomic impacts of cationic polymers**

Despite plethora of investigations on application of polymers in drug/gene delivery, surprisingly, little attention has been devoted about possible biofunction of polymer *per se* in particular genomic effects. Many researchers have now consensus upon functionalities of polymers, and accordingly new domains of polymer science such as "polymer genomics", "polymer genocompatibly" and "polymer genotoxicity" have been arisen. To examine the polymer genocompatibly concept, we have previously reported that starburst PAMAM dendrimers (i.e., PF and SF) as well as polypropylene imine (PPI) dendrimers (e.g., DAB8 and DAB16) can inadvertently induce alterations in gene expression (Hollins et al., 2007; Omidi et al., 2005b). These dendrimers have been successfully exploited for delivery of gene based medicines. Of these dendrimers, we have previously shown dramatic alteration in gene expression induced by DAB16 dendrimer in A431 and A549 cells (Omidi et al., 2005b). Table 2 represents the gene expression changes by DAB polymers in A431 and A549 cells. Of the altered genes in A431 cells, some are related to cell defense and response to stress (e.g., ALOX5, TNFRSF7) and apoptosis (e.g., TNFRSF7). In A549 cells, some of the altered genes

therapy. The CSK along with some other genes were upregulated in A549 cells treated with cationic lipids similar to what we observed previously in A431 cells (Omidi et al., 2003) and is mainly involved in cell growth and/or cell maintenance. The SEP6 and PSMA4 were downregulated genes by OF in both cell lines. The SEP6 gene is a member of the septin family of GTPases. Members of this family are required for cytokinesis. One version of pediatric acute myeloid leukemia is the result of a reciprocal translocation between chromosomes 11 and X, with the breakpoint associated with the genes encoding the mixedlineage leukemia and septin 2 proteins. This gene encodes four transcript variants encoding three distinct isoforms. An additional transcript variant has been identified, but its biological validity has not been determined. The PSMA4 is a multicatalytic proteinase complex with a highly ordered ring-shaped 20S core structure. They are distributed throughout eukaryotic cells at a high concentration and cleave peptides in an

Because of the gene expression commonalities and distinctions between the two cell lines, we conceptualized that these cells may respond to the cationic lipid "OF" differently upon their cellular characteristics. These cells appeared to undergo somewhat adaptation upon exposure to xenobiotics, as a result of which they could dynamically respond as expressing/activating related cellular elements for recognition and internalization of the cationic lipid. Of interest, we found that the genotoxicity elicited by the cationic lipid nanosystems were largely dependent upon the structural architecture and/or physicochemical properties of the cationic lipid since no extensive overlap was observed in the gene expression profile induced by either LF or OF in A431 cells. Besides, the responsiveness of the target cells to the lipids could be different since the transfection efficiency is significantly depended upon the target cells and lipids used. Likewise, Filion and Phillips (1997) reported high toxicity rate elicited by some cationic lipids in phagocytic

cells such as macrophages and U937 cells, but not in non-phagocytic T lymphocytes.

Taken all these findings together, it seems that for attaining detailed characterization of the toxicogenomics of these lipid delivery systems (based on their molecular structure), the gene expression patterns/profiles need to be determined in different cell types perhaps with

Despite plethora of investigations on application of polymers in drug/gene delivery, surprisingly, little attention has been devoted about possible biofunction of polymer *per se* in particular genomic effects. Many researchers have now consensus upon functionalities of polymers, and accordingly new domains of polymer science such as "polymer genomics", "polymer genocompatibly" and "polymer genotoxicity" have been arisen. To examine the polymer genocompatibly concept, we have previously reported that starburst PAMAM dendrimers (i.e., PF and SF) as well as polypropylene imine (PPI) dendrimers (e.g., DAB8 and DAB16) can inadvertently induce alterations in gene expression (Hollins et al., 2007; Omidi et al., 2005b). These dendrimers have been successfully exploited for delivery of gene based medicines. Of these dendrimers, we have previously shown dramatic alteration in gene expression induced by DAB16 dendrimer in A431 and A549 cells (Omidi et al., 2005b). Table 2 represents the gene expression changes by DAB polymers in A431 and A549 cells. Of the altered genes in A431 cells, some are related to cell defense and response to stress (e.g., ALOX5, TNFRSF7) and apoptosis (e.g., TNFRSF7). In A549 cells, some of the altered genes

ATP/ubiquitin-dependent process in a non-lysosomal pathway.

known cell surface architecture.

**8. Genomic impacts of cationic polymers** 


Toxicogenomics of Nonviral Cationic Gene Delivery Nanosystems 565

inflammatory cytokine TNF and IL1, which suggests a cyto-protective function that is essential for lymphocyte activation as well as cell survival; reader is directed to see following citations (May et al., 1994; Ruvolo et al., 2001). The upregulation of TGFβ1 and BCL2α conceivably imply incitement of apoptosis in A549 cells upon treatments with

It was also found that the altered genes induced by PF, DAB16 and OF in A431 cells shows some commonalities and differences in pattern, presumably due to their positive charge and structural architecture. In A431 cells, treated with either DAB8 or DAB16 resulted in ~13% and ~7% similar and opposite patterns of gene expression changes, respectively. For example, BCL2α1 which acts as anti- and pro-apoptotic regulator was largely affected by DAB16 compared to DAB8. This could be due to higher surface charge and/or interaction capacity of DAB16. Similar pattern was seen for proteasomeα4, but Met proto-oncogene revealed opposite pattern. Once DAB16 was tested in different cell line (i.e., A549 cells), similar and opposite patterns of gene expression changes were ~11% and ~9%, respectively. Intriguingly, upregulation of some important genes (e.g., IL9R, TGFα) was seen solely in A549 cells, but not in A431 cells. It can be speculated that A549 cells can show greater response than A431 cells. Hence, these dendrimers could potentially affect cell growth and immune response of cells by altering the expression of some related genes at doses which did not distinctly modify cell viability (Table 2). It should be also evoked that the identity of the genes whose expression was significantly altered (i.e. the "gene signature" of the delivery system) was markedly different in the two cell lines, despite the similar expression of the majority of the genes (80%) that remained unaffected (Akhtar &

Table 3 shows the gene expression of some selected genes induced by branched and linear polyethylenimine (BPEI and LPEI, respectively) in A431 cells. These data solely present the upregulated and downregulated genes, similarly induced by these cationic polymers, while there are a large number of genes showed opposite pattern (data not shown). Based on these results, it was found that the alterations in gene expression by BPEI were significantly greater than LPEI. We contemplate that this could be because of the greater interaction of

To examine the late effect of BPEI in target cells, we evaluated gene expression pattern of caspases genes in A431 cells as a time series approache (i.e., immediately after transfection, 24 h and 48 h after transfection). Fig. 5 represents the gene expression profile of selected caspase pathway genes in A431 cells treated with BPEI, showing significant impacts of BPEI even 48 h after treatment. Of these genes, as previously mentioned, caspase 8 play a key role

These findings directed us to examine some other cationic polymers such as PAMAM and PEI. Upon our examination on SF and PF, we found that PF induced gene expression changes much greater than SF. This could be due to differences in dendrimers architecture. Significant decrease in gene expression changes were observed upon PF complexation with a DNA at the supplier recommended ratio of 10:1 (w/w) of PF:DNA. Reduced in number, but not in nature and magnitude, of expressed genes were observed upon PF:DNA complexation. In treated A431 cells with cationic dendrimer PF or cationic lipid OF, opposite and similar patterns of gene expression changes were 20% and 16%, respectively (Barar et

DAB16:DNA polyplexes.

Benter, 2007).

in apoptosis.

al., 2009).

BPEI with subcellular biomolecules.


Table 2. Gene expression changes by DAB polymers in A431 and A549 cells. NC: no changes; +/-: up/down regulation; adapted with permission from (Omidi et al., 2005b).

were in association with cell defense, DNA repair/damage and apoptosis (e.g., CCNH; ERCC1; PCNAM, CD14).

With a particular interest on toxicogenomic of the DBA16:DNA nanoparticles in A549 cells, expression changes (upregulation/downregulation) were found for some important genes (i.e., TGFβ1,BCL2α1, IL5, CXCR4 and PCKα). Of these, TGFβ1 is a member of a super-family of multifunctional cytokines that regulate cell proliferation, differentiation, and apoptosis (Chiarugi et al., 1997; Haufel et al., 1999), while the BCL2 protein family is involved in a wide variety of cellular activities that also act as anti- and pro-apoptotic regulators. The protein encoded by BCL2 is able to reduce the release of pro-apoptotic cytochrome c from mitochondria and block caspase activation which is the main apoptosis pathway. Further, this gene is a direct transcription target of NF-KAPPAβ in response to inflammatory mediators, and has been shown to be upregulated by different extracellular signals, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), CD40, phorbol ester and

NM\_002737 Protein kinase c, alpha; PRKCα NC NC NC ─ NM\_006083 Red protein; IK NC ─ NC NC

NM\_002947 Replicationprotein a3 (14kd); RPα3 NC ─ NC NC NM\_002957 Retinoid x receptor, alpha; RXRα NC NC + NC NM\_007209 Ribosomal protein l35; RPL35 NC NC NC + NM\_033301 Ribosomal protein l8; RPL8 NC ─ NC NC

NM\_006087 Tubulin, beta, 5; TUBβ5 NC + NC NC

Table 2. Gene expression changes by DAB polymers in A431 and A549 cells. NC: no changes; +/-: up/down regulation; adapted with permission from (Omidi et al., 2005b).

were in association with cell defense, DNA repair/damage and apoptosis (e.g., CCNH;

With a particular interest on toxicogenomic of the DBA16:DNA nanoparticles in A549 cells, expression changes (upregulation/downregulation) were found for some important genes (i.e., TGFβ1,BCL2α1, IL5, CXCR4 and PCKα). Of these, TGFβ1 is a member of a super-family of multifunctional cytokines that regulate cell proliferation, differentiation, and apoptosis (Chiarugi et al., 1997; Haufel et al., 1999), while the BCL2 protein family is involved in a wide variety of cellular activities that also act as anti- and pro-apoptotic regulators. The protein encoded by BCL2 is able to reduce the release of pro-apoptotic cytochrome c from mitochondria and block caspase activation which is the main apoptosis pathway. Further, this gene is a direct transcription target of NF-KAPPAβ in response to inflammatory mediators, and has been shown to be upregulated by different extracellular signals, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), CD40, phorbol ester and

subunit, beta type, 4; PSMβ4 NC NC ─ NC

(40kd); RFC2 NC NC NC ─

('docking protein'); SRPR NC NC NC +

activated met oncogene); TPR NC NC + NC

TGFβ1 NC NC NC +

TRA1 NC NC + NC

regulator of chromatin, SMARCA4 NC ─ NC ─

**A431 cells A549 cells** 

NC NC + NC

NC ─ NC NC

DAB1 6:DN A

DAB8 DAB16 DAB16

**Description** 

subunit, alpha type, 4; PSMα4

NM\_002796 Proteasome (prosome, macropain)

NM\_002914 Replication factor c (activator 1) 2

NM\_003139 Signal recognition particle receptor

NM\_003072 Swi/snf related, matrix associated

NM\_003236 Transforming growth factor, alpha;

NM\_000660 Transforming growth factor, beta 1;

NM\_003292 Translocated promoter region (to

NM\_003299 Tumor rejection antigen (gp96) 1;

TGFα

Tyrosine 3 monooxygenase/tryptophan 5 monooxygenase activation protein, theta polypeptide; YWHAQ

**Gene ID (Accession No.)** 

NM\_006826

ERCC1; PCNAM, CD14).

inflammatory cytokine TNF and IL1, which suggests a cyto-protective function that is essential for lymphocyte activation as well as cell survival; reader is directed to see following citations (May et al., 1994; Ruvolo et al., 2001). The upregulation of TGFβ1 and BCL2α conceivably imply incitement of apoptosis in A549 cells upon treatments with DAB16:DNA polyplexes.

It was also found that the altered genes induced by PF, DAB16 and OF in A431 cells shows some commonalities and differences in pattern, presumably due to their positive charge and structural architecture. In A431 cells, treated with either DAB8 or DAB16 resulted in ~13% and ~7% similar and opposite patterns of gene expression changes, respectively. For example, BCL2α1 which acts as anti- and pro-apoptotic regulator was largely affected by DAB16 compared to DAB8. This could be due to higher surface charge and/or interaction capacity of DAB16. Similar pattern was seen for proteasomeα4, but Met proto-oncogene revealed opposite pattern. Once DAB16 was tested in different cell line (i.e., A549 cells), similar and opposite patterns of gene expression changes were ~11% and ~9%, respectively. Intriguingly, upregulation of some important genes (e.g., IL9R, TGFα) was seen solely in A549 cells, but not in A431 cells. It can be speculated that A549 cells can show greater response than A431 cells. Hence, these dendrimers could potentially affect cell growth and immune response of cells by altering the expression of some related genes at doses which did not distinctly modify cell viability (Table 2). It should be also evoked that the identity of the genes whose expression was significantly altered (i.e. the "gene signature" of the delivery system) was markedly different in the two cell lines, despite the similar expression of the majority of the genes (80%) that remained unaffected (Akhtar & Benter, 2007).

Table 3 shows the gene expression of some selected genes induced by branched and linear polyethylenimine (BPEI and LPEI, respectively) in A431 cells. These data solely present the upregulated and downregulated genes, similarly induced by these cationic polymers, while there are a large number of genes showed opposite pattern (data not shown). Based on these results, it was found that the alterations in gene expression by BPEI were significantly greater than LPEI. We contemplate that this could be because of the greater interaction of BPEI with subcellular biomolecules.

To examine the late effect of BPEI in target cells, we evaluated gene expression pattern of caspases genes in A431 cells as a time series approache (i.e., immediately after transfection, 24 h and 48 h after transfection). Fig. 5 represents the gene expression profile of selected caspase pathway genes in A431 cells treated with BPEI, showing significant impacts of BPEI even 48 h after treatment. Of these genes, as previously mentioned, caspase 8 play a key role in apoptosis.

These findings directed us to examine some other cationic polymers such as PAMAM and PEI. Upon our examination on SF and PF, we found that PF induced gene expression changes much greater than SF. This could be due to differences in dendrimers architecture. Significant decrease in gene expression changes were observed upon PF complexation with a DNA at the supplier recommended ratio of 10:1 (w/w) of PF:DNA. Reduced in number, but not in nature and magnitude, of expressed genes were observed upon PF:DNA complexation. In treated A431 cells with cationic dendrimer PF or cationic lipid OF, opposite and similar patterns of gene expression changes were 20% and 16%, respectively (Barar et al., 2009).

Toxicogenomics of Nonviral Cationic Gene Delivery Nanosystems 567

Caspase 8a Caspase 8b Caspase 8e Caspase 1b Caspase 2 Caspase 5

Fig. 5. Gene expression ratio of selected caspase pathway genes in A431 cells treated with

Likewise, Pluronic® block copolymers were shown to cause various functional alterations in cells through interacting with cellular biomolecules and thus affecting various cellular functions such as mitochondrial respiration, ATP synthesis, activity of drug efflux transporters, apoptotic signal transduction, and transcriptional activation of gene expression both *in vitro* and *in vivo* (Batrakova & Kabanov, 2008). This polymer is able to enhance expression of reporter genes under the control of cytomegalovirus promoter and NF-KB response element in stably and transiently transfected mouse fibroblasts and myoblasts *in vitro*. It has been shown that these block copolymers are able to act as biological response modifying agents through upregulating the transcription of genes via activation of selected

4 24 48

**Time (h)**

Furthermore, Pluronic® P85 (P85) was reported to promote transport of the pDNA to the nucleus in cells transiently transfected with DNA/PEI polyplex (Kabanov, 2006). It has also been successfully exploited for DNA vaccine delivery, however some investigations revealed that P85 simultaneously increase transgene expression and activate immunity, in which P85 alone and P85:DNA complexes were shown to increase the systemic expansion of CD11c+ (DC), and local expansion of CD11c+, CD14+ (macrophages) and CD49b+ (natural killer) cell populations. DNA/P85 polyplex can also increase maturation of local DC (CD11c+ CD86+, CD11c+ CD80 +, and CD11c+ CD40+ (Gaymalov et al., 2009). Thus, the activation of immunogenes in the antigen-presenting cells by P85:DNA complexes can

In addition, Pluronic® can cause some alterations in HSP68 expression, suggesting that this polymer may affect stress-related pathways or there is a cross-talk between the stress and other pathways activated by the copolymer (Sriadibhatla et al., 2006). These results are in accord with what we have observed for some other cationic polymers or lipids. Pluronic®

BPEI after 4, 24 and 48 h (our unpublished data produced by Omidi et al.).

signaling pathways such as NF-KB (Sriadibhatla et al., 2006).

0.00

0.50

1.00

1.50

**Expression ratio (T/UT)**

2.00

2.50

highlight new insights for these kinds of polymers.


Table 3. Gene expression changes of selected genes induced by branched and linear polyethylenimine (BPEI and LPEI, respectively) in A431 cells (our unpublished data produced by Omidi et al.). +/-: up/down regulation

gi:407955 - membrane-associated protein hem-1 M58285 4.10 1.95 + gi:7106883 - HSPC247 AF151081 2.74 1.98 + gi:13569894 - diaphanous homolog 3; DIAPH3 NM\_030932 2.23 2.44 + gi:14010613 - methylmalonyl-coa epimerase AF364547 2.13 2.21 + gi:14248538 - STONIN2 AF255309 2.10 2.13 + gi:188560 - prepro-mullerian inhibiting substance K03474 2.10 2.69 + gi:285915 - epimorphin D14582 2.07 3.17 + gi:7109206 - four alpha helix cytokine; ZCYTO10 AF224266 1.99 2.00 + gi:558098 - protein kinase c-theta; PRKCT L01087 1.97 1.93 +

PGPEP1 AJ278828 1.93 2.82 +

gi:14588660 - histidase; hal AB042217 0.57 0.27 -

clone hsi07088; unnamed protein product. AK026297 0.53 0.26 gi:10944321 - myozenin; MYOZ AF240633 0.53 0.26 -

receptor AF030512 0.52 0.26 gi:20278870 - delta 4 progesterone receptor; pr AB084248 0.46 0.26 gi:7020101 - cdna clone unnamed protein product AK000183 0.45 0.27 gi:7209599 - melatonin 1b receptor AB033598 0.44 0.26 gi:307425 - nerve terminal protein; SNAP L19760 0.43 0.25 gi:18182679 - nkg2d AF461811 0.41 0.25 -

subunit; SDH L21936 0.39 0.23 gi:2738815 - p2y1 receptor; p2yr1 AF018284 0.28 0.26 gi:21928730 - seven transmembrane helix receptor AB065731 0.26 0.27 -

spermatogenic protein; cres AF059244 0.24 0.24 -

KIAA1393 XM\_050793 0.21 0.22 -

Table 3. Gene expression changes of selected genes induced by branched and linear polyethylenimine (BPEI and LPEI, respectively) in A431 cells (our unpublished data

BPEI LPEI

XM70908 0.58 0.25 -

**Function Gene ID T/UT ratio** 

gi:9843747 - putative pyroglutamyl-peptidase i;

gi:22041589 - similar to data source:sptr, source

gi:10439114 - homo sapiens cdna: flj22644 fis,

gi:2613124 - small cell vasopressin subtype 1b

gi:347133 - succinate dehydrogenase flavoprotein

gi:3088552 - cystatin-related epididymal

gi:22048232 - similar to riken cdna 2610027o18;

produced by Omidi et al.). +/-: up/down regulation

key:q9h4b3, evidence:iss~homolog to mucolipidin~putative; loc255231

Fig. 5. Gene expression ratio of selected caspase pathway genes in A431 cells treated with BPEI after 4, 24 and 48 h (our unpublished data produced by Omidi et al.).

Likewise, Pluronic® block copolymers were shown to cause various functional alterations in cells through interacting with cellular biomolecules and thus affecting various cellular functions such as mitochondrial respiration, ATP synthesis, activity of drug efflux transporters, apoptotic signal transduction, and transcriptional activation of gene expression both *in vitro* and *in vivo* (Batrakova & Kabanov, 2008). This polymer is able to enhance expression of reporter genes under the control of cytomegalovirus promoter and NF-KB response element in stably and transiently transfected mouse fibroblasts and myoblasts *in vitro*. It has been shown that these block copolymers are able to act as biological response modifying agents through upregulating the transcription of genes via activation of selected signaling pathways such as NF-KB (Sriadibhatla et al., 2006).

Furthermore, Pluronic® P85 (P85) was reported to promote transport of the pDNA to the nucleus in cells transiently transfected with DNA/PEI polyplex (Kabanov, 2006). It has also been successfully exploited for DNA vaccine delivery, however some investigations revealed that P85 simultaneously increase transgene expression and activate immunity, in which P85 alone and P85:DNA complexes were shown to increase the systemic expansion of CD11c+ (DC), and local expansion of CD11c+, CD14+ (macrophages) and CD49b+ (natural killer) cell populations. DNA/P85 polyplex can also increase maturation of local DC (CD11c+ CD86+, CD11c+ CD80 +, and CD11c+ CD40+ (Gaymalov et al., 2009). Thus, the activation of immunogenes in the antigen-presenting cells by P85:DNA complexes can highlight new insights for these kinds of polymers.

In addition, Pluronic® can cause some alterations in HSP68 expression, suggesting that this polymer may affect stress-related pathways or there is a cross-talk between the stress and other pathways activated by the copolymer (Sriadibhatla et al., 2006). These results are in accord with what we have observed for some other cationic polymers or lipids. Pluronic®

Toxicogenomics of Nonviral Cationic Gene Delivery Nanosystems 569

to gene transfer vectors potentially improve the specific problem by permitting lower and

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

Aardema, M.J. & MacGregor, J.T. (2002). Toxicology and genetic toxicology in the new era

Aberle, A.M.; Tablin, F.; Zhu, J.; Walker, N.J.; Gruenert, D.C. & Nantz, M.H. (1998). A

Akhtar, S. & Benter, I. (2007). Toxicogenomics of non-viral drug delivery systems for

of "toxicogenomics": impact of "-omics" technologies. *Mutat.Res.*, Vol.499, No.1,

novel tetraester construct that reduces cationic lipid-associated cytotoxicity. Implications for the onset of cytotoxicity. *Biochemistry*, Vol.37, No.18, (1998), pp.

RNAi: potential impact on siRNA-mediated gene silencing activity and

safer vector doses while facilitating tissue targeting.

expression intensities with functional genomics.

6533-6540, ISSN 0006-2960

(January 2002), pp. 13-25, ISSN 0027-5107

**10. References** 

**9. Concluding remarks** 

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

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 as imidazolium or pyridinium.

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

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 al., 2008).

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) to gene transfer vectors potentially improve the specific problem by permitting lower and safer vector doses while facilitating tissue targeting.
