Key modifications to increase polymeric gene delivery efficiency will incorporate molecules that have the ability to discriminate between differences in biologic microenvironments, including pH, ionic or redox potentials [[35
. One type of modification has taken advantage of changes in pH to assist in targeting through the incorporation of acid-labile linkages to facilitate polymer degradation within the local environment of a tumor [36
], or to assist in endosomal release of stabilizing complexes [37
] or polyplex degradation [43
]. The latter becomes important as polyplex unpacking is seen as a rate-limiting step for non-viral mediated gene delivery, especially for large polymer constructs [46
]. These acid-labile polymers may exhibit poor transfection efficiency due to either rapid hydrolysis or poor endosomal escape, demonstrating that in the design of hydrolytically degradable carriers, a balance needs to exist between extracellular hydrolytic stability and rapid intracellular degradation. This is further compounded by difficulties in vector handling and post-modifications of these hydrolysis sensitive polymers [45
]. A more rational approach is to have the complex escape the endosome and decomplex within the cytoplasm.
Bioreducible polymers use disulfide bonds instead of ester linkages to take advantage of the differences in reductive potential between the extracellular and intracellular microenvironments in normal as well as disease states. Several novel gene carriers incorporate disulfide bonds into their structure for cargo release upon entry into the subcellular space(s) [47
]. Delivery of nucleotides by means of redox-sensitive gene carriers has included mRNA [51
], antisense oligonucleotides [39
], siRNA [53
] and plasmid DNA [54
]. These studies have incorporated numerous cell lines and several disease states, but until recently have not included cardiac tissues [8
Poly(amido amine)s, (PAA’s) are considered a family of peptidomimetic polymers that are generated by Micheal type addition of primary and secondary aliphatic amines to bisacrylamide monomers [59
]. As such, poly(amido amine)s (PAAs) are more hydrolytically stable than poly(amino ester)s, and have good water solubility and biodegradability. Such polymer characteristics have garnered interest for biomedical applications, including drug and gene delivery [60
]. Modifications of these PAA’s with carboxylic acid side groups provide transfection efficiencies similar to that of 25 kDa branched polyethylenimine (pEI) [61
]. Upon protonation in the endosome, a conformational change is induced to allow escape of the polyplexes, but amide hydrolysis is too slow to completely free the DNA from the complexes in the cytoplasm [62
]. Work using poly(glycoamidoamine)s has demonstrated transgene expression similar to PEI and low cytotoxicity in BHK and H9C2 cell lines, but further investigation exploiting the reductive nature of cardiac cells was never initiated [63
Recently, novel poly(amido amine)s were generated containing disulfide linkages to create a highly efficient degradable gene carrier [66
]. Differences exist in the polymer backbone, but most of the novel poly(amido amine)s have similar toxicity profiles and degradative profiles in reductive environments. Although it is difficult to directly compare transfection efficiencies amongst the polymers due to the different cell lines used, the difference in transfection between PEI and pABOL in COS-7 cells resembles that of reducible histidine and poly-lysine vectors [69
]. Most relevant to this discussion, however, is the use of poly(amido ethylenimine)s to deliver DNA to cardiovascular cells, such as arterial endothelial cells and smooth muscle cells.
Reducible poly(amido ethylenimine)s (SS-PAEI)s utilize multiple disulfide linkages to decrease toxicity while exploiting the characteristics of PEI to protect and condense DNA [66
]. This condensation/protection begins at w/w ratios of polymer to DNA of 6:1 and maintains a stable particle size of under 200nm and zeta-potential of +32mV over a broad range of w/w ratios (6/1 to 48/1). Reported results indicate that regardless of the cell type, the SS-PAEI’s exhibited similar or higher transfection levels than bPEI 25k, with some as high as 20 times higher, with negligible toxicity. In depth fluorescent studies suggest that there may not be a difference in cellular uptake of the complexes by flow cytometry analysis [53
], although the cellular distribution of the fluorescent nucleotides was significantly more diffuse in the reducible poly(amido ethylenimine)s [53
]. Further experiments were conducted to determine if this reductive potential could be extended to other cardiovascular cell lines such as H9C2.
SS-PAEI with CMV driven eGFP transfected greater than 50% of H9C2 cells as opposed to only 11% of H9C2 cells when bPEI 25k was the polymer carrier [8
]. This result correlated well when comparisons of a hypoxia responsive promoter (RTP801) driving luciferase yielded a significant difference in expression between the two polymers. This effect also translated over to the more highly branched version of SS-PAEI, triethylenetetramine (TETA) (). TETA transfected H9c2 cells had significantly higher luciferase expression (p<0.01) than bPEI25k (0.75:1 w/w) without the associated toxicity (). Interestingly, while the difference of expression among the polymers remained similar when using the RTP801 promoter driving VEGF expression, it was the difference between normoxic and hypoxic environments that was striking. Typically, a 2 to 3 fold increase under hypoxic conditions is seen regardless of the polymer used, but here VEGF expression in H9C2 cells was striking, with a 76-fold increase in expression with the SS-PAEI carrier and 22-fold increase using the bPEI25k carrier. This result was attributed to post-translational modifications by the protein kinase C pathway [[72–70. Direct myocardial injections of SS-PAEI were compared against that of WSLP, and VEGF expression with the SS-PAEI carrier was twice as high as WSLP with only half of the injected dose (100ug SS-PAEI to 200ug WSLP). Confirmation of these findings was carried out using 200ug of naked DNA, WSLP, and TETA in infarcted myocardium expressing luciferase under control of the RTP801 promoter (See ). The TETA treated myocardium expressed a greater than 5-fold increase in expression compared to WSLP and a ten-fold increase compared to naked DNA. These results demonstrate the importance of both the carrier and the plasmid on expression within the myocardium and that the combination of both should be given careful consideration.
The bioreducible polymer, TETA, shows superior transfection characteristics over bPEI25k in cardiovascular cells
TETA exhibits significant reporter gene expression in infarcted rabbit myocardium