The novel polycations generated in this work were biodegradable and should be able to compete with the ‘golden standard’ PEI22lin (Boussif 1995, Zuber 2001) in terms of gene delivery. Ideally, they possess at least equivalent transfection efficiency while being less toxic than the non-degradable HMW-PEI. In order to obtain optimum transfection activity for each polymer, the whole library was screened at varying weight ratios of conjugate to plasmid (C/P-ratios) in different cell lines. Polymers based on small polyamines like e.g. spermidine or triethylentetramine were overall characterized by poor transfection activity or had to be applied at very high concentrations. These findings were in accordance with transfection studies described by Anderson et al. (2003) where weight ratios (polymer / DNA) up to 100 were needed for efficient gene delivery. Polyamines with a higher number of protonable nitrogens like pentaethylenhexamine (PH) or oligoethylenimine (OEI 800) achieved luciferase expression levels similar to PEI22lin when applied at their optimal C/P-ratios. It is important to note that differences in DNA binding activity of oligoamidines and oligoamides (IP- vs. SP-polymers) are not reflected in their gene transfer efficiency. This suggests that, depending on the polycation, effective DNA binding is important but does not necessarily correlate with high transfection activity in vitro. For example, DNA binding of polyphosphoramidates was strongly influenced by the type of charge group, however it did not correlate with transfection efficiency (Wang 2004).
Discussion 95 Structural differences like hydrophobicity of the HD-linker, its reactivity towards primary and secondary amines and the ester/amide linkages were the reason why HD-polymers could not be compared directly with IP- and SP-polymers. While OEI- HD-1 and OEI-HD-0.5 showed very promising transfection results even at low charge ratios, HD-linked small polyamines SD-HD or TT-HD possessed poor gene transfer activity. But, exceptions confirmed the rule since, for example TT-HD-1 showed very high transfection efficiency on HUH-7 cells. This clearly demonstrated that transfection results strongly depend on the cell type and that optimal C/P-ratios have to be investigated for every cell line.
With regard to degradation and biological properties we were interested to find out whether a polymer consisting of disulfide and ester linkages had any functional advantages in comparison to standard IP-, SP-, or HD-linked polymers. Cystamine, a disulfide bond containing LMW diamine was oligomerized by HD-crosslinking and the originated polycation was included into the screening of the library. DNA binding experiments revealed that Cys-HD-1 did not form stable complexes with DNA. This lack of DNA binding affinity is in accordance with poor DNA condensation properties of other small polyamines. It is even better explicable when taking into account that each cystamine unit only provides two protonable nitrogens, whereas e.g. OEI 800 is equipped with ~16 nitrogens within one unit. Moreover, poor transfection rates even at high C/P-ratios confirmed its inability to provide enough polyplex stability for potent gene delivery.
In contrast, OEI-IP-HD-1 (via the IP-linker dimerized OEI 800 that was further oligomerized by HD-crosslinking) showed high DNA binding activity and provided polyplex stability at low C/P-ratios. With regard to transfection efficiency, this polymer had no advantages in comparison to standard OEI-HD or OEI-IP polyplexes. However, the idea of such combined polymers seemed interesting and optimization of the OEI-IP-HD-1 synthesis could result in improved biological properties.
Taken together, it became clear that some novel polycations, e.g. OEI-based polymers, possessed very promising transfection activity in all analyzed cell lines and that they provided an interesting basis for further analysis. Recently published work
Discussion 96 highlighted the overall interest to develop degradable gene carriers. Furthermore, the strategy to oligomerize LMW amines via reversible crosslinkers as performed in this thesis turned out to be followed by many other groups (Table 7).
Polymer Labile bond Potential as gene carrier
Petersen 2002
Poly(ethylenimine-co-L- lactamide-co-
succinamide
amide Low toxicity; improved TE compared to PEI 1.2 kDa
Lim 2000 Poly(α-(4-aminobutyl)-L-
glycolic acid (PAGA) ester
No toxicity; 2-fold higher TE than PLL 4 kDa
Lim 2002 Network poly(amino
ester) ester Low toxicity; similar TE as PEI25br
Jon 2003 Poly(amino alcohol esters) ester Less toxic than PEI; no transfection experiments Akinc 2003 Linear poly(ß-amino
esters) ester
Higher TE than PEI25br and Lipofectamine 2000
Forrest 2003 Crosslinked 800-Da PEI ester Low toxicity; 2-16-fold higher TE than PEI25br Anderson 2004 Library of linear poly(ß-amino esters) ester Higher TE than PEI25br and Lipofectamine 2000
Li 2004 Poly(D,L-lactide-co-4-hydroxy-L-proline) ester
Lower toxicity than PEI25br and PLL; higher TE than PEI25br after 5 days (sustained activity)
Ahn 2004 Multi-block copolymers of PLL and PEG ester Low toxicity; similar TE as PLL Kim 2005
Branched poly(ß-amino esters) mod. with amino-
hexanoic acid or lysine ester
Low toxicity; high TE in primary cells Thomas 2005 Crosslinked 2-kDa & 423- Da PEI ester/amide Low toxicity; 2-fold higher TE than PEI25br; activity in vivo Zhong 2005 Hyperbranched poly(ester
amine)s ester
Low cytotoxicity; TE comparable to that of PEI25br or pDMAEMA
Kim 2005 Crosslinked 1.8 kDa PEI acid labile imines Low toxicity; lower TE than PEI25br
Oupický 2001 Crosslinked 19.6 kDa PLL (crosslinking after polyplex formation) disulfide
Similar TE for crosslinked and standard PLL polyplexes; extended circulation time in vivo
Gosselin 2001
Crosslinked 800-Da PEI via amide or amidine
bonds disulfide Low toxicity; lower TE than PEI25br
Pichon 2002 Poly(Lys-(AEDTP)) disulfide 10-50-fold higher TE than PLL Read 2005
Reducible polycations based on His and pLys residues
disulfide Higher TE than PEI25br, activity in mediating mRNA and siRNA delivery
Table 7: Overview: biodegradable polymers for gene delivery
Currently developed degradable polymers are summarized in terms of degradability and their potential as nontoxic gene carriers. Abbreviation: TE Æ Transfection Efficiency
Discussion 97 For example, Langer and co-workers generated a library of 2350 structurally unique degradable cationic polymers. In a combinatorial approach, various diacrylates were added to different amine-containing monomers via Michael addition, and the resulting structures were characterized in large-scale transfection screenings (Anderson 2003 & 2004). Further studies with two promising candidates were carried out to investigate the effect of end group, molecular weight and polymer/DNA ratio (w/w) on transfection efficiency and cytotoxicity (Akinc 2003). Recently, a new library of 486 second-generation poly(ß-amino esters) was synthesized and structure activity studies were performed in order to elucidate the role of chemical properties on gene transfer efficiency (Anderson 2005). High-throughput syntheses and transfection screening allowed the generation of these impressive libraries. However, transfection activity was only evaluated for a single cell line and there was a lack of detailed biocompatibility studies.
Potential degradability, efficient DNA binding and a high gene transfer activity are very important features of novel nonviral vectors. Nevertheless, it is highly desirable to develop low toxic vector systems which do not interact unspecifically with e.g. blood cells in vivo.