DNA binding ability was investigated using agarose gel electrophoresis to determine the polymer concentrations that each material could effectively bind DNA
54
(Figure 2.3). Transfection efficiency (Figure 2.4) was also evaluated and then related to ionization/buffering and complex formation to establish a target for material design. In the DNA binding experiments complexes were formed at various N/P ratios (0.1 – 100; depending on the charge neutralization). For the transfection experiments, a 6000 base pair pCMV-luc plasmid containing the luciferase reporter gene was used, and each material was tested at N/P ratios of 2, 4, and 10.
At an ionization of 76%, PMAI was shown to have efficient complex formation with quenching at an N/P ratio of 1. Meanwhile, it had low buffering capacity and only nominal transfection, which may be from the inability of the material to serve as an efficient ‘proton sponge’. PPAI, on the other hand, with an ionization of 2.4% had poor complex formation with quenching at an N/P of 20, but simultaneously maintained high buffering capacity. With the inability to form a neutral complex until high polymer concentrations, this material had poor transfection efficiency. When looking at the MePr copolymer series, the percent ionization was shifted to examine whether a balance between complex formation and buffering capacity could be reached. In this series, as the DMAI incorporation decreased the complexation efficiency decreased. Much like PPAI, MePr-40 (16.6% ionization) and MePr-20 (7.6% ionization) possessed high buffering capacity, but had the lowest binding ability, with complexation occurring above an N/P of 4. These materials showed low transfection, with the exception of MePr-40 which had some efficiency at an N/P of 10. MePr-80 and MePr-60 had ionizations of 50 and 33%, respectively, and showed DNA binding near an N/P ratio of 2. These materials had drastically improved transfection at this low polymer concentration, with the efficiencies 100 fold higher than any of the diene homopolymers or PEI. MePr-60 was
55
the highest performing polymer in this study, and with 33% ionization, balances complex formation with strong buffering ability.
PEAI was less efficient at DNA binding than PMAI with quenching at an N/P ratio of 2, and similarly, the complexation efficiency in the EtPr series decreased as DEAI incorporation decreased. PEAI, with ionization of 24%, showed transfection twenty fold higher than plasmid DNA at an N/P ratio of 4, but at an N/P of 2 and 10 there was low efficiency. On the other hand, EtPr-80, with an ionization of 20%, had low efficiency until an N/P of 10. This followed the same trend as the MePr series where the materials below 16% ionization did not effectively complex the DNA until an N/P of 10 or greater and also showed no transfection ability at the lower concentrations tested. Based on these results it appears that a window above 24% and below 50% charge optimizes the balance between complex formation and buffering capacity and is a reasonable target for designing materials that transfect at low polymer concentrations.
MePr-54 was designed to mimic the pKa of PEAI which was 6.9. PEAI had more efficient binding than its analogue, MePr-54, despite having the same degree of ionization. The transfection efficiency however, for MePr-54 and PEAI were similar with only small variations in efficiency at low concentrations. At an N/P of 10, MePr-54 maintained higher transfection efficiency than PEAI which is possibly the result of requiring slightly higher concentrations to form a neutral complex with DNA. Despite having an identical charge and buffering capacity, the structural property of the material played an important role, and the design of future materials will probe the influence of binding affinity on transfection; taking into account contributions from electrostatic, steric, hydrophobic, hydrogen bonding, and van der Waals interactions.
56
Figure 2.3. Agarose gel electrophoresis assay; Lanes correspond to various N/P ratios; Lane L: DNA molecular ladder; lane P: Naked DNA; All other lanes correspond to various N/P ratios; a) MePr series and b) EtPr series
a)
Figure 2.4. Transfection experiments of selected materials with HeLa cells at N/P ratios of 2,4, and 10; Values represe
3.4 Complex Studies
The polyplexes were also characterized by surface charge measurements ( potential; Appendix A). For nonspecific
maintain a positive surface charge to interact with
revealed that the surface charge of the complexes closely followed the pK
materials and transfection only occurred at concentrations for each material that resulted in positive surface charge; however,
than the percent ionization would have predicted. This shows that the effective charge is not only governed by the pKa
binding which results in further protonation of the amines. It was previously reported that binding can induce proton
1 10 100 1000 10000 100000 1000000 10000000 2 lo g (R L U / µµµµ g p ro te in ) 57
experiments of selected materials with HeLa cells at N/P ratios Values represent mean + SD (n = 3)
3.4 Complex Studies
The polyplexes were also characterized by surface charge measurements ( ). For nonspecific transfection, it is important for the complex to maintain a positive surface charge to interact with the cell surface. The zeta potential revealed that the surface charge of the complexes closely followed the pKa
only occurred at concentrations for each material that resulted in positive surface charge; however, the isoelectric point occurred at lower concentrations than the percent ionization would have predicted. This shows that the effective charge is a of the materials but also that there is a cooperative effect of ults in further protonation of the amines. It was previously reported that binding can induce proton transfer from the surrounding media. This occurs from a
4 10
N/P Ratio
experiments of selected materials with HeLa cells at N/P ratios
The polyplexes were also characterized by surface charge measurements (ζ- it is important for the complex to the cell surface. The zeta potential a trends of the only occurred at concentrations for each material that resulted the isoelectric point occurred at lower concentrations than the percent ionization would have predicted. This shows that the effective charge is of the materials but also that there is a cooperative effect of ults in further protonation of the amines. It was previously reported fer from the surrounding media. This occurs from a
MePr-40 MePr-54 MePr-60 MePr-80 EtPr-80 PMAI PEAI PPAI PEI
decrease of electrostatic repulsion upon cation effectively increasing the pK
Figure 2.5. Cytotoxicity of polyplexes
3.5 Cytotoxicity
The in vitro cytotoxicity was then evaluated using an MTS assay (
The polyplexes were tested at N/P ratios ranging from 1 to 100 with a DNA concentration of 0.5 µg/mL. PPAI was shown to have the lowest toxicity followed by the copolymers with higher incorporation of DPAI. The DEAI series also showed less toxicity when compared to the DMAI series. As expected, the materials with the highest charge density had the highest toxicity potentially stemming from membrane perturbation of the cell.19,39,40 However, the toxicity of PEAI was slig
PEAI was also slightly more efficient at complexation
-20 0 20 40 60 80 100 120 2 C e ll V ia b il it y ( % ) 58
decrease of electrostatic repulsion upon cation-phosphate charge neutralization, easing the pKa of the polymer.37,38
of polyplexes in vitro; Values represent mean ± SD
cytotoxicity was then evaluated using an MTS assay (
The polyplexes were tested at N/P ratios ranging from 1 to 100 with a DNA g/mL. PPAI was shown to have the lowest toxicity followed by the copolymers with higher incorporation of DPAI. The DEAI series also showed less toxicity when compared to the DMAI series. As expected, the materials with the highest highest toxicity potentially stemming from membrane perturbation However, the toxicity of PEAI was slightly higher than that of MePr
more efficient at complexation than MePr-54, which may result
4 10
N/P Ratio
phosphate charge neutralization,
SD (n = 3)
cytotoxicity was then evaluated using an MTS assay (Figure 2.5). The polyplexes were tested at N/P ratios ranging from 1 to 100 with a DNA
g/mL. PPAI was shown to have the lowest toxicity followed by the copolymers with higher incorporation of DPAI. The DEAI series also showed less toxicity when compared to the DMAI series. As expected, the materials with the highest highest toxicity potentially stemming from membrane perturbation htly higher than that of MePr-54.
54, which may result
Propyl Ethyl Methyl EtPr-20 EtPr-40 EtPr-60 EtPr-80 MePr-20 MePr-40 MePr-54 MePr-60 MePr-80 PEI
59
from the incorporation of the dipropylamine that may decrease surface interactions compared to the diethylamine functionality.
4. Conclusions
A series of tertiary amine functionalized homopolymers and copolymers have been synthesized with high control over their ionization potential. A combination of the buffering capacity as well as binding affinity governs the transfection efficiency of these diene based delivery vehicles. We have shown that delivery is possible with materials that have distinct buffering ranges as opposed to the broad range that occurs with PEI. MePr-60 maintains 33% amine protonation at a pH of 7.4, whereas PEI has only 20% protonation.22 This enabled MePr-60 to form a complex at low concentrations while maintaining a high buffering capacity. Ultimately, this led to the ability of MePr-60 to transfect at a concentration (N/P of 2) that PEI was not able. In this study a window from 24% to 50% ionization was found to be optimal for transfection with the best material having 33% amine protonation. This range may serve as a target for designing future delivery systems. While the facile acid/base titration proved useful as an initial test, based on the differences between PEAI and MePr-54 continued work is needed to examine the ionization properties of these materials in an environment that better mimics that of the endosome. To this end, more sophisticated titration experiments have been developed41 and will be utilized for further biophysical characterization. Future studies will also focus on quantifying DNA binding affinity in order to identify a desired binding strength, determine the contribution of various forces involved in complex formation, and ultimately further establish a guideline in the design of new materials.
60 References
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