Bioconjugate Chem. 1999, 10, 589−597
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Structure-Activity Relationships of Water-Soluble Cationic Methacrylate/Methacrylamide Polymers for Nonviral Gene Delivery Petra van de Wetering,† Ed E. Moret,‡ Nancy M. E. Schuurmans-Nieuwenbroek,† Mies J. van Steenbergen,† and Wim E. Hennink*,† Department of Pharmaceutics, Department of Medicinal Chemistry, Utrecht Institute for Pharmaceutical Sciences (UIPS), Faculty of Pharmacy, Utrecht University, Utrecht, The Netherlands. Received December 12, 1998; Revised Manuscript Received March 31, 1999
A number of water-soluble cationic carriers was evaluated as transfectant. Almost all studied cationic methacrylate/methacrylamide polymers were able to condense the structure of plasmid DNA, yielding polymer/plasmid complexes (polyplexes) with a size of 0.1-0.3 µm and a slightly positive ζ-potential, which can be taken up by cells, e.g., via endocytosis. However, the transfection efficiency and the cytotoxicity of the polymers differed widely: the highest transfection efficiency and cytotoxicity were observed for poly[2-(dimethylamino)ethyl methacrylate], p(DMAEMA). Assuming that polyplexes enter cells via endocytosis, p(DMAEMA) apparently has advantageous properties to escape the endosome. A possible explanation is that, due to its average pKa value of 7.5, p(DMAEMA) is partially protonated at physiological pH and might behave as a proton sponge. This might cause a disruption of the endosome, which results in the release of both the polyplexes and cytotoxic endosomal/lysosomal enzymes into the cytosol. On the other hand, the analogues of p(DMAEMA) studied here have a higher average pKa value and have, consequently, a higher degree of protonation and a lower buffering capacity. This might be associated with a lower tendency to destabilize the endosome, resulting in both a lower transfection efficiency and a lower cytotoxicity. Furthermore, molecular modeling showed that, of all studied polymers, p(DMAEMA) has the lowest number of interactions with DNA. We therefore hypothesized that the superior transfection efficiency of p(DMAEMA) containing polyplexes can be ascribed to an intrinsic property of p(DMAEMA) to destabilize endosomes combined with an easy dissociation of the polyplex once present in the cytosol and/or the nucleus.
INTRODUCTION
In previous studies, it has been shown that poly(2(dimethylamino)ethyl methacrylate), p(DMAEMA), is able to bind plasmid DNA, yielding polymer/plasmid complexes (also called polyplexes) (1). The size and ζ-potential of the polyplexes were dependent on the polymer/plasmid ratio. We demonstrated that polyplexes with a slightly positive ζ-potential (25-30 mV) and a size around 0.2 µm possessed the highest transfection potential (2, 3). However, as observed for many other polycationic compounds (4, 5), e.g., the frequently used transfectant poly(l-lysine) (6), p(DMAEMA) is cytotoxic (3). We also reported, upon the synthesis and characterization of random copolymers of DMAEMA with hydrophilic, noncharged comonomers. These copolymers showed a better efficacy/cytotoxicity ratio as compared to p(DMAEMA) of comparable molecular mass (7). In this study, the transfection capability of other watersoluble cationic polymers with structures closely related to p(DMAEMA) is evaluated to obtain insight into the relationship between their structure and activity. For cationic lipids, peptides, and cationic amphiphiles, a number of studies has been published concerning the relationship between structure and their transfection * To whom correspondence should be addressed. Phone: +31 30 253 6964. Fax: +31 30 251 7839. E-mail: W.E.Hennink@ pharm.uu.nl. † Department of Pharmaceutics. ‡ Department of Medicinal Chemistry.
characteristics. For lipids, the effects of the nature of the lipid headgroup (8, 9), the length of the hydrophobic tail (10), and the length of the head-tail linker (11) on their transfection characteristics have been studied. The influence of structural modifications in peptoids (12), peptides (13-15), and cationic facial amphiphiles (16) on transfection efficiency has been investigated. For synthetic cationic polymers, no systematic studies have been reported so far, to our knowledge. The polymers in this study were evaluated on their ability to bind plasmid DNA using dynamic light scattering (DLS), and the resulting polyplexes were characterized by ζ-potential measurements. The transfection efficiency and the cytotoxicity of the polyplexes were studied in vitro in OVCAR-3 cells. Furthermore, molecular modeling was used to obtain insight into the characteristics of possible DNA-polymer interactions. Polymer Selection. The structure of p(DMAEMA) is shown in Figure 1a. The effect of a propyl side chain instead of an ethyl side chain is studied with poly[3(dimethylamino)propyl methacrylate] [p(DMAPMA), Figure 1b]. Substitution of the ester group in p(DMAEMA) by an amide group is evaluated with poly[2-(dimethylamino)ethyl methacrylamide] [p(DMAEMAm), Figure 1c]. The effect of both a longer side chain and an amide group is studied with poly[3-(dimethylamino)propyl methacrylamide] [p(DMAPMAm), Figure 1d]. P(DMAEMA) contains tertiary amino groups, with an average pKa of 7.5, implying that around 50% of the side groups is positively charged at physiological pH. Poly[2-(trimethy-
10.1021/bc980148w CCC: $18.00 © 1999 American Chemical Society Published on Web 06/04/1999
590 Bioconjugate Chem., Vol. 10, No. 4, 1999
Figure 1. Chemical structure of (a) poly(2-(dimethylamino)ethyl methacrylate), p(DMAEMA); (b) poly(3-(dimethylamino)propyl methacrylate), p(DMAPMA); (c) poly(2-(dimethylamino)ethyl methacrylamide), p(DMAEMAm); (d) poly(3-(dimethylamino)propyl methacrylamide), p(DMAPMAm); (e) poly(2-(trimethylamino)ethyl methacrylate chloride), p(TMAEMA); (f) poly(2-(diethylamino)ethyl methacrylate), p(DEAEMA); and (g) poly(2-(dimethylamino)ethyl acrylate), p(DMAEA).
lamino)ethyl methacrylate chloride] [p(TMAEMA), Figure 1e] is used to study the effect of structurally almost identical polymers with permanently positively charged side chains. Another modification of the amino group is studied with poly[2-(diethylamino)ethyl methacrylate] [p(DEAEMA), Figure 1f]. This polymer contains diethylamino groups instead of dimethylamino groups. Finally, the effect of a more hydrophilic, and probably more flexible, polymer backbone is investigated with poly[2(dimethylamino)ethyl acrylate] [p(DMAEA), Figure 1g]. MATERIALS AND METHODS
Materials. The following compounds were used as received: 2-(dimethylamino)ethyl methacrylamide (DMAEMAm, Polysciences), 3-(dimethylamino)propyl methacrylamide (DMAPMAm, Aldrich, Bornem, Belgium), 2-(trimethylamino)ethyl methacrylate chloride (TMAEMA, Aldrich), 2-(dimethylamino)ethyl acrylate (DMAEA, Aldrich), 2,2′-azobisisobutyronitrile (AIBN, Fluka, Bornem, Belgium), and ammonium peroxodisulfate (APS, Fluka). All other chemicals were of analytical grade. Water purified by reversed osmosis was applied throughout the study. 2-(Dimethylamino)ethyl methacrylate (DMAEMA, Fluka) and 2-(diethylamino)ethyl methacrylate (DEAEMA, Aldrich) were purified by distillation under reduced pressure before use. 3-(Dimethylamino)propyl methacrylate (DMAPMA) was synthesized from methacryloyl chloride and 3-(dimethylamino)propanol as described elsewhere (17). Cell culture flasks and microtiter plates were obtained from Falcon (Micronic, Lelystad, The Netherlands). Dulbecco’s Modified Eagle’s Medium (DMEM) and RPMI 1640 medium were from Gibco (Life technologies, Breda, The Netherlands). Fetal calf serum (FCS) was from Integro (Zaandam, The Netherlands). Biotinylated, methoxypsoralen inactivated adenovirus dl1014 (18, 19) (stock solution 1.9 × 1011 particles/mL) was kindly provided by Dr. E. Wagner (Bender, Austria). Hepes-buffered saline (HBS) was composed of 150 mM NaCl, 20 mM Hepes, pH 7.4. The plasmid pCMVLacZ containing a bacterial LacZ gene preceded by a nuclear localization signal under control of a CMV promoter was amplified and purified as reported before (2). Synthesis and Characterization of Polymers. The polymers were synthesized by radical polymerization of
van de Wetering et al.
the corresponding monomers essentially as described in detail previously for the synthesis of p(DMAEMA) (3). Polymers of DMAEMA, DMAEA, and DEAEMA were synthesized in toluene (20% w/v) using AIBN as initiator (M/I 100-250, mol/mol). After 22 h at 60 °C the polymers were isolated by precipitation in a 5-10-fold excess cold petroleum ether and dried in vacuo. The polymers were dissolved in water, dialyzed against water, and isolated by lyophilization. Polymers of DMAEMAm, DMAPMA‚ HCl, DMAPMAm‚HCl, and TMAEMA were synthesized in aqueous hydrochloric acid (20% w/v) using APS as initiator (M/I 100-500, mol/mol). Polymerizations were stopped after 22 h at 60 °C, by transferring the reaction mixture into dialysis tubes. After extensive dialysis against water at 4 °C, the polymers were obtained by lyophilization. The weight and number average molecular masses of the polymers were determined by gel permeation chromatography (GPC). The GPC measurements were performed with two thermostated (35 °C) columns in series (Shodex OHpak KB-802 and KB-80M, Showa Denko, Japan) equipped with a refractive index detector. The eluent was an aqueous solution of 0.7 M NaNO3, 0.1 M Tris (pH 7.2), and the flow rate was 1 mL/min. The columns were calibrated using dextran standards of known molecular masses (Fluka). pKa Measurements. The pKa values of monomers and polymers were determined by titration with a Metrohm combi-titrator, equipped with a 614 pulsomat. The compounds were dissolved in 0.9 w/v % NaCl solutions (to a final concentration of 0.2-0.3 w/v %), acidified with 0.1 M HCl and titrated with 0.25 M NaOH. Particle Size and ζ-Potential Measurements. For dynamic light scattering (DLS) measurements, the stock solutions of the polymers (2 mg/mL in HBS) were diluted with “fake” RPMI medium (RPMI 1640 medium without phenol red and vitamins) to 2.4-78 µg/mL. The plasmid pCMVLacZ was diluted to 25 µg/mL in fake RPMI. Polymer solution (400 µL) was added to 100 µL of plasmid solution, resulting in 0.39/1 to 12.5/1 (w/w) polymer/ plasmid ratio. After 30-45 min complex formation time, Z-average particle size and polydispersity index (PI) of the polyplexes were determined by dynamic light scattering (DLS) at 25 °C with a Malvern 4700 system using an argon-ion laser (488 nm) operating at 10.4 mW (Uniphase). The PCS (photon correlation spectrometry) software for Windows (version 1.34, Malvern, U.K.) was used. For the data analysis, the viscosity and refractive index of pure water at 25 °C were used. The instrument was checked with a polystyrene dispersion containing particles of 100 nm. For the ζ-potential measurements, the polymer stock solutions were diluted with 20 mM Hepes. Next, 680 µL of polymer solution was added to 170 µL of plasmid solution (75 µg/mL in 20 mM Hepes), resulting in 0.39/1 to 12.5/1 (w/w) polymer/plasmid ratio. After 30-45 min of complex formation time, the ζ-potential was measured at a temperature of 25 °C in an aqueous DTS5001 cell with a Zetasizer 2000 (Malvern, U.K.) and PCS software for Windows (version 1.34, Malvern, U.K.). The instrument was checked using a polystyrene dispersion with a known ζ-potential. Molecular Modeling. Molecular modeling was performed with Insight II 97.0 (20) on a Silicon Graphics workstation. Eight adenine-thymine (AT) basepairs were built in canonical B-conformation with Insight II. To choose a starting orientation of the flexible polymer backbone, either in the major or in the minor groove of octa(DNA), a Monte Carlo simulated annealing docking
SAR of Cationic Polymers for Nonviral Gene Delivery
was used. Therefore, the backbone of octa(DMAEMA) was docked flexibly five times to DNA with AutoDock 2.2 (21). The results were clustered and the best cluster was used as a starting conformation for building syndiotactic octa(DMAEMA). This was also done for the other polymers. The number of protonated side groups in the octamers was varied. To mimic the situation at pH 7.4, the fraction of protonated groups was calculated from the average pKa values of the polymers. To exclude end effects, the outer groups of the octamers were protonated preferentially. Minimization of the structure was performed with the CVFF force field and conjugate gradients in Discover 2.98. Minimization was stopped after 105 iterations or when the maximum derivative of the potential energy was less than 0.001 kcal/Å. Throughout the minimization and molecular dynamics, the DNA was held fixed, and the nonbonded energy (representing the hydrogen bonds) was computed within a cutoff radius of 12.5 Å at a default dielectric constant of 1. The minimized structure was subjected to 500 ps of molecular dynamics at 300 K with 1 fs steps. The number of hydrogen bonds formed during the simulation were counted every ps and used as a measure of binding. All simulations were performed at least twice. Cell Culture. The human ovarian cancer cell line NIH:OVCAR-3 originated from Dr. Hamilton (National Cancer Institute, Bethesda, MD) (22). OVCAR-3 cells were cultured in DMEM supplemented with 10% (v/v) FCS, l-glutamine (2 mM), l-glucose (4.5 g/L), penicillin (100 IU/mL), streptomycin (100 µg/mL), and amphotericin B (0.25 µg/mL). Before performing a transfection experiment, the cells were grown to 60-80% confluency at 37 °C in a humidified atmosphere containing 5% CO2. Preparation of Polymer/Plasmid Complexes for Transfection Experiments. The stock solutions of the polymers (2 mg/mL in HBS) were diluted to 2.4-(6 × 102) µg/mL with plain RPMI medium. The plasmid pCMVLacZ was diluted to 25 µg/mL in RPMI. Complexes of polymer and plasmid were prepared by adding 400 µL of polymer solution to 100 µL of plasmid solution, resulting in 0.39/1 to 100/1 (w/w) polymer/plasmid ratio. After 3060 min complexation time, the complexes were added to the cells. Transfection Studies. Transfection experiments were performed with OVCAR-3 cells using the plasmid pCMVLacZ as reporter gene essentially as described before (2, 3, 7). In brief, cells were seeded at a concentration of 3 × 104/cm2 in 96-well plates 24 h before transfection. At the day of transfection, cells were rinsed and incubated for 1 h with 200 µL of polyplexes/well. Transfection experiments in the presence of adenovirus were performed essentially as described above and analogous to Cotten et al. (23). Polyplexes were prepared by adding 5 µL (9.4 × 108 particles) of adenovirus and 50 µL of polymer solution (78 and 156 µg/mL) to 50 µL of plasmid solution (50 µg/mL). After 30 min, 400 µL of RPMI was added and the complexes were applied to the cells. All transfection experiments were performed in two identical series in separate 96-well plates. After removal of the transfection complexes, 100 µL of fresh culture medium was added, and the cells were further cultured for about 48 h. Then, one series was tested for reporter gene expression (β-galactosidase) by histochemical staining with X-Gal (24). To determine the cytotoxic effect of polyplexes, the number of viable cells was measured using a XTT colorimetric assay (25).
Bioconjugate Chem., Vol. 10, No. 4, 1999 591 Table 1. Molecular Masses of the Studied Cationic Polymers, and pKa Values of the Polymers and Their Monomers polymer compd
Mw (kDa)
Mn (kDa)
DMAEMA DMAEMAm DMAPMA DMAPMAm DMAEA DEAEMA TMAEMA
550 ( 200a 120 ( 60b 275 ( 90b 220 ( 180b 25 ( 4a 90 ( 20a 845 ( 145b
50 ( 30 27 ( 12 27 ( 6 30 ( 19 7(1 8(2 70 ( 24
average pKa monomer pKa ((0.1) ((0.1) 7.4 7.8 8.4 8.8 8.2 7.5 no pKa
8.3 8.5 9.1 9.2 8.3 8.8 no pKa
a Synthesized in toluene (M/I 100-250, mol/mol). b Synthesized in aqueous hydrochloric acid (M/I 100-500, mol/mol).
Figure 2. Particle size of complexes of plasmid (final concentration of 5 µg/mL) with different polymers as a function of the polymer/plasmid (w/w) ratio. The results are shown as mean values ((SD) of three experiments. (9) p(DMAPMA), (1) p(DMAPMAm), ([) p(TMAEMA), (4) p(DMAEMAm), (O) p(DMAEMA). RESULTS
Polymer Characterization. The different (meth)acrylate/methacrylamide polymers (Figure 1) were synthesized by radical polymerization of the corresponding monomers. Since the transfection efficiency of p(DMAEMA) depended on the molecular mass (3), the aim was to synthesize high molecular mass polymers, which are also able to condense DNA to particles with a size of about 0.1-0.3 µm. Table 1 shows the molecular masses of the synthesized polymers. Except for p(DMAEA), polymers with a weight average molecular mass of g90 kDa were obtained. Further, Table 1 shows that the average pKa (which is defined as the pH at which 50% of the amino groups in the polymers are protonated) is 0.11.3 pH units below the pKa of the monomers. A likely explanation is that the presence of protonated side chains reduced the protonability of the remaining di(m)ethylamino groups; a phenomenon that has been reported before for other polymers (26). Particle Size and ζ-Potential Measurements. The characteristics of the polyplexes in terms of particle size and ζ-potential were studied as a function of polymer/ plasmid ratio. As reference polymer, p(DMAEMA) with good condensing properties (Mw 550 kDa) was used. Like p(DMAEMA), polymers of DMAEMAm, DMAPMA, DMAPMAm, and TMAEMA were able to condense plasmid to particles with a size of 0.1-0.3 µm at a polymer/plasmid ratio of 1.5-3/1 (w/w) (Figure 2). P(DMAEA) (Figure 1g) was not able to condense plasmid to
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Table 2. Results of the Molecular Dynamic Analysis of Octa(DNA) and the Octamers in 500 ps Using 1 fs Steps
poly- or octa- pKaa
DNA-polymer intrapolymer interactions interactions calcd no. of no. of H+ H+ at pH 7.4b in octamerc (PO4-)DNA-(amino)poly (NH)DNA-(CdO)poly (amino)poly-(CdO)poly (amide)poly-(CdO)poly
DMAEMA
7.5
4.5
DMAEMAm
7.8
5.5
DMAPMA
8.4
7.3
DMAPMAm
8.8
7.7
DMAEA
8.2
6.9
DEAEMA
7.5
4.5
4 5 6 7 8 6 8 7 8 7 8 4 7 4 5
791 ( 28 961 ( 35 963 ( 87 984 ( 45 1174 ( 108 1300 ( 5 1214 ( 151 2738 ( 766 2516 ( 52 2874 ( 602 3869 ( 840 1411 ( 308 1483 ( 317 1317 ( 3 1276 ( 118
74 ( 61 670 ( 180 103 ( 91 259 ( 153 398 ( 103 147 ( 58 55 ( 42 1(1 1(1 2(1 4(4 3(4 173 ( 207 294 ( 50 622 ( 274
0(0 0(0 1(1 31 ( 13 3(1 619 ( 22 452 ( 128 53 ( 92 2(3 10 ( 14 0(0 2(2 0(0 0(0 5(7
406 ( 59 141 ( 176 354 ( 15 262 ( 370
a Average pK values of the polymers. b Calculated number of protonated amino groups in the octamers at pH 7.4. c The number of a protonated amino groups with which the modeling was performed.
Table 3. Maximum Value of the Zetapotential at Polymer/Plasmid (w/w) Ratio g2/1 and the IC50 Values (µg/mL) of the Different Polymers and of the Polyplexes (plasmid concentration 5 µg/mL)
polymer
ζ-potential max (mV)
IC50 polymer (mg/mL polymer)
IC50 polyplex (mg/mL polymer)
p(DMAEMA) p(DMAEMAm) p(DMAPMA) p(DMAPMAm) p(TMAEMA)
24 ( 2 26 ( 2 28 ( 2 30 ( 2 30 ( 2
4.5 ( 0.5 14 ( 2 39 ( 1 12 ( 1 21 ( 2
11.0 ( 0.5 30 ( 2 58 ( 1 20 ( 1 45 ( 2
small particles; a heterogeneous mixture (particle size 1-2 µm) with a high polydispersity was formed. This might be explained by the low molecular mass of this polymer (Table 1). At room temperature, only large aggregates were formed by incubation of p(DEAEMA) (Figure 1f) and plasmid. Particles with a relatively smaller size were obtained by incubation of p(DEAEMA) and plasmid at 4 °C (polymer/plasmid 6/1, particle size 0.6-0.7 µm). However, when the dispersion was brought at ambient temperature, aggregates of about 2 µm were formed within 5 min. The relatively low molecular mass of p(DEAEMA) combined with the low solubility of this polymer around pH 7 (27) could be the reason for the inability to form small complexes with DNA. It was shown that at a polymer/plasmid ratio of g2/1 (w/w), the ζ-potential of the polyplexes composed of p(DMAEMAm), p(DMAPMA), p(DMAPMAm), or p(TMAEMA) and plasmid was 26-30 mV, which is comparable with the ζ-potential of polyplexes composed of p(DMAEMA) and plasmid (Table 3) and comparable with other systems [poly(l-lysine) (28), liposomes (9, 29, 30)]. Modeling. With molecular dynamics, the hydrogen bonding between octamers of all polymers [octa(repeating unit)] and a DNA octamer was studied. The octamers were built in a syndiotactic form, which is the preferred configuration for p(DMAEMA) prepared by radical polymerization (31). As an example, Figure 3 shows that the docking of octa(DMAEMA) to a DNA octamer resulted in a stable orientation in the major groove. Minimization and molecular dynamics performed on the complex showed that octa(DMAEMA) remained in the major groove, which was also observed for poly(N-vinyl pyrrolidone), PVP, by Mumper et al. (32). PVP was placed in a minimized conformation near a DNA oligomer. In their study, two kinds of interactions could be distinguished. The vinyl backbone of PVP provided a hydro-
Figure 3. Octa(DMAEMA) docked into the major groove of octa(DNA).
phobic surface on DNA and hydrogen bonding was observed between the hydrophilic pyrrolidone groups and the base pairs of DNA. In our study, using molecular dynamics, three (Figure 4) and four (Figure 5) types of interactions were observed between DNA and the methacrylate octamers and methacrylamide octamers, respectively. First, interaction occurred between the positively charged amino groups and the negatively charged phosphate groups in DNA: (PO4-)DNA-(amino)poly interaction. Second, interaction between a NH of adenine and the CdO group of the polymers was observed ((NH)DNA-(CdO)poly, see Figure 6). Third, interaction between the positively charged amino
SAR of Cationic Polymers for Nonviral Gene Delivery
Figure 4. Interactions between a DNA octamer and octa(DMAEMA) (PO4-)DNA-(amino)poly and (NH)DNA-(CdO)poly.
group and the carbonyl group within a polymer chain occurred [intrapolymeric interaction: (amino)poly-(Cd O)poly]. The fourth type of interaction could only occur between methacrylamide compounds, because it involved the interaction of the amide proton with the carbonyl: (amide)poly-(CdO)poly interaction. First, the effect of the degree of protonation of amino groups on the interaction with DNA was studied for octa(DMAEMA) (Figure 4). Table 2 shows that, with an increasing degree of protonation, the total number of (PO4-)DNA-(amino)poly interactions increased. However, the number of these interactions per protonated group slightly decreased with an increasing degree of protonation. Besides (PO4-)DNA-(amino)poly interactions, also (NH)DNA(CdO)poly interactions were observed. With exception of octa(DMAEMA) bearing five protonated groups, octa(DMAEMA)5+, the number of interactions between the (NH)DNA and (CdO)poly increased with the degree of protonation. Taking both types of interactions together, it can be concluded that, with an increasing degree of protonation, the octamer comes into a more fixed position in the major groove of DNA. Next, the number and types of interactions between the different fully protonated octamers and octa(DNA) were compared. Table 2 shows that replacement of the ester group in octa(DMAEMA)8+ by an amide group, i.e. octa(DMAEMAm)8+, had no significant effect on the number of interactions between (PO4-)DNA and (amino)poly. Due to the presence of the amide group, octa(DMAEMAm)8+ showed additional (amide)poly-(CdO)poly interactions as well as an increased number of (amino)poly-(Cd O)poly interactions (Figure 5). In contrast, the number of interactions between (CdO)poly and (NH)DNA was lower in comparison with octa(DMAEMA)8+, which might be
Bioconjugate Chem., Vol. 10, No. 4, 1999 593
Figure 5. Interactions between a DNA octamer and octa(DMAEMAm) (PO4-)DNA-(amino)poly, (NH)DNA-(CdO)poly, (amino)poly-(CdO)poly, and (amide)poly-(CdO)poly.
Figure 6. Interaction between an adenine-thymine base pair (R ) sugar/phosphate backbone) and adenine and (the carbonyl group of) the polymer.
due to the large increase in the number of (amino)poly(CdO)poly interactions. When the behavior of octa(DMAEMA)8+ is compared with octa(DMAPMA)8+, the number of interactions between (PO4-)DNA and (amino)poly was higher for octa(DMAPMA)8+, whereas (NH)DNA-(Cd O)poly interactions were absent. As was observed for octa(DMAEMA)8+, the number of intrapolymeric interactions within octa(DMAPMA)8+ were marginal. Replacing the ester group in octa(DMAPMA)8+ with an amide group, i.e. octa(DMAPMAm)8+, resulted in an increase of (PO4-)DNA-(amino)poly interactions while the number of (NH)DNA-(CdO)poly and intrapolymer interactions remained very low. Similar as observed for octa(DMAEMAm)8+, octa(DMAPMAm)8+ also showed additional (amide)poly-(CdO)poly interactions. However, in contrast to the ethyl compound octa(DMAEMAm)8+, octa(DMAP-
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MAm)8+ had no intrapolymeric (amino)poly-(CdO)poly interactions. Finally, we compared the types and the number of interactions at pH 7.4. For each octamer, the fraction protonated with amino groups was calculated from the average pKa of the polymers (Table 2). Comparison of the interactions of the different octamers with DNA at pH 7.4 shows similar trends, as were observed for fully protonated octamers. However, the only observed difference is that for p(DMAEMA)4-5+, the number of (PO4)DNA and (amino)poly interactions, was 1.5, 2.8-3.5, and 4-5 times lower in comparison with octa(DMAEMAm)6+, octa(DMAPMA)7+, and octa(DMAPMAm)8+, respectively. For octa(TMAEMA), which permanently bears positively charged amino groups, interaction with DNA is expected. Because the (PO4-)DNA-(amino)poly interaction does not involve hydrogen bonds, it was not possible to quantify the number of interactions between octa(DNA) and octa(TMAEMA). However, due to its permanent positive charge, relatively strong electrostatic interactions between octa(TMAEMA) and DNA can be expected. Although complexing problems prevented evaluation in transfection, octa(DMAEA) and octa(DEAEMA) could be studied in terms of interaction with DNA. As can be seen in Table 2, for octa(DMAEA) and octa(DEAEMA), similar numbers of DNA-octamer interactions were observed. However, in comparison with the results of octa(DMAEMA), the number of (PO4-)DNA-(amino)poly interactions was higher than the number of interactions found for octa(DMAEMA). Transfection Efficiency and Influence on Cell Viability. The influence of the polymer concentration on the cell viability was evaluated in cell culture for the different polymers. From Figure 7a, which shows the effect of the polymer concentration on the cell viability, the IC50 values [defined as the concentration resulting in 50% inhibitory activity (cell-death)], which give an indication about the cytotoxicity, were determined (Table 3). Both Figure 7a and Table 3 show that all evaluated polymers had a lower cytotoxicity than p(DMAEMA). P(DMAEMAm) was about 3-fold less toxic than p(DMAEMA). The analogue with a propyl instead of an ethyl side chain, p(DMAPMA), was almost 9-fold less toxic than p(DMAEMA). Combination of both structural differences (amide and propylene side chain) in p(DMAPMAm) did not result in a synergistic reduction of the cytotoxicity: p(DMAPMAm) was only 3-fold less toxic than p(DMAEMA). Finally, the permanently positively charged p(TMAEMA) had about a 5-fold lower toxicity as compared to p(DMAEMA). Next, the influence of the polymer/plasmid ratio on the cell viability and transfection efficiency was evaluated for the different polymers. Figure 7b shows the relation between the polymer/plasmid ratio and the cell viability. The same trend as found for the free polymers was observed (Figure 7a). The IC50 values, calculated from Figure 7b, give an indication about the cytotoxicity of the transfection mixture, consisting of polyplexes and free polymer which is likely present above a certain polymer/ plasmid ratio. Comparison of the IC50 values of the polymers with the IC50 values of the polyplexes shows that the presence of plasmid masked part of the cytotoxic effect of the polymer. This phenomenon has also been reported for, e.g., R-helical peptides (14) and liposomes (33). For p(DMAEMA), a bell-shaped relation between polymer/plasmid ratio and transfection efficiency was found (Figure 7c). At low polymer/plasmid ratios (
