Poly(3-guanidinopropyl methacrylate): A Novel Cationic Polymer for

Bioconjugate Chem. 2004, 15, 1212−1220

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Poly(3-guanidinopropyl methacrylate): A Novel Cationic Polymer for Gene Delivery Arjen M. Funhoff, Cornelus F. van Nostrum, Martin C. Lok, Marjan M. Fretz, Daan J. A. Crommelin, and Wim E. Hennink* Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, P.O. Box 80082, 3508 TB, Utrecht, The Netherlands. Received June 11, 2004; Revised Manuscript Received September 15, 2004

A cationic polymethacrylate with a guanidinium side group was designed in order to create a polymer with cell membrane-penetrating properties such as Tat or other arginine-rich peptides. The polymer, poly(3-guanidinopropyl methacrylate), abbreviated as pGuaMA, was synthesized by free radical polymerization. The DNA-condensing properties of pGuaMA (Mw 180 kDa) were investigated via dynamic light scattering and zeta potential measurements, and small, positively charged particles (110 nm, +37 mV) were found. It was shown that polyplexes based on pGuaMA were able to transfect COS-7 cells efficiently in the absence of serum, while under the same conditions poly(arginine) (pArg) polyplexes did not show detectable transfection levels. Addition of a membrane-disrupting peptide, INF 7, derived from the influenza virus, to preformed pGuaMA polyplexes did result in ∼2 times increased transfection levels. DLS, zeta potential measurements, gel electrophoresis, and ethidium bromide displacement measurements indicated that serum induced aggregation of the polyplexes at high polymer/plasmid ratios, while at low polymer/plasmid ratios the polarity of the polyplexes reversed likely due to adsorption of negatively charged proteins on their surface. Likely, the unfavorable interactions of pGuaMA polyplexes with serum proteins is the reason for the absent transfection activity of these polyplexes in the presence of serum. Confocal laser scanning microscopy indicated cellular internalization via endocytosis of both polyplexes and free polymer. Thus, pGuaMA polyplexes enter cells, as reported for other polyplexes, by endocytosis and not, as hypothesized, via direct membrane passage.

INTRODUCTION

The use of genetic material as medicines to cure lifethreatening diseases is currently under investigation. Several potential vehicles for gene delivery (called vectors) into target cells are investigated, each with its own advantages and disadvantages. There exist however a number of physical and biological barriers (1-5) which limit the potential transfection activity of the presently investigated nonviral () synthetic) gene delivery systems. It is widely accepted that complexes of the carrier system and DNA (called polyplexes in the case of polymers or polypeptides (such as pEI, pDMAEMA, or pLL), and lipoplexes in the case of lipids (such as DOTAP)) (6-9), are taken up by cells via endocytosis (2) and end up in endosomes/lysosomes. The content of the endosomes/ lysosomes is eventually degraded by the enzymes present in these organelles. Therefore, poly-/lipoplexes have to escape from the endosomes/lysosomes before this degradation occurs. The overall efficiency of this process is usually low, resulting in low gene-expression levels, but it has been demonstrated that the transfection efficiency of polyplexes can be increased using endosomal escape agents (10). However, completely circumventing endocytosis as the pathway of uptake and making use of an alternative route to enter the cell cytoplasm is an attractive strategy to improve the overall efficiency of nonviral gene delivery systems. * Corresponding author. Tel. +31-30.2536964. Fax +3130.2517839. E-mail address: [email protected].

In the past few years, it was reported that some peptides are taken up by cells by a route other than endocytosis. Uptake of these peptides is seemingly receptor- or protein-independent and already occurs at 4 °C, although the actual mechanism is not clarified. These peptides are called cell-penetrating peptides or protein transduction domains, as they easily pass cellular membranes. Two examples of such peptides are the basic domain from the HIV-1 Tat protein (11) and a sequence from the Drosophila melanogaster antennapedia protein (Ant) (12). A common feature of these two peptides is that they consist of a large number of positively charged amino acids, mainly arginine and lysine. It was shown that other arginine-rich peptides (13) as well as oligoarginines (14) show the same cellular uptake as the Tat peptide. These peptides were coupled to macromolecular therapeutics to facilitate the cellular uptake of these compounds. The group of Kopec¸ ek showed that when the Tat peptide was coupled to a N-(2-hydroxypropyl)methacrylamide copolymer, cellular uptake and nuclear localization of the copolymer was substantially improved (15). Coupling of the Tat or the Ant peptide to antisense oligonucleotides also increased the cellular uptake (16). It was even shown that if the backbone of peptide nucleic acids (PNAs) was modified with guanidinium groups, cellular uptake of the PNAs was much higher and directed toward the nucleus (17). Since the guanidinium group is highly basic (pKa ) 12.5), it is fully protonated at physiological pH. Therefore, synthetic polymers (e.g., poly(meth)acrylates) bearing guanidinium side chains should be able to complex and

10.1021/bc049864q CCC: $27.50 © 2004 American Chemical Society Published on Web 10/23/2004

Arginine-like Polymethacrylate for Gene Delivery

condense DNA into small particles. On the basis of the above-mentioned literature, we initially expected that such polymers and/or polyplexes based thereon will be taken up by cells via another pathway than endocytosis and localize in the nucleus. This may result in increased transfection levels. For poly(arginine), the ability to condense DNA into small particles has been shown by Pouton et al. (18). However, the transfection efficiency of poly(arginine)-based polyplexes was the lowest of the tested cationic polypeptides. When compared to polymethacrylates, polypeptides have a much lower transfection efficiency (19), thus changing the backbone of poly(arginine) to a methacrylate may be advantageous. In more recent publications the spontaneous passage of arginine-rich peptides over the plasma membrane has been questioned. Richard et al. (20) suggested that the observed ‘spontaneous’ membrane passage by these peptides might be an artifact created by the fixation of the cells with formaldehyde, as diffuse fluorescence in the cytosol was only found when cells were fixated, whereas a punctuate distribution was found in living cells. Kra¨mer et al. reported that the Tat peptide is not able to penetrate the lipid bilayer of liposomes or the cell membrane of living cells (21). Both Richard et al. and Kra¨mer et al. concluded therefore that endocytosis is the main pathway of internalization of these cationic peptides. Although the cell-penetrating properties of these peptides are questionable, they may still be useful for drug delivery purposes. Console et al. showed that the Ant or Tat peptide promotes cellular uptake of macromolecules and liposomes through endocytosis (22). The amount of internalized FITC-labeled avidin or streptavidin was higher when it was bound to biotinylated Ant or Tat peptide. Thus, although our initial approach to synthesize a polymer with cell membrane-translocating properties is probably not feasible, an increase in cellular uptake can also promote the transfection efficiency and may therefore be of interest. This paper reports on the synthesis of the monomer 2-methacrylic acid 3-guanidinopropyl ester and the corresponding polymer. The transfection activity and mechanism of cellular uptake of polyplexes based on this polymer are studied as well. EXPERIMENTAL PROCEDURES

Materials. The following materials were used as received: 3-amino-1-propanol (Janssen Chimica), 2-ethyl2-thiopseudourea hydrobromide (Aldrich), N-(aminopropyl)methacrylamide hydrochloride (Polysciences), rhodamine B isothiocyanate (Aldrich), poly(L-aspartic acid) sodium salt (pAspA) (Sigma), poly(L-arginine hydrochloride) (pArg) (Sigma, Mw ) 42.4 kDa). Methacryloyl chloride purum g 97% (Fluka) was distilled before use. The plasmid pCMVLacZ, containing a bacterial LacZ gene preceded by a nuclear localization signal under control of a CMV promoter, was purchased from Sanvertech (Heerhugowaard, The Netherlands). INF-7, a 24 amino acid containing peptide with fusogenic activity derived from the influenza virus, was synthesized via standard Fmoc solid-phase synthesis (23). The reference polymers: poly((dimethylamino)ethyl methacrylate) (pDMAEMA, Mn ) 92 kg/mol) was synthesized via radical polymerization (24), and PEI (branched 25 kDa) was purchased from Sigma. Synthesis of 2-Methacrylic Acid 3-Guanidinopropyl Ester (GuaMA). 2-Guanidinopropanol was synthesized according to Fishbein and Gallaghan (25). In detail, 3-aminopropanol (0.641 g, 8.54 mmol) was added

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dropwise to 2-ethyl-2-thiopseudourea hydrobromide (1.58 g, 8.54 mmol) in a 10 mL flask. After 5 min of stirring, 1 mL water was added. The resulting solution was stirred overnight at room temperature. Next, water was removed under reduced pressure to obtain a white solid, which was further dried (yield 1.67 g, 8.43 mmol, 99.8%). NMR spectra were recorded on a Varian G-300 300 MHz spectrometer (Varian, Palo Alto, CA). 1H NMR (DMSO): δ ) 7.6-6.6 (multiple bs, 4H, NH’s), 4.7 (bs, 1H, OH), 3.44 (t, 2H, HOCH2CH2CH2N), 3.14 (t, 2H, HOCH2CH2CH2N), 1.60 (dt, 2H, HOCH2CH2CH2N). 13C NMR (DMSO): δ ) 157.1 (CH2NHC(NH)NH2), 58.0 (HOCH2CH2CH2), 38.2 (CH2CH2NHC), 31.6 (CH2CH2CH2NH). 2-Methacrylic acid 3-guanidinopropyl ester was synthesized as follows. Methacryloyl chloride (0.892 g, 8.50 mmol) was added to guanidinopropanol (1.67 g, 8.43 mmol) dissolved in acetonitrile (10 mL) and stirred at room temperature for 2 days. Acetonitrile was removed under reduced pressure, resulting in a white solid. The crude product was purified by column chromatography over silica (eluent EtOAc/EtOH 1/1, Rf ) 0.68), and the solvent from the pooled fractions with the desired product was removed under reduced pressure, resulting in a white solid, the HCl salt of the monomer. This was recrystalized in EtOAc, yielding 1.04 g (46%). 1H NMR (DMSO): δ ) 7.6 and 7.2 (bs, 4H, NH’s), 6.04 (s, 1H, H2CdC), 5.68 (s, 1H, H2CdC), 4.12 (t, 2H, OCH2CH2), 3.19 (m, 2H, CH2CH2NHC), 1.87 (s, 3H, CdC(CH3)), 1,82 (m, 2H, CH2CH2CH2). 13C NMR (DMSO): δ ) 157.4 (CH2NHC(NH)NH2), 136.4 (H2CdC), 126.6 (H2CdC), 62.3 (OCH2CH2), 38.3 (CH2CH2NC), 28.3 (CH2CH2CH2NH), 18.6 (CdC(CH3)). Polymerization. pGuaMA was synthesized via radical polymerization as described before for pDMAEMA (24). In brief, 0.500 g of monomer was dissolved in 2 mL of 1 M hydrochloric acid, and the pH was adjusted to 5. Polymerization was carried out under a nitrogen atmosphere at 60 °C overnight with ammonium peroxodisulfate as initiator at a M/I ratio of 100. After cooling to room temperature, the solution was poured into a dialysis tube (MWCO 3.5 kDa) and extensively dialyzed against water. The polymer was collected as a white fluffy solid after freeze-drying (yield 0.366 g, 73%). Molecular weights were determined via gel permeation chromatography (GPC) relative to dextran standards (Fluka) (24). GPC measurements were performed with two thermostated (30 °C) columns in series (Shodex OHpak K80P and KB80M, Showa, Denko, Japan) on a system consisting of a pump Model 600, an autoinjector Model 486, and a refractive index detector Model 410 (all Waters Associates, Milford, MA). 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. A fluorescently labeled polymer was synthesized by copolymerization of GuaMA with 5 mol % N-(aminopropyl)methacrylamide using the same synthetic procedure as described above. Rhodamine isothiocyanate (1 mg, 1.87 µmol) was coupled to the free amines of the polymer (50.0 mg, 17.3 µmol free NH2) in 2.50 mL of 20 mM HEPES buffer, pH 7.4, overnight at 4 °C. Unreacted rhodamine was removed by gel permeation chromatography over a Sephadex G25 column (Pharmacia) using water as an eluent. The fractions with the polymer were collected, and the fluorescently labeled polymer was obtained via lyophilization of the combined fractions (yield 39.7 mg, 78%). Physical Characterization of Polyplexes. Polyplexes with different polymer-to-plasmid ratios (expressed as N/P ratios, where N is the amount of protonated nitrogens in the polymer (for pGuaMA/pArg: one

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per side chain) in moles and P is the amount of plasmid phosphate in moles) were made by adding a pGuaMA or pArg solution (700 µL, various concentrations) in buffer (HEPES (20 mM) buffered saline (HBS) for particle size measurements, 20 mM HEPES buffer pH 7.4 for ζ-potential measurements) to a plasmid DNA solution (175 µL, 75 µg/mL). After incubation of the polyplex dispersions for 30 min at room temperature, Z-average diameters were measured with dynamic light scattering at 25 °C with a Malvern 4700 system using an argon-ion laser (488 nm) operating at 10.4 mW (Uniphase) and PCS (photon correlation spectrometry) software for Windows version 1.34 (Malvern, UK). Viscosity and refractive index of pure water at 25 °C were used. The system was calibrated with an aqueous dispersion of polystyrene particles with a 100 nm diameter. The ζ-potential of the polyplexes was determined at 25 °C in a DTS5001 cell with a Zetasizer 2000 unit (Malvern). The instrument was calibrated with a polystyrene dispersion with known ζ-potential. In Vitro Transfection and Cell Viability Studies. Transfection and cell viability studies were performed in COS-7 cells as described before (26). The plasmid pCMVLacZ was used as reporter gene. Plates (96-well) were seeded with 3 × 104 cells/cm2 24 h before transfection. At the day of transfection, 250 µL pGuaMA/pArg polyplex dispersions in HBS were made essentially as described above with the exception that the DNA concentration used was 50 µg/mL (instead of 75 µg/mL). INF-7 coated pGuaMA polyplexes were made by mixing 150 µL of polymer solution and 50 µL of DNA solution in HBS. After 30 min of incubation, 50 µL of INF-7 solution (150 µg/mL in HBS) was added, and the polyplexes were incubated for an additional 15 min. Previous studies showed that positively charged polyplexes were coated with the negatively charged INF-7 peptide (27). After washing of the cells with HBS, 100 µL of polyplex dispersion and 100 µL of culture medium (with or without fetal bovine serum) were applied to the cells for 1 h, after which the cells were washed and fresh culture medium was applied. All studies were performed in two identical series in two different 96-well plates. After 48 h of incubation, one plate was evaluated for reporter gene (βgalactosidase) expression with o-nitrophenyl-β-D-galactopyranoside, the other plate was used to determine cell viability using a XTT colorimetric assay (28). As a reference, pDMAEMA and PEI polyplexes prepared at the same DNA concentration and an N/P ratio of 6 were used. The transfection activity of the pDMAEMA polyplex formulation was set at one. Influence of Serum on the Biophysical Properties of Polyplexes. To determine the effect of serum on the biophysical properties of the polyplexes, their size in the presence or absence of serum was measured. In detail, 500 µL polyplex dispersions in HBS were made as described above. After 30 min incubation, the polyplex dispersions were split into two equal volumes and 1 to 1 diluted with either buffer solution or buffer solution containing 10% serum. These dispersions were incubated for another hour before Z-average diameters were measured using DLS. For zeta potential measurements, a 2.5 mL polyplex dispersion (30 µg/mL DNA) in 20 mM HEPES buffer, pH 7.4, was diluted with 2.5 mL of 20 mM HEPES buffer containing 10% serum. To study whether serum proteins induced polyplex dissociation, two different tests were performed. In the first test, DNA condensation (by pGuaMA/pArg) and poly(aspartic acid) (pAspA)-mediated dissociation of the formed complexes in the absence or presence of 5% serum

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was monitored using agarose gel electrophoresis. Polyplexes were made by mixing 10 µL plasmid solution (80 µg/mL in HBS) with 10 µL of polymer solution (various concentrations in HBS), and the dispersions were incubated for 30 min at room temperature. Next, 5 µL of buffer or buffer containing 30% serum was added, and the dispersions were incubated for 15 min. Subsequently, 5 µL of pAspA (10 mg/mL) (or 5 µL buffer as a control) was added, and the dispersions were incubated for another 30 min. After addition of 3 µL of sample buffer (containing bromophenol blue 0.4% (w/v), 10 mM EDTA, and 50% (v/v) glycerol in water), 30 µL of this mixture was applied onto a 0.7% agarose gel containing 0.5 µg/ mL ethidium bromide. After development of the gel, DNA was visualized with a UV lamp using a GelDoc system (BioRad) (26). In the second test, free DNA was detected via fluorescence with ethidium bromide that was added to a DNA solution (25 µg/mL in HBS) in a 1:10 molar ratio to the DNA phosphates. A solution of pGuaMA (400 µL, various concentrations in HBS) was added to 100 µL of DNA, vortexed for 5 s, and incubated for 30 min. This was done in two identical series. To one series, 500 µL buffer was added, and to the other series 500 µL of buffer containing 10% serum. The dispersions were incubated at room temperature for 1 h after which the fluorescence was measured using a Spex Fluorlog III spectrofluorimeter (Spex, Edison, NJ). The excitation and emission wavelength were 520 and 600 nm, respectively, and the slits were 1.26 and 4.52 nm, respectively. The relative fluorescence (Fr) values were determined as follows:

Fr ) (Fobs - Fe)/(F0 - Fe) where Fobs is the fluorescence of the polyplex dispersion, Fe is the fluorescence of ethidium bromide in the absence of DNA, and F0 is the initial fluorescence of DNA/ ethidium bromide in the absence of pGuaMA. Cellular Uptake of Polyplexes and Free Polymer. The cellular uptake of polyplexes and free polymer was investigated via confocal laser scanning microscopy (CLSM) in living cells in the absence or presence (only polyplexes) of an endocytosis inhibitor (1 mM iodoacetamide) or at 4 °C. DNA was labeled with AlexaFluor 647 C2 maleimide (Molecular Probes) using the Fasttag FL labeling kit from Vector Laboratories. Polyplexes were made with the rhodamine-labeled polymer, and for experiments in the presence of inhibitor nonlabeled DNA was used. In a 16-well glass plate, cells were seeded (3 × 104 cells/cm2) and culture medium was added. The cells were grown at 37 °C in a humidified 5% CO2-containing atmosphere. Twenty-four or 47 h after seeding, 100 µL of serum-free culture medium and 100 µL of either polyplexes with an N/P ratio of 2.7 (10 µg/mL DNA, polymer 15 µg/mL) or free pGuaMA (15 µg/mL) were applied for 1 h to the cells. As control, pDMAEMA based polyplexes (N/P ) 6) were applied to the cells 24 h after seeding. Thereafter, the polyplex dispersion and the free polymer were removed and culture medium was applied. The cells were cultured for 48 h in total after seeding, after which the cells were rinsed with PBS, and covered with a glass slide. Directly afterward, fluorescent and transmitted light microscope images were taken simultaneously on a Leica TCS-SP microscope equipped with an argon 488 nm, a krypton 568 nm and a HeNe 647 nm laser and were analyzed with Leica TCS-SP Power Scan software (Leica Microsystems, Rijswijk, The Netherlands).



Figure 1. Synthetic route toward 2-methylacrylic acid 3-guanidinopropyl ester. RESULTS AND DISCUSSION

Monomer and Polymer Synthesis. The synthesis of 2-methacrylic acid 3-guanidino-propyl ester (GuaMA) was performed in two steps (Figure 1). In the first step, 3-aminopropanol was converted into guanidinopropanol using 2-ethyl-2-thiopseudourea hydrobromide with the release of ethanethiol (25). This reaction yielded the desired product in quantitative yield and good purity (>97% by NMR). In the second step, guanidinopropanol was esterified with methacryloyl chloride and purified via column chromatography and recrystallization from ethyl acetate. This resulted in the desired monomer in reasonable yield (46%) and good purity (>97% by NMR, melting point 65 °C). Free radical polymerization of this monomer in an aqueous HCl solution and subsequent dialysis and lyophilization gave poly(3-guanidinopropyl methacrylate) (yield 73%) with a weight average molecular weigh (Mw) of 180 kDa and a number average molecular weight (Mn) of 25 kDa as determined with GPC relative to dextran standards. A pGuaMa Mw 180 kDa was synthesized since high molecular weight cationic polymers have, generally speaking (as demonstrated, for example, for pDMAEMA (24)), the best DNA-condensing properties. Biophysical Characterization of Polyplexes. The biophysical properties of pArg- or pGuaMA-based polyplexes were investigated by DLS and zeta potential measurements. Figure 2 shows the average size and zeta potential of these polyplexes as a function of the polymerto-DNA ratio (in the absence of serum proteins). As can be seen in Figure 2A, pArg was able to condense DNA in small particles of approximately 80 nm at all tested N/P ratios. Zeta potential measurements showed negatively charged polyplexes at low N/P ratios. With increasing N/P ratios the zeta potential of the polyplexes raised to approximately + 20 mV. As shown in Figure 2B, pGuaMA condensed DNA into small particles of approximately 100 nm when an excess of the polymer was used. The particles have a zeta potential of approximately +37 mV at these ratios, which is rather high when compared to the pArg polyplexes and other polyplexes (29-31). This might suggest that under the conditions where small and stable particles are formed (N/P > 5) DNA binds more pGuaMA than pArg in a polyplex. pGuaMA polyplexes prepared at an N/P ratio of 2 were neutral, and consequently, due to the absence of electrostatic repulsion, large aggregates were formed in the polyplex formulation. At lower ratios negatively charged polyplexes were formed. Similar DNA condensation behavior has been found for other cationic polymers (29-31). In Vitro Transfection and Cell Viability Studies. Transfection studies were performed in COS-7 cells (Figure 3). The transfection activity and cytotoxicity of the pGuaMA/pArg polyplexes was compared with those of pDMAEMA and PEI systems. When serum was present during the incubation of the polyplexes and the cells, no transfection was found for both pGuaMA- and pArg-based polyplexes. Compared to pDMAEMA-based

Figure 2. Physical characteristics of pArg (A)- and pGuaMA (B)-based polyplexes: particle size (2) and zeta potential (b) as a function of the N/P ratio. All experiments were done in triplicate.

polyplexes, the pGuaMA and pArg polyplexes had a low toxicity since the concentration of polymer resulting in 50% cell death was 100 µg/mL for pArg and 150 µg/mL for pGuaMA (Table 1), respectively, whereas it was 30 µg/mL for pDMAEMA. For the pGuaMA polyplexes prepared at N/P ratios above 22, it was observed by visual inspection that a precipitate was deposited onto the cells. These precipitates likely are aggregates of the free pGuaMA present in the polyplex formulation and serum proteins (see section ‘Influence of Serum on pGuaMA Based Polyplexes’). Therefore, transfection experiments were also done in the absence of serum. As can be seen in Figure 3 and Table 1, this had a major effect on both the transfection activity and the cytotoxicity of the pGuaMA polyplexes. Transfection efficiencies of 0.20 relative to the reference polymers (pDMAEMA and PEI) were found at an N/P ratio of 5.4. As has been found for other polymer (24, 30), small, positively charged polyplexes were formed at ratios where transfection occurred. Above an N/P ratio of 5.4, the transfection levels decreased most likely due to the increasing toxicity of the polyplexes as can be seen in Figure 3B. Thus serum proteins mask the toxicity of the polyplexes, as has been observed previously for other polyplex systems (31, 32). In contrast, omitting serum proteins did not result in detectable transfection levels or polyplexes based on pArg, while the cytotoxicity of the polyplexes was comparable to pGuaMA-based polyplexes (Figure 3). Pouton et al. compared different cationic polypeptides, among which pArg and poly(L-lysine) (pLL), as vectors for gene delivery in B16 cells and they showed that pArg had the lowest transfection efficiency (18). It is known that different polymers, albeit having related structures, may


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Figure 3. Transfection efficiency (A) and cell viability (B) of pGuaMA (closed symbols)- and pArg (open symbols)-based polyplexes in the presence (square) or absence (circle) of serum and of pGuaMA-based polyplexes in the absence of serum and presence of INF-7 (triangle) in COS-7 cells. All experiments were done in triplicate. Both reference polyplex formulations (pDMAEMA or PEI/DNA 6/1 (N/P) had comparable transfection activity, respectively 1.0 (pDMAEMA) and 1.05 (PEI), in the absence of serum. Table 1. IC50 Values of pGuaMA and pArg Polyplexes under Different Transfection Conditionsa transfection conditions polymer

+ serum - INF

- serum - INF

- serum + INF

pGuaMA pArg

152 ( 5 105 ( 5

42 ( 8 26 ( 2

51 ( 4 42 ( 4

a The IC value is defined as the concentration polymer (in µg/ 50 mL) resulting in 50% cell death.

Figure 5. Ethidium bromide fluorescence (relative intensity) of pGuaMA-based polyplexes in the absence (9) or presence (0) of 5% serum. All experiments were done in triplicate.

Figure 4. Gel electrophoresis of pGuaMA-based polyplexes in the absence (-) or presence (+) of serum or poly(aspartic acid) at various N/P ratios. Ref ) pDMAEMA-based polyplexes at N/P ) 6.

show substantial differences in transfection activity (33). Probably, changes in the polymeric backbone, such as

from amino acid (pArg) to methacrylate (pGuaMA) can also affect the transfection activity. Figure 3 shows that when the membrane-disrupting peptide INF-7 was added to pGuaMA-based polyplexes, an increase in transfection was observed by a factor of approximately 2. In contrast, addition of the peptide to pArg-based polyplexes did not result in increased transfection levels (data not shown). Probably, one of the following processes (e.g., polyplex destabilization, nuclear uptake) in gene delivery is rate limiting. As observed for other polyplexes (30, 33, 34), the cell viability was not influenced by the addition of the peptide. To investigate whether an endocytosis inhibitor had effect on the transfection, experiments in the presence of 1 mM iodoacetamide were performed. However, no detectable expression of the transgene was found at all pGuMA/ DNA ratios tested. Both experiments indicated that the polyplexes are taken up via endocytosis rather than direct membrane passage. Influence of Serum on pGuaMA-Based Polyplexes. Free polymer present in the polyplex dispersions prepared at high N/P ratios probably binds to negatively charged serum proteins, causing large aggregates, as has been demonstrated for pDMAEMA polyplexes (35). Incubating free polymer with serum in the same concentrations indeed resulted in large aggregates (data not shown). Because the pGuaMA polyplexes did not show



Figure 6. CLSM pictures of pGuaMA-based polyplexes (N/P ratio of 2.7) (A-C 1 h, D-F 24 h) and free polymer (15 µg/mL) (G-H 1 h, I-J 24 h) in the absence and polyplexes in the presence (K, L) of endocytosis inhibitor or at 4 °C (M, N). pDMAEMA-based polyplexes (N/P ratio of 6) (O-Q, 24 h) were used as control.


detectable transfection in COS cells and simultaneously large aggregates were visible, possible adverse interactions of serum with these polyplexes were investigated. It is known that serum proteins can cause aggregation of polyplexes or cause polyplex destabilization (36, 37). DLS measurements showed that polyplexes prepared at N/P ratios 1 µm was observed when pGuaMA polyplexes (N/P > 10) were incubated with serum. At this ratio free polymer is probably present. When serum was added to a pGuaMA solution with the same concentration as used for the preparation of the N/P ) 10 polyplexes, large aggregates were formed. Thus serum proteins adsorb on the positively charged polyplexes by which their zeta potential becomes negative. It has been shown before that polyplexes with a negative zeta potential had low aspecific cellular binding and consequently low transfection activity (33). Moreover, it has been reported that serum proteins can also induce polyplex destabilization (36, 37). Figure 4 shows the gel electrophoretic patterns of pGuaMA polyplexes prepared at three different N/P ratios in the presence or absence of serum and poly(aspartic acid) (pAspA), a known and strong polyplex destabilization agent (26, 38) pDMAEMA-based polyplexes (N/P ) 6) were used as a reference. In the absence of pAspA, all DNA was found in the starting slots at N/P ratios 5 and 21, demonstrating that the DNA was bound to the polymer. A small amount of free DNA could be detected at an N/P ratio of 1.3. This is in agreement with DLS data (Figure 2), which showed small, positively charged particles at N/P ratios of 5 and 21, but aggregates at N/P ratio of 1.3. The electrophoretic patterns in the presence of serum gave the same results, suggesting that serum was not able to dissociate the polyplexes. When pAspA was present, free DNA could be detected at all ratios, indicating that the polyplexes were dissociated. Also here, no influence of serum was found. Figure 5 shows the change in ethidium bromide fluorescence when pGuaMA is added to DNA in the absence or presence of serum. This figure shows that serum does not affect the complex formation between DNA and pGuaMA. Considering all the data, it can be concluded that not polyplex destabilization but interaction of serum proteins with the polyplexes is probably the main reason for the low transfection activity of the pGuaMA polyplexes in the presence of serum. These adverse interactions of polyplexes with serum proteins may be prevented by pegylation of the polyplexes. Moreover, pegylation of polyplexes has the additional advantage that the circulation times in vivo are increased, thereby increasing the accumulation of the polyplexes at the desired site (39-41). Cellular Uptake of Polyplexes. Polyplexes based on previously described polymers (e.g., pDMAEMA, pEI) are taken up by cells via endocytosis (2). As pointed out in the Introduction, for Tat and other arginine-rich peptides another pathway than endocytosis that leads to the direct introduction of the peptide in the cytoplasm, has been suggested. However, this has been questioned in latter publications and uptake by endocytosis was suggested for these peptides. To investigate the pathway by which pGuaMA polyplexes are taken up by cells, confocal laser scanning microscopy (CLSM) studies were done in living cells, to avoid fixation artifacts (20, 21). Cells in serum-

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free medium were incubated with polyplexes made with a red fluorescent labeled polymer and (blue labeled) DNA, or with free labeled polymer. CLSM and transmission pictures are shown in Figure 6. From these photographs it can be seen that the polyplexes and free polymer were associated with the cellular membrane 1 h after polyplex addition, whereas hardly any fluorescence was observed intracellularly, in agreement with previous observations (33). Twenty-four hours after administration, the polyplexes and free polymer were intracellularly located in small, punctuated spots lying around the nucleus of the cells. These spots most likely represent endosomes or lysosomes, as has also been found for other cationic polymers (30, 33). When iodoacetamide, an endocytosis inhibitor, was added during incubation of the polyplexes with cells, or when the cells were incubated at 4 °C, only membrane binding of the polyplexes was found. The diffuse fluorescent pattern in the cytoplasm, due to cell membrane passage found for Tat peptide (conjugates) in the studies mentioned in the Introduction, was not observed in our experiments. This indicates that the route of uptake of pGuaMA polyplexes is, as for other polyplexes, mainly endocytosis. As pGuaMA-based polyplexes do transfect cells to some extent, escape from the endosomes has to occur. For some cationic polymeric gene delivery systems, the ‘proton sponge mechanism′ has been proposed as an explanation for their endosomal escape (42, 32). However, this mechanism is suggested for polymers having a buffering capacity around physiological and lysosomal pH. As pGuaMA is already fully protonated at physiological pH (pKa ) 12.5) and consequently does not have buffering capacity, the proton sponge mechanism cannot be the reason for endosomal escape for polyplexes based on this polymer. However, indications that the proton sponge mechanism is not generally valid were reported recently because polymers having buffering capacities around physiological pH were not able to transfect cells after endosomal uptake (30, 43). Still, pGuaMA-based polyplexes, because they give transfection, obviously can escape from the endosomes and thus must have some membrane destabilizing activity. CONCLUSION

A new polymethacrylate with a guanidinium side group, abbreviated as pGuaMA, was synthesized. The polymer was able to transfect cells, but only when serum was absent during transfection, as absorption of serum proteins changed the polarity of the polyplexes. When compared to the polypeptide analogue pArg, a substantial better transfection was observed. Polyplexes based on pGuaMA were internalized in small, punctuate spots as visualized via CLSM. No evidence was found with CLSM that polyplexes were taken up via an endocytosisindependent way, as has been suggested for argininerich peptides and other guanidine-containing structures, but disputed in recent publications. ACKNOWLEDGMENT

This research was supported by a grant from the Dutch ministry of Economic Affairs (project number: BTS98150). LITERATURE CITED (1) Zabner, J., Fasbender, A. J., Moninger, T., Poellinger, K. A., and Welsh, M. J. (1995) Cellular and molecular barriers to gene transfer by a cationic lipid. J. Biol. Chem. 270, 1899719007.

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