Endosomal Escape of Polymeric Gene Delivery Complexes Is Not

One of the crucial steps in gene delivery with cationic polymers is the escape of the polymer/DNA complexes (“polyplexes”) from the endosome. A po...
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Biomacromolecules 2004, 5, 32-39

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Endosomal Escape of Polymeric Gene Delivery Complexes Is Not Always Enhanced by Polymers Buffering at Low pH Arjen M. Funhoff, Cornelus F. van Nostrum, Gerben A. Koning, Nancy M. E. Schuurmans-Nieuwenbroek, 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 February 6, 2003; Revised Manuscript Received July 25, 2003

One of the crucial steps in gene delivery with cationic polymers is the escape of the polymer/DNA complexes (“polyplexes”) from the endosome. A possible way to enhance endosomal escape is the use of cationic polymers with a pKa around or slightly below physiological pH (“proton sponge”). We synthesized a new polymer with two tertiary amine groups in each monomeric unit {poly(2-methyl-acrylic acid 2-[(2(dimethylamino)-ethyl)-methyl-amino]-ethyl ester), abbreviated as pDAMA}. One pKa of the monomer is approximately 9, providing cationic charge at physiological pH, and thus DNA binding properties, the other is approximately 5 and provides endosomal buffering capacity. Using dynamic light scattering and zeta potential measurements, it was shown that pDAMA is able to condense DNA in small particles with a surface charge depending on the polymer/DNA ratio. pDAMA has a substantial lower toxicity than other polymeric transfectants, but in vitro, the transfection activity of the pDAMA-based polyplexes was very low. The addition of a membrane disruptive peptide to pDAMA-based polyplexes considerably increased the transfection efficiency without adversely affecting the cytotoxicity of the system. This indicates that the pDAMA-based polyplexes alone are not able to mediate escape from the endosomes via the proton sponge mechanism. Our observations imply that the proton sponge hypothesis is not generally applicable for polymers with buffering capacity at low pH and gives rise to a reconsideration of this hypothesis. Introduction Gene therapy is a potentially attractive approach to treat patients suffering from life-threatening diseases caused by genetic deficiencies. Over the years, two types of delivery systems have been developed: viral and nonviral vectors. Although viral vectors are more efficient in delivering the therapeutic gene into cells,1-4 they possess some serious disadvantages, among which immune responses upon repeated administrations and difficulties with large scale production of pharmaceutically acceptable batches. Therefore, recently the attention is also focused on the development of nonviral vectors. Cationic lipids such as DOTAP and cationic polymers such as poly(L-lysine) (pLL), poly(ethylene imine) (pEI), chitosan, and poly(2-dimethylamino ethyl)methacrylate (pDMAEMA) are presently under investigation as DNA delivery systems.5-11 However, improvements are necessary as the present generations of nonviral carriers are far less efficient in transfection than viral vectors. One important reason for the low transfection efficiency of nonviral vectors can be found in the cellular processing of poly/lipoplexes. It has been shown that polymer/DNA complexes (“polyplexes”) adhere to the cell membranes and are subsequently taken up by cells via endocytosis.12 After fusion of endosomes with lysosomes, degradation of polyplexes will take place and no functional activity is observed. Viral vectors have fusion peptides at their surface. These * To whom correspondence should be addressed. Phone: +3130.2536964. E-mail: [email protected].

allow them to escape from endosomes by membrane fusion.13 Cationic polymers do not have fusogenic activity and are therefore unable to leave the endosome via such a process. Degradation of the content of endosomes/lysosomes is triggered by a decrease in the pH, thereby activating enzymes. For pEI, the “proton sponge” hypothesis has been proposed to explain the high transfection activity of this polymer.14,15 It has been demonstrated that pEI has buffering capacity over a broad pH range.16 Once pEI-based polyplexes are present in the endosome, they can absorb protons that are pumped into this organelle. Because of repulsion between the protonated amine groups, swelling of the polymer occurs. Moreover, to prevent the build up of a charge gradient due to the influx of protons, an influx of Cl- ions also occurs. The influx of both protons and Cl- ions increases the osmolarity of the endosome and causes water absorption. Combination of swelling of the polymer and osmotic swelling of the endosome leads to a destabilization of the endosome and release of its content into the cytoplasm.15 Subsequently, transport to and uptake in the nucleus as well as dissociation of the polyplex have to occur before transcription of the DNA can take place. It is thought that the proton sponge mechanism is not only valid for pEI but is more generally applicable for polymers containing amine groups with a pKa at or below physiological pH, like pDMAEMA,17 polyamidoamine dendrimers,18 and histidylated polylysine.19 Our group has mainly focused on the cationic polymer pDMAEMA (structure given in Figure 1). This polymer condenses DNA into small, positively charged particles that

10.1021/bm034041+ CCC: $27.50 © 2004 American Chemical Society Published on Web 11/21/2003

Buffering Polymers as Gene Delivery Vectors

Figure 1. Structures of pDMAEMA and pDAMA.

are able to transfect various cell types.20,21 Expression of the plasmid demonstrates that endosomal escape must have occurred. However, confocal laser scanning microscopy studies revealed that after 24 h of incubation a major part of the polyplexes was still present in endosomes.22 Consequently, a polymer with an increased ability to destabilize the endosome might positively affect the transfection levels. We therefore synthesized a new polymer, resembling pDMAEMA, but with two tertiary amine groups in each monomeric unit. This polymer is abbreviated as pDAMA (“polydiamine methacrylate”; Figure 1). One amine group of the monomer has a relatively high pKa, giving the polymer a positive charge and thus DNA binding capacities at physiological pH. The other amine group has a lower pKa, suggesting that the polymer would have endosomal buffering capabilities. In this paper, the DNA binding characteristics of (co)polymers of DAMA and the mechanism of transfection of the resulting polyplexes have been investigated. Experimental Section Materials. 2-Methacryloyloxyethyl phosphorylcholine (MPC)23,24 was kindly donated by Dr. K. Ishihara, University of Tokyo. The following compounds were used as received: 2-[(2-(dimethylamino)ethyl) methylamino]ethanol (Aldrich), mono-2-(methacryloyloxy)ethyl succinate (succinated 2-hydroxyethyl methacrylate, HEMASA, Aldrich), N-(3-aminopropyl) methacrylamide hydrochloride (Polysciences), rhodamine-B-isothiocyanate (Aldrich), poly-Laspartic acid sodium salt (pAspA, Sigma), picogreen dsDNA quantitation reagent (Molecular Probes), and p-formaldehyde solution (37%, for histology, Merck). Water purified by reverse osmosis was used throughout the study. Methacrylic acid (MA, Janssen Chimica) was purified by distillation under reduced pressure before use. 4-(Dimethylamino)pyridinium 4-toluenesulfonate (DPTS) was synthesized by mixing solutions of 4-(dimethylamino) pyridine or p-toluenesulfonic acid in toluene and collecting the precipitate.25 pDMAEMA (Mn ) 92 kg/mol) was synthesized via radical polymerization.21 INF-7, a 24 amino acid containing peptide with fusogenic activity derived from the influenza virus, was synthesized via standard Fmoc solid-phase synthesis.26 The crude peptide was precipitated in ether and subsequently collected after centrifugation. The peptide was dissolved in a 20 mM ammonium bicarbonate buffer, pH 8.5, and lyophilized. Synthesis of 2-Methyl-acrylic Acid 2-[(2-(Dimethylamino)-ethyl)-methyl-amino]-ethyl Ester (DAMA Monomer). Findlay et al. described 2-methyl-acrylic acid 2-[(2-

Biomacromolecules, Vol. 5, No. 1, 2004 33

(dimethylamino)-ethyl)-methyl-amino]-ethyl ester (further abbreviated as DAMA) as an unexpected side reaction from a transesterfication of glycidyl methacrylate and 2-[(2(dimethylamino)ethyl) methylamino]ethanol.27 Here, we describe a direct route to synthesize this monomer. A dry round-bottom flask was charged with 1,3-dicyclohexylcarbodiimide (DCC, 12.8 g, 62.1 mmol), DPTS (1.8 g, 6.2 mmol), and 125 mL of dichloromethane. This suspension was put under a nitrogen atmosphere. Methacrylic acid (3.56 g, 3.51 mL, 41.4 mmol) was added dropwise while stirring. Then 2-[(2-(dimethylamino)ethyl)methyl-amino]ethanol (5.89 g, 6.51 mL, 41.4 mmol) was added, the flask was closed with a septum, and the mixture was subsequently stirred for 24 h. The solid (DCU, unreacted DCC) was filtered off, and hydroquinone monomethyl ether (approximately 30 mg) was added to prevent premature polymerization. The solvent was evaporated under reduced pressure. Next, diethyl ether (100 mL) was added, insoluble products were filtered off, and the solvent was again evaporated. This was repeated with hexane. To purify the monomer from its contaminations (DCC and DCU), it was subjected to vacuum distillation at 70 °C/0.7 mbar. The desired product was obtained in a good yield (5.48 g, 25.6 mmol, 62%), and its purity was checked via RP-HPLC (eluens: water/acetonitrile 95/5, 10 mM triethylamine, pH 2 with perchloric acid, 1 mL/min, detection UV 210 nm). 1 H NMR (CDCl3, δ in ppm): 6.07 (1H, s, dCH2), 5.53 (1H, s, dCH2), 4.22 (2H, t, O-CH2-CH2), 2.70 (2H, t, O-CH2-CH2), 2.52 (2H, t, N-CH2-CH2-N), 2.37 (2H, t, N-CH2-CH2-N), 2.30 (3H, s, N(CH3)), 2.21 (6H, s, N(CH3)2). 13C NMR: (CDCl3, δ in ppm): 167.2 (CdO), 136.2 (H2CdC), 125.5 (H2CdC), 62.6 (O-CH2-CH2N(CH3)), 57.4 (O-CH2-CH2-N(CH3)), 56.1 (N(CH3)CH2-CH2-N(CH3)2), 55.8 (N(CH3)-CH2-CH2-N(CH3)2), 45.8 (N(CH3)-CH2-CH2-N(CH3)2), 42.8 (N(CH3)-CH2CH2-N(CH3)2), 18.3 (dC(CH3)). Titration of (p)DAMA. A known amount of the monomer (25 µL; 0.12 mmol) was dissolved in 20 mL of 0.15 M NaCl solution acidified with 5 mL of 0.1 M HCl. This solution was titrated with a 0.1 M NaOH solution using a Metrohm Titroprocessor 636 with a Metrohm Dosimat E635. pKa values were calculated from the inflection points of the obtained titration curve. pDAMA (34 mg, corresponding with 0.16 mmol monomeric units) was dissolved in 20 mL of 0.15 M NaCl solution, acidified with 0.5 mL of 1 M HCl, and titrated with 0.1 M NaOH. For comparison, pDMAEMA and pEI (Fluka, 50% solution in water) were also titrated. Synthesis and Characterization of DAMA (Co)Polymers. Homopolymers of DAMA (monomer/initiator ratio (M/I) ) 100, 20, and 5 respectively) and copolymers with HEMASA or MPC (M/I ) 100) were synthesized via radical polymerization under a nitrogen atmosphere, essentially as described before for DMAEMA.21 In brief, the monomers (around ∼1 g) were added to 4 mL of an aqueous hydrochloric acid solution (1 M) and the pH was adjusted to 5. Polymerization was carried out at 60 °C under shaking conditions with ammonium peroxodisulfate as initiator. After 20 h, the polymerization mixture was cooled to room

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temperature and transferred into a dialysis tube (MWCO 12kDa). After extensive dialysis against water at 4 °C, the polymer was collected after freeze-drying. A fluorescently labeled DAMA polymer was synthesized by the coupling of rhodamine B isothiocyanate to the free amine groups of a copolymer with 5% N-(3-aminopropyl) methacrylamide (synthesized as described above). The copolymer was dissolved in a 20 mM HEPES buffer pH 7.4 (5 mg/mL), rhodamine was added (40 mol % relative to the amount of free amines), and the solution was stirred overnight in the dark at 4 °C. Free rhodamine was removed by gel filtration using Sephadex G-25M. Molecular weights of the copolymers relative to dextran standards (Fluka) were determined by gel permeation chromatography (GPC) using a method described previously.21 The composition of the copolymers in D2O was determined by 1H NMR using a Gemini 300 MHz spectrometer (Varian Associates Inc. NMR Instruments, Palo Alto, CA). Preparation and Characterization of Polyplexes. The plasmid pCMVLacZ, containing bacterial LacZ gene, encoding β-galactosidase, preceded by a nuclear localization signal under control of a CMV promoter, was used.28 Stock solutions of polymer (5.0 mg/mL) and dilutions were made in 20 mM HEPES buffer, pH 7.4. In general, polyplexes were made by adding polymer solution (200 µL, various concentrations) to a plasmid solution (50 µL, 75 µg/mL in HEPES buffer), mixing thoroughly, and incubating the polyplexes for 30 min. When INF-7 peptide coated polyplexes were made, 150 µL of the polymer solution (in stead of 200 µL) was added to the plasmid solution, and after a 30 min incubation, 50 µL of the peptide solution was added and the polyplexes were incubated for another 15 min. To study the effect of serum proteins, a polyplex dispersion (DNA concentration was 30 µg/mL) was 1:1 diluted with a 10% fetal calf serum solution in 20 mM HEPES buffer, pH 7.4, and incubated for 60 min. Z-average diameters of polyplexes were determined by dynamic light scattering (DLS) at 25 °C with a Malvern 4700 system using an argonion laser (488 nm) operating at 10.4 mW (Uniphase) and PCS (photon correlation spectrometry) software for Windows version 1.34 (Malvern, U.K.). For data analysis, the viscosity and refractive index of water was used. The system was calibrated with a polystyrene dispersion containing particles of 100 nm. For the zeta potential, measurements were done at 25 °C in an aqueous DTS5001 cell with a Zetasizer 2000 unit (Malvern, U.K.) and PCS software version 1.34 (Malvern, U.K.). For instrument calibration, a polystyrene dispersion with known zeta potential was used. Transfection Efficiency and Cell Viability. Transfection experiments were performed with COS-7 cells using the plasmid pCMVLacZ as reporter gene essentially as described before.29 In brief, 96 well plates were seeded with cells at a concentration of 3 × 104/cm2 24 h before transfection. At the day of transfection, polyplexes were made as described above, with the exceptions that Hepes buffered saline (HBS, 20 mM Hepes, 130 mM NaCl) was used for dilutions and the plasmid concentration was 50 µg/mL instead of 75 µg/ mL. After rinsing the cells with HBS, 100 µL of the polyplex dispersion and 100 µL of culture medium were applied to

Funhoff et al.

the cells for 1 h. In another series of experiments, the incubation time of the polyplexes with the cells was varied from 1 to 48 h. To investigate the effect of serum and chloroquine on the transfection activity of polyplexes of pDAMA, transfection studies with culture medium without serum and/or 100 µM chloroquine were also carried out. After removal of the polyplex dispersion, fresh culture medium was added and the cells were incubated for another 48 h. All transfection experiments were performed in two identical series in separate 96 well plates. One series was tested for reporter gene expression (β-galactosidase) by ONPG colorimetric assay, and the other series was used to determine the number of viable cells using a XTT colorimetric assay. As reference, pDMAEMA polyplexes with a nitrogen/phosphate (N/P) ratio 6 were used. The transfection efficiency of polyplexes of this polymer was set at 1. Association and Dissociation of Polyplexes Studied by Agarose Gel Electrophoresis. Gel electrophoresis was performed essentially as described before.29 In general, polyplexes were formed as described above. Subsequently, samples were incubated with an excess of poly-L-aspartic acid (12.5 µg pAspA/µg DNA). Polyplex dispersions with or without pAspA were analyzed by electrophoresis in an agarose gel containing 0.5 µg/mL ethidium bromide, in TAE buffer pH 8 (40 mM Tris, 20 mM Acetic acid, 10 mM EDTA). Naked DNA was used as a marker. After electrophoresis, DNA in the gels was visualized by exposure to UV light. Picogreen Quenching Assay. Plasmid was diluted in 20 mM HEPES buffer pH 7.4 to a concentration of 1 µg/mL and picogreen was added (2.5 µL of stock solution in DMSO per mL). One mL of this mixture was transferred into a cuvette. The fluorescence was measured on a Perkin-Elmer LS50B luminescence spectrometer with Perkin-Elmer FL WinLab software (Perkin-Elmer, Wellesly, U.S.A.), with an excitation wavelength of 503 nm and an emission wavelength of 521 nm. The intensity of the fluorescence was measured after the additions of aliquots of 10 µL of polymer solution (20 µg/mL). Confocal Laser Scanning Microscopy (CLSM). Plasmid DNA was covalently labeled with fluorescein via the Fasttag FL labeling kit (Vector laboratories, Inc., Burlingame, U.S.A.). The rhodamine B labeled polymer was used. With labeled DNA and polymer, a normal transfection study was performed with COS-7 cells, using 16-well glass plates. Polyplexes were prepared at a N/P ratio of 70/1. Polyplexes were coated with INF-7 peptide (30 µg/mL) as described above. The cells were grown during 24 h after polyplex incubation, rinsed with phosphate buffered saline (PBS), fixated with a 2% p-formaldehyde solution in PBS for 1 h at 4 °C, and rinsed again with PBS. The cells were embedded in FlourSave Reagent (Calbiochem, San Diego, U.S.A.) and covered with a cover glass. Confocal fluorescent and transmitted light microscope images of cells 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).

Biomacromolecules, Vol. 5, No. 1, 2004 35

Buffering Polymers as Gene Delivery Vectors Table 1. Characteristics of the Synthesized Polymers

polymer pDAMA219a pDAMA20b pDAMA5c pDAMA-co-MPC pDAMA-co-MA pDAMA-co-MA pDAMA-co-MA pDAMA-co-HEMASA pDAMA-co-HEMASA pDAMA-co-HEMASA pDAMA-co-HEMASA

monomer feed ratio (mol %)

50/50 90/10 80/20 70/30 95/5 90/10 85/15 80/20

NMR (mol %)

Mw (kDa)

Mn (kDa)

yield

55/45 e e e 89/11 84/16 80/20 81/19

219 20 5 d 1050 1000 420 900 845 626 452

49 7 2 d 51 52 38 38 42 33 29

93 30 68 86 54 55 50 48 47 51 46

a M/I ) 100. b M/I ) 20. c M/I ) 5. d Molecular weights could not be determined by GPC. e The composition of pDAMA-co-MA polymers could not be determined by 1H NMR.

Figure 3. Particle size ([) and zeta potential (O) as a function of the ratio pDAMA219 to plasmid DNA (n ) 3). Table 2. Biophysical Characteristics, Transfection Efficiency and Toxicity of PDAMA Polyplexes polymer

particle size (nm)a

ζ-potential (mV)a

transfection efficiencyb

IC50c

pDAMA219 pDAMA20 pDAMA5

103 ( 3 94 ( 9 105 ( 4

20 ( 3 16 ( 2 18 ( 4

0.03 ( 0.01 (30) 0.04 ( 0.01 (25) 0

120 550 .500

a N/P > 5. b Relative to pDMAEMA polyplexes N/P ) 6. The N/P ratio at maximum transfection is between brackets. c Defined as the polymer concentration (in µg/mL) where the relative cell viability was 50%.

Figure 2. Titration of an acidified solution of pDAMA (9), pDMAEMA (2), or pEI (O) in 0.9% NaCl with NaOH. The amount of monomeric units was 0.16 mmol (34, 25, and 6.7 mg, respectively).

Results Polymer Synthesis and Characterization. The synthesis of the DAMA monomer was performed via a DCC mediated condensation of methacrylic acid and 2-[(2-(dimethylamino)ethyl)methylamino]ethanol. The product was obtained in a reasonable yield (62%) and a good purity (>97% by RPHPLC analysis). Table 1 summarizes the results of synthesis of the different DAMA (co)polymers. Most synthesized polymers were obtained in a reasonable to good yield (5095%). For the DAMA homopolymer (pDAMA), the weight average molecular weight could be tailored by the monomerto-initiator ratio. The weight average molecular weights of the copolymers ranged from 400 to 1000 kDa. Inherent to the polymerization method, the polydispersity ()Mw/Mn) for all polymers was high (5-20). The copolymer compositions were almost the same as the feed ratio, which is to be expected for high conversion polymers. It was shown by titration that DAMA in 0.9% NaCl solution has two pKa’s (5.5 and 9.3). Figure 2 shows the titration curves of pDAMA, pDMAEMA, and pEI in 0.9% NaCl. This figure shows that the three polymers possessed buffering capacity from pH 7.4 to 5. Using this figure, it was calculated that going from pH 7.4 to 5.0 pDMAEMA, pDAMA, and pEI can bind 0.35, 0.38, and 0.50 mol H+ per mol monomeric unit, respectively.

Biophysical Properties and Transfection Activity of Polyplexes Based on Homopolymers of DAMA. Figure 3 shows a typical curve for particle size and zeta potential as a function of the polymer nitrogen to plasmid phosphate ratio (N/P) for pDAMA with Mw of 219 kDa. At N/P ) 3, where the polyplex charge is close to neutral, severe aggregation occurred. At higher N/P ratios, a plateau value was reached for both particle size and zeta potential being 100 nm and +20 mV, respectively. When the polymer/DNA dispersions prepared at a N/P ratio of 5, the residual fluorescence remained constant (data omitted from figure). Thus, the lower residual fluorescence with pDAMA indicates a higher binding strength, in accordance with gel electrophoresis data. Biophysical Properties and Transfection Activity of Polyplexes Based on Copolymers of DAMA. To modulate the association/dissociation characteristics of pDAMA and thereby probably increasing its transfection properties once complexed with plasmid DNA, copolymers of DAMA with acid (MA or HEMASA) or zwitterionic (MPC) comonomers were synthesized. Table 3 reports the biophysical characteristics of complexes of these copolymers with DNA, as well as their transfection activity and toxicity. A general trend was observed that increasing amounts of comonomer in the polymer resulted in an increase in particle size. When the amount of MA or HEMASA in the copolymers was greater than 40 and 30 mol %, respectively, the copolymers were not able to condense DNA (results not shown in Table 3). Table 3 shows that the zeta-potential of the polyplexes is independent of the characteristics (type and amount of comonomer) of the investigated copolymers. Polyplexes based on copolymers of DAMA with MA or HEMASA showed some transfection activity (around 0.10.4 relative to pDMAEMA N/P ) 6 polyplexes). However, transfection was only found for polyplexes prepared at very high polymer-to-plasmid ratios (N/P values of 75 or higher), but still no toxicity was observed. The MPC/DAMA 50/50 copolymer showed some transfection after complexation with plasmid DNA. Copolymers richer in MPC showed no activity (not shown in Table 3). Figure 6 shows the association and dissociation behavior of polyplexes based on p(DAMA-coMA) studied with gel electrophoresis. The copolymers were

Table 3. Physical Characteristics, Transfection Efficiency, and Toxicity of Polyplexes Based on Copolymers of DAMA comonomer

fraction of comonomer (mol %)

particle size (nm)a

ζ-potential (mV)a

transfection efficiencyb (N/P)

IC50c

MPC MA MA MA HEMASA HEMASA HEMASA HEMASA

50 10 20 30 5 10 15 20

230 ( 30 85 ( 8 89 ( 10 119 ( 28 90 ( 13 96 ( 8 115 ( 21 113 ( 19

23.7 ( 2.2 23.0 ( 2.3 23.4 ( 2.9 19.7 ( 4.0 24.1 ( 1.4 22.4 ( 1.8 20.3 ( 2.4 23.8 ( 2.1

0.16 ( 0.05 (200/1) 0.10 ( 0.04 (140/1) 0.27 ( 0.05 (140/1) 0.39 ( 0.06 (500/1) 0.16 ( 0.02 (75/1) 0.15 ( 0.04 (75/1) 0.13 ( 0.02 (150/1) 0.11 ( 0.04 (150/1)

1900 1300 1300 >2300 300 500 >600 600

a N/P > 5. b Relative to pDMAEMA polyplexes N/P ) 6. The N/P ratio at maximum transfection is between brackets. c Defined as the polymer concentration (in µg/mL) where the relative cell viability was 50%.

Buffering Polymers as Gene Delivery Vectors

Figure 6. Gel electrophoresis of complexes of p(DAMA-co-MA) and plasmid DNA.

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of these polyplexes were similar to the uncoated polyplexes (data not shown). Confocal Laser Scanning Microscopy (CLSM). DNA and pDAMA were fluorescently labeled with FITC and rhodamine, respectively, and used in a transfection study comparing the effects of coating of the polyplexes with INF-7 on the intracellular localization. Polyplexes were prepared at a N/P ratio of 70 with or without INF-7 coating. This ratio was selected since under these conditions maximum transfection was observed (Figure 7). To ensure that not only the uptake of free polymer was visualized with these high ratios, DNA was also fluorescently labeled. Figure 8 shows that after 24 h incubation the green DNA label (Figure 8A) and the red polymer label (Figure 8B) colocalized in small punctate fluorescent spots, likely representing an endosomal or lysosomal localization. When polyplexes were coated with the INF-7 peptide, the same pattern was observed (Figure 8D,E). Discussion

Figure 7. Transfection (upper plot) and toxicity (lower plot) of pDAMA polyplexes ([) and polyplexes coated with INF-7 (3 µg/well) (9) as function of the N/P ratio. Transfection data are normalized with respect to transfection of the reference polymer pDMAEMA (N/P ) 6).

able to bind DNA at N/P ratios g3. This figure also shows that dissociation of the polyplexes with pAspA was not complete, but the amount of DNA remaining in the starting slot was less than with a DAMA homopolymer with comparable molecular weight (pDAMA 219, see Figure 4). Coating of pDAMA Based Polyplexes with INF-7. Figure 7 shows the transfection and toxicity of polyplexes of pDAMA219 and plasmid DNA that were coated with a fusion peptide derived from the influenza virus (INF-7).26 Polyplexes coated with this peptide showed a substantial increase in transfection activity, without (adversely) affecting the toxicity. The influence of the peptide on particle size and zeta potential of complexes was also determined. Addition of the peptide to polyplexes prepared at a N/P ratio of 9 resulted in aggregation of the complexes and in a decrease of the zeta potential of the complexes to negative values. After the addition of the peptide, the size of polyplexes prepared at a high polymer-to-plasmid ratio (N/P ) 70) increased from 100 to 170 nm, whereas the zeta potential slightly decreased (from +20 to +16 mV). Binding of pDAMA to DNA and pAspA mediated dissociation of INF-7 coated polyplexes was not influenced by the presence of the peptide, as the electrophoretic patterns

In this paper, a cationic polymer (pDAMA, see Figure 1) with two different amino groups was evaluated for DNA binding and transfection potential. The homopolymer of DAMA was indeed able to condense DNA in small and positively charged polyplexes when the N/P ratio was above 4/1 (Figure 3). At lower N/P ratios, large aggregates were observed due to absence of electrostatic repulsion between the particles. This DNA binding/condensation behavior has also been found for other polymers such as high molecular weight pDMAEMA.20 However, in contrast to pDMAEMA,21 pDAMA with low molecular weight was able to condense DNA into small particles (Table 2). This difference might indicate that pDAMA binds stronger to DNA than pDMAEMA. Moreover, the size of the polyplexes formed with pDAMA (around 100 nm, see Table 2) is smaller than polyplexes formed with pDMAEMA (around 150-200 nm),20 also indicating a stronger DNA binding. Despite being able to efficiently condense DNA, pDAMA based polyplexes had a very low transfection activity (Table 2) as compared to pDMAEMA based systems. The toxicity of the pDAMA-based polyplexes was substantially lower than the pDMAEMA-based systems. A possible reason for the low transfection activity of the pDAMA-based polyplexes might be that the serum proteins present induce aggregation or destabilize the polyplexes, as observed for other lipo/ polyplex formulations.31-33 However, in the absence of serum proteins, no improved transfection activity of the pDAMA polyplexes was observed. CLSM (Figure 8A,B) showed that the pDAMA-based polyplexes are taken up by the cells, probably via an endocytic process. The low levels of transfection may be ascribed to a lack of endosomal escape and/or intracellular dissociation of the complexes. Lauffenberg et al.34 described a computational model for intracellular gene delivery and they showed that a low dissociation rate of the polyplex resulted in relatively low levels of transgene expression. To study the dissociation of pDAMA-based polyplexes, they were subjected to gel electrophoresis in the presence or absence of an excess of poly(aspartic acid)

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Funhoff et al.

Figure 8. CLSM pictures of pDAMA polyplexes at a N/P ratio of 70 without (A-C) and with (D-F) INF peptide added. A and D are DNA label, B and E are polymer label, and C and F are the transmission pictures.

(pAspA).29 Figure 4 shows that the polyplexes incubated with pAspA showed a higher fluorescence in the starting slot as compared to untreated polyplexes. This indicates that ethidium bromide had better access to the DNA in the pAspA treated polyplexes than in control polyplexes, suggesting that the pAspA decreases the binding strength of pDAMA and DNA. Only with pDAMA of a low molecular weight (Mw 5kD), pAspA was able to substantially dissociate the corresponding polyplexes (Figure 4). The high binding strength of the pDAMA219 was also suggested by the picogreen fluorescence quenching assay (Figure 5). All techniques used in this study (DLS, picogreen fluorescence, and pAspA induced polyplex destabilization) indicate that the pDAMA binds (much) stronger to DNA than pDMAEMA, which in turn might explain the low transfection activity of the pDAMA-based polyplex formulations. To reduce the binding strength between DNA and pDAMA, copolymers of DAMA with neutral (MPC) and anionic monomers (MA and HEMASA) were synthesized and evaluated as transfectant. DLS measurements showed that, as with copolymers of DMAEMA with NVP or triEGMA,35 the resulting copolymers were still able to bind to plasmid DNA when the amount of comonomer was not too high. When the results of Figures 4 and 6 are compared, it is shown that the polyplexes with p(DAMA-co-MA) show a slightly better, although still not complete, destabilization when incubated with pAspA. This indeed indicates that the copolymers possessed a reduced affinity for DNA as compared with the homopolymers of DAMA. Polyplexes prepared with these copolymers showed some transfection activity (Table 3). It should however be mentioned that transfection was only observed at high N/P ratios. A possible

explanation is that the maximum transfection efficiency is associated with some toxicity (cell viability was 70%), as described before for pDMAEMA.20,21 Addition of chloroquine to the pDAMA based polyplexes did not result in an increase in transfection activity. In contrast, chloroquine improves the transfection efficiency of poly(lysine) based polyplexes.36,37 It has been proposed that chloroquine prevents the acidification of endosomes and their fusion with lysosomes, by which the possible enzymatic degradation of DNA is retarded. Further, it has been suggested that chloroquine promotes the dissociation of carrier/DNA complexes.38 However, chloroquine does not always result in higher transfection levels, as has been shown for nonbuffering polymers39 and lipids.40 We therefore used a membrane disrupting peptide derived from the influenza virus (INF-7) to destabilize endosomes. Previous studies have shown that this peptide significantly enhanced the transfection activity of poly(lysine)-26 and pDMAEMA-based systems.22 When this peptide was added to pDAMA-based polyplexes, significant transfection activity was observed (Figure 7), whereas the DNA binding properties remained the same as shown by electrophoresis. This indicates that lack of endosomal escape and not the binding strength of pDAMA complexes with DNA is the main reason for their low transfection activity. Polyplexes with or without INF-7 peptide coating showed the same cellular localization (Figure 8). This indicates that, although for the INF-7 coated polyplexes endosomal escape and nuclear localization must have occurred, the overall transfection efficiency of these systems may still be susceptible to improvement. The results presented in this paper strongly suggest that the very low transfection activity of the pDAMA system,

Buffering Polymers as Gene Delivery Vectors

despite the buffering capacity in the pH range from 7 to 5 (Figure 2), is due to lack of endosomal escape and indicates that the proton sponge hypothesis is at least not valid for pDAMA polyplexes. This hypothesis is also debated in other recent papers. For example, using confocal laser scanning microscopy with pH dependent fluorescent probes, both Godbey et al. and Forrest et al. showed that lysosomes are not buffered during transfection with complexes formed with pEI.41,42 Moreover, Godbey et al. showed that, when fluorescently labeled pEI was used, no merging of endosomes containing the pEI/DNA complexes with lysosomes was observed. Also, Re´my-Kristensen et al. were not able to find evidence of endosomal disruption and/or delivery of fluorescently labeled pEI/DNA into the cytoplasm or nucleus of L929 cells.43 Conclusion We have shown that polyplexes from a polymer containing two amino groups with different pKa’s, and with a buffering capacity at low pH, do not escape from the endosome. These results question the validity of the proton sponge hypothesis. Acknowledgment. This research was supported by a grant from the Dutch ministry of Economic Affairs (Project No. BTS-98150). The authors thank Dr. R. Verrijk (OctoPlus Technologies, Leiden, The Netherlands) for valuable discussions and Dr. K. Ishihara (University of Tokyo, Japan) for his gift of the MPC monomer. References and Notes (1) Robbins, P. D.; Ghivizzani, S. C. Pharmacol. Ther. 1998, 80, 3547. (2) Rots, M. G.; Curiel, D. T.; Gerritsen, W. R.; Haisma, H. J. J. Controlled Release 2003, 87, 159-165. (3) Blits, B.; Boer, G. J.; Verhaagen, J. Cell Transplant. 2002, 11, 593613. (4) McTaggart, S.; Al-Rubeai, M. Biotechnol. AdV. 2002, 20, 1-31. (5) Brown, M. D.; Schatzlein, A. G.; Uchegbu, I. F. Int. J. Pharm. 2001, 229, 1-21. (6) Moret, I.; Peris, J. E.; Guillem, V. M.; Benet, M.; Revert, F.; Dasi, F.; Crespo, A.; Alino, A. F. J. Controlled Release 2001, 76, 169181. (7) Godbey, W. T.; Mikos, A. G. J. Controlled Release 2001, 72, 115125. (8) Mao, H. Q.; Roy, K.; Troung-Le, V. L.; Janes, K. A.; Lin, K. Y.; Wang, Y., August, J. T.; Leong, K. W. J. Controlled Release 2001, 70, 399-421. (9) Merdan, T.; Kopecek, J.; Kissel, T. AdV. Drug DeliVery ReV. 2002, 54, 715-758. (10) Wang, D. A.; Narang, A. S.; Kotb, M.; Gaber, A. O.; Miller, D. D.; Kim, S. W.; Mahato, R. I. Biomacromolecules 2002, 3, 1197-1207. (11) De Smedt, S. C.; Demeester, J.; Hennink, W. E. Pharm. Res. 2000, 17, 113-126.

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