Buffering Properties of Cationic Polymethacrylates Are Not the Only

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Buffering Properties of Cationic Polymethacrylates Are Not the Only Key to Successful Gene Delivery Peter Dubruel,† Bart Christiaens,‡ Maryvonne Rosseneu,‡ Joe¨l Vandekerckhove,‡,§ Johan Grooten,| Vera Goossens,|,⊥ and Etienne Schacht*,†,# Polymer Materials Research Group, Department of Organic Chemistry, Ghent University, Ghent, Belgium, Laboratory for Lipoprotein Chemistry, Department of Biochemistry, Ghent University, Ghent, Belgium, Flanders Interuniversity Institute for Biotechnology (VIB), Department of Medical Protein Research, Ghent University, Ghent, Belgium, Department of Molecular Biomedical Research, Molecular Immunology Unit, Flanders Interuniversity Institute for Biotechnology and Ghent University, Institute for Biomedical Technology (IBITECH), Ghent University, Ghent, Belgium Received October 30, 2003; Revised Manuscript Received December 7, 2003

Recently, we have shown that polymethacrylates containing imidazole side groups (HYMIMMA) or acid functions (MA), which have similar buffering properties as polyethyleneimine, were not able to transfect Cos-1 cells, whereas polymers containing only tertiary amines (DMAEMA) do transfect Cos-1 cells (Dubruel, P. et al. Eur. J. Pharm. Sci. 2003, 18 (3-4), 211-220). In the present work, we investigated to what extent the differences in transfection activity are related to differences in cellular internalization and/or subcellular localization. Therefore, we synthesized a series of polymethacrylates containing primary amine functions, used for the coupling of the fluorescent Oregon Green probe. The polymers containing acid functions were labeled with an amine containing fluorescein derivative (5-aminomethyl)fluorescein hydrochloride. It is demonstrated that the endosomal release of the MA and HYMIMMA-based complexes might be the limiting step in the gene transfer process in Cos-1 cells. 1. Introduction In the last two decades, gene delivery has attracted considerable interest as a possible therapy for the treatment of diseases caused by genetic defects.1-3 One of the advantages of this therapy is its potential application for the treatment of acquired diseases such as cancer and AIDS. In a classical gene delivery protocol, DNA, the carrier of the genetic information, is used as a drug for corrective treatment. Since spontaneous DNA internalization is hampered by its physicochemical properties (i.e., a negative charge and a large hydrodynamic volume4), it has to be combined with a vector. Virusses have the natural ability to infect cells and their use as biological gene delivery systems seems straightforward.5 However, the different types of virusses (retrovirusses, adenovirusses, adeno-associated virusses, ...) suffer several drawbacks depending on the type of viral vector used.6-7 Alternative DNA vectors are the nonviral vectors covering both liposomal and polymeric gene delivery systems. These two systems have in common that DNA condensation occurs through electrostatic interaction between the negative charges * To whom correspondence should be addressed. Address: Krijgslaan 281 (S4 Bis), B-9000 Ghent, Belgium. Tel.: 0032-(0)9-2644497. Fax.: 0032-(0)9-2644998. E-mail: [email protected]. † Department of Organic Chemistry. ‡ Department of Biochemistry. § Department of Medical Protein Research. | Flanders Interuniversity Institute for Biotechnology and Ghent University. ⊥ Current adress: Innogenetics, Therapeutics R&D, Industriepark 7, 9052 Zwijnaarde, Belgium. # Institute for Biomedical Technology (IBITECH).

of the DNA phosphate groups and the positive charges of the amine containing liposomes or polymers.8-9 A large number of research groups, including our own, have focused on the synthesis and evaluation of cationic polymers as gene delivery systems.10-15,27-30 Polymers have the advantage that a large variety of functional groups can be introduced by an accurate monomer selection or by polymer functionalization. In addition, large scale production of polymers is feasible, opening perspectives for industrial exploitation. However, at present, polymeric gene delivery systems have not yet met the high expectations (cell specific high transfection efficiency and low cytotoxicity) despite the large number of polymers that have been evaluated as gene carriers in recent years: poly-L-lysine16-18 and other poly-R-amino acids,19-20 polyethyleneimine (PEI)21-23 and its derivatives,24-25 poly(2-N,N-(dimethylaminoethyl) methacrylate)26 (PDMAEMA) and DMAEMA based (block)-copolymers,27-31 gelatin,32,33 and polyphosphazenes.34 PEI has a high transfection efficiency in a large number of cell lines. A possible explanation for the good transfection properties of PEI is the socalled “proton sponge theory”, proposed by Behr et al.35 This theory is based on the fact that PEI undergoes further protonation when PEI-DNA complexes are localized in the endosomal compartment. To maintain the endosomal pH, proton pumps in the endosomal membrane, are transferring protons (and chloride counterions) into the endosomes. This transfer leads to an osmotic effect resulting in a swelling of the endosomes. Finally, this would result in a rupture of the endosomes. In addition to this osmotic effect, further PEI

10.1021/bm034438d CCC: $27.50 © 2004 American Chemical Society Published on Web 01/15/2004

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protonation would also result in an expansion of the polymer (and thus the complexes) due to internal charge repulsion. Both synergistic effects are thought to shorten the endosomal lifetime drastically. However, at present, there is some controversy regarding the validity of this theory. In addition, PEI-DNA complexes possess a high cytotoxicity and haemolytic activity.30 Therefore, we synthesized polymethacrylates with similar transfection efficiency and lower toxicity compared to PEI.27,29-30 We showed that these polymers condense DNA and that copolymers containing tertiary amines (DMAEMA) and acid functions (MA) and copolymers containing DMAEMA and imidazole functions (HYMIMMA) are not able to transfect cells in vitro.30 In the present work, we want to elucidate the mechanisms underlying these findings by studying the cellular internalization and localization of a series of fluorescently labeled polymer-DNA complexes by confocal microscopy. 2. Materials and Methods 2.1. Chemicals. Ethanolamine, linear calf thymus DNA (15-23 kb), boric acid, bromophenol blue, and agarose (electrophoresis grade) were from Sigma (Bornem, Belgium). Ethanolamine was distilled before use. EDTA and 2-N,N(dimethylaminoethyl) methacrylate (DMAEMA) were purchased from Fluka (Bornem, Belgium). DMAEMA was distilled before use. Glycerol was from Vel (Leuven, Belgium). Di-tert-butyl dicarbonate, methacrylic anhydride, 2,2′-azobis(2-methyl-propionitrile), calcium hydride, and triethylamine were purchased from Acros (Geel, Belgium). All chemicals, except triethylamine which was distilled, were used as received. Branched polyethyleneimine (Mw ) 25 000 g/mol), EDC, MTT, and trifluoro acetic acid were from Aldrich (Bornem, Belgium) and were used as received. Lysotracker Red DND-99, Oregon Green 488-X succinimidyl ester, and 5-(aminomethyl)fluorescein hydrochloride were obtained from Molecular Probes (Leiden, The Netherlands) and were used as received. Toluene, the solvent used for the polymerization reactions, was dried over calcium hydride and distilled before use. Penicillin, Streptomycin, Lipofectamine, and DMEM were obtained from Gibco BRL (Life Technologies) (Merelbeke, Belgium). pCMVbeta plasmid (7.2 kb) was obtained from Clontech (Erembodegem, Belgium). FBS was obtained from Perbio (Helsingborg, Sweden). The β-gal ELISA kit was obtained from Roche (Vilvoorde, Belgium). The BCA Protein Assay kit was obtained from Pierce (Erembodegem, Belgium). The endotoxin-free plasmid Maxiprep kit was obtained from Qiagen (Leusden, The Netherlands). The water used throughout this study was double distilled. 2.2. Monomer Synthesis. 2.2.1. Synthesis of t-Boc Protected 4-Methyl-5-imidazoyl Methyl Methacrylate. To a solution of 4-methyl-5-imidazoyl methanol hydrochloride (1.5 g, 11.15 mmol) in dioxane (20 mL) and H2O (10 mL) was added 0.1 N NaOH (12 mL, 24 mmol) and di-tert-butyl dicarbonate (2.4 g, 11 mmol). The reaction mixture was cooled in an ice bath and stirred for 24 h under N2 atmosphere. The solution was then concentrated to 15 mL

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by evaporation of the solvent under reduced pressure. The aqueous phase was saturated with NaCl and extracted three times with EtAc. After drying over MgSO4, the solvent was evaporated under reduced pressure. t-Boc protected 4-methyl5-imidazoyl methanol was isolated after addition of cold pentane (40 mL). The product was filtered off, washed three times with pentane, and dried under reduced pressure for 24 h. To a solution of t-Boc protected 4-methyl-5-imidazoyl methanol (10.65 g, 50 mmol) and phenothiazine (20 mg, 0.1 mmol) in CH2Cl2 (30 mL) was added Et3N (7.0 mL, 50 mmol). The reaction mixture was cooled in an ice bath, and methacryloyl chloride (6.5 mL, 66.53 mmol) was added. The reaction was stirred for 1 h under N2 atmosphere and then poured into H2O (100 mL). The product was extracted with ether. The organic phase was washed three times with a saturated NaHCO3 and dried over MgSO4. After addition of phenothiazine (to avoid premature polymerization), the monomer was obtained as an oil by evaporation of the solvent under reduced pressure. The chemical structure was determined by 1H NMR spectroscopy. 1 H NMR: (500 MHz, CDCl3) δ ) 8.1 and 8.0 ppm (imidazole proton), δ ) 6.2 ppm, 6.1, 5.8, and 5.55 ppm (CH2dC), δ ) 5.3 and 5.1 ppm (CH2-O), δ ) 2.45 and 2.35 ppm (CH3 on imidazole), δ ) 1.9 ppm (CH3-C), δ ) 1.5 and 1.6 ppm ((CH3)3-C). 2.2.2. Synthesis of t-Boc Protected Aminoethyl Methacrylate. To an ice-cooled solution of ethanolamine (3 mL, 50.7 mmol) in CH2Cl2 (30 mL) was added a solution of di-tertbutyl dicarbonate (9.23 g, 42 mmol) in CH2Cl2 (15 mL) under continuous stirring. The reaction was left stirring overnight at room temperature. Then, the reaction mixture was extracted one time with a 10% KHSO4 solution and two times with water. After drying the organic phase over MgSO4, t-Boc protected ethanolamine was isolated as an tranparent oil after evaporation of the solvent under reduced pressure. The chemical structure was confirmed by 1H NMR spectroscopy. 1 H NMR: (500 MHz, CDCl3) δ ) 5.0 ppm (NH-CdO), δ ) 3.8 ppm (CH2-O), δ ) 3.25 ppm (CH2-N), δ ) 2.4 ppm (CH2-OH), δ ) 1.5 ppm ((CH3)3-C). In a subsequent step, triethylamine (3.1 mL, 22 mmol) was added to an ice-cooled solution of t-Boc protected ethanolamine (3.55 g, 22 mmol) in CH2Cl2. To this was added a solution of methacrylic anhydride (3.9 mL, 24 mmol) in CH2Cl2 (5 mL). After stirring overnight at room temperature, the reaction mixture was extracted three times with a 10% NaHCO3 solution and one time with water. After drying the organic phase over MgSO4, the solvent was partially removed under reduced pressure. t-Boc protected aminoethyl methacrylate was isolated after precipitation in ice-cooled pentane. After filtration, the product was washed three times with pentane and dried for 24 h under reduced pressure. The chemical structure of the monomer was determined by 1H NMR spectroscopy. 1 H NMR: (500 MHz, CDCl3) δ ) 6.1 and 5.6 ppm (CH2d C), δ ) 4.8 ppm (NH-CdO), δ ) 4.2 ppm (CH2-O), δ ) 3.4 ppm (CH2-N), δ ) 1.9 ppm (CH3-C), δ ) 1.5 ppm ((CH3)3-C).

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Figure 1. Synthesis scheme of P(DMAEMA0.48-co-AEMA0.42-co-HYMIMMA0.10). t-Boc-AEMA, t-Boc-HYMIMMA, and DMAEMA were polymerized for 24 h in toluene at 65 °C using AIBN as the radical initiator.

2.3. Polymer Synthesis. 2.3.1. Synthesis of PDMAEMA and P(DMAEMA0.65-co-MA0.35). The synthesis of poly(2-N,N(dimethylaminoethyl) methacrylate), PDMAEMA, and poly(2-N,N-(dimethylaminoethyl) methacrylate0.65-co-methacrylic acid0.35), P(DMAEMA0.65-co-MA0.35), was described before. As an example, the synthesis of PDMAEMA will be given. To a solution of DMAEMA (5 g; 29.67 mmol) in dry toluene (20 mL) was added 319 µl (3.19 mg/19.4 µmol) of a AIBN stock solution (10 mg/mL) in toluene. The solution was degassed three times and then heated to 65°C. The reaction mixture was left stirring at 65 °C for 24 h under N2 atmosphere. Next the PDMAEMA homopolymer was precipitated in 200 mL of ice-cooled pentane, filtered, and washed. The polymer was then redissolved in 0.1 N HCl and dialyzed during 48 h against water. Finally, the polymer was obtained by freeze-drying. The chemical structure of the polymer obtained was determined by 1H NMR spectroscopy. 2.3.2. Synthesis of P(DMAEMA0.96-co-AEMA0.04) and P(DMAEMA0.48-co-AEMA0.42-co-HYMIMMA0.10). The synthesis of P(DMAEMA0.96-co-AEMA0.04) and P(DMAEMA0.48co-AEMA0.42-co-HYMIMMA0.10) was carried out in a similar

way as described in section 2.3.1 and in ref 27. As an example, the synthesis of P(DMAEMA0.48-co-AEMA0.42-coHYMIMMA0.10) is described below. The general reaction scheme is shown in Figure 1. t-Boc protected HYMIMMA (303.5 mg, 1.084 mmol), DMAEMA (365 µL, 2.168 mmol) and t-Boc protected AEMA (496 mg, 2.168 mmol) were dissolved in 7 mL of dry toluene. The solution was degassed 3 times before polymerization. Finally, AIBN (0.4688 mg, 2.85 µmol) was added as a radical initiator. After 24 h at 65 °C under nitrogen atmosphere, the polymer was isolated by precipitation in icecooled pentane. The polymer was filtered and washed three times with pentane. The t-Boc protecting group from the HYMIMMA and AEMA units was removed by acidolysis in trifluoro acetic acid as described earlier (for a detailed protocol, see ref 27). The chemical composition of the polymer was determined by 1H NMR spectroscopy, taking into account the characteristic peaks from the different monomers used. The composition of all polymers used in the present study is shown in Table 1. The chemical structure of the compounds is given in Figure 2.

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Table 1. Chemical Composition, Molecular Weight, Refractive Index Increment, Polydispersity, and Mass Per Charge of the Cationic Polymers Used in This Worka chemical composition

weight average molecular weight (g/mol)

refractive index increment

polydispersity (Mw/Mn)

mass/charge (g/mol)

mol % fluorophore

PDMAEMA P(DMAEMA0.96-co-AEMA0.04) P(DMAEMA0.48-co-AEMA0.42-co-HYMIMMA0.10) P(DMAEMA0.65-co-MA0.35) PEI

201 000 273 000 47 000 316 000b 25 000

0.201 0.162 0.163 0.155 0.282

1.4 1.7 2.4

194 193 201 520 208

0.7 1.7 0.4 0.5

2.0

a

Finally, the coupling efficiency of Oregon Green to P(DMAEMA0.96-co-AEMA0.04), P(DMAEMA0.48-co-AEMA0.42-co-HYMIMMA0.10), and PEI and the coupling efficiency of 5-(aminomethyl)fluorescein hydrochloride to P(DMAEMA0.65-co-MA0.35) are also given. b The molecular weight of P(DMAEMA0.65co-MA0.35) was not determined by gel permeation chromatography due to adsorption phenomena on the GPC column. Therefore, the molecular weight of this polymer was determined by static light scattering as reported earlier (see ref 30).

Figure 2. Chemical structure of the different polymethacrylates and fluorophores used in this work.

2.4. Molecular Weight Determination. The molecular weight of the polymers was determined by gel permeation chromatography (GPC) on an Ultrahydrogel-1000 column (7.8 mm × 300 mm) from Waters. The eluent used was a phosphate buffer (5% NaH2PO4, 3% acetonitrile, pH ) 4). The flow rate was 0.750 mL/min. GPC analysis was performed at concentrations of 10-15 mg/mL. The detector was a combination of a differential refractometer (Waters) and a MALLS light scattering detector (Meyvis). The refractive index of all polymers was first determined with a double beam differential refractometer (DRM 1020, Polymer Laboratories). The molecular weight, the polydispersity, and

the refractive index increment of all polymers used in this study are presented in Table 1. 2.5. Coupling of Oregon Green to P(DMAEMA0.96-coAEMA0.04), P(DMAEMA0.48-co-AEMA0.42-co-HYMIMMA0.10) and PEI. The coupling of Oregon Green to P(DMAEMA0.96-co-AEMA0.04), P(DMAEMA0.48-co-AEMA0.42-co-HYMIMMA0.10) and PEI was carried out in a similar way. The general protocol for the different polymers is given below. The polymer (20 mg) was dissolved in 0.2 mL pf NaHCO3 buffer (pH ) 8.3). To this solution was added dropwise an amount of Oregon Green (dissolved in DMF) under continu-


Keys to Successful Gene Delivery Table 2. DNA Condensation Properties as Studied by Agarose Gel Electrophoresisa chemical composition

charge ratio (()

PDMAEMA P(DMAEMA0.96-co-AEMA0.04) P(DMAEMA0.48-co-AEMA0.42-co-HYMIMMA0.10) P(DMAEMA0.65-co-MA0.35) PEI

1.6/1 1.2/1 1.4/1 1.6/1 0.4/1

a Polymer-DNA complexes were prepared at different charge ratios and applied on a 1% agarose gel. The DNA in the gels was stained by incubating the gel in an EtBr solution (0.5 µg/mL) for 1 h.

ous stirring. The amount of Oregon Green used corresponds to a maximum conversion of 2 mol % of the primary amines. After reacting for 1 h in the dark, the reaction mixture was dialyzed against 1 M NaCl to remove any unreacted Oregon Green. Any residual Oregon Green in the dialysis water (outer part) was detected by UV (absorbance measurements at 496 nm). Finally, the product was dialyzed against water to remove traces of NaCl. The product was obtained by freeze-drying. The amount of fluorophore coupled was determined by UV (496 nm). The coupling efficiency for the different polymers are summarized in Table 1. 2.6. Coupling of 5-(Aminomethyl)fluorescein Hydrochloride to P(DMAEMA0.65-co-MA0.35). 65 mg of P(DMAEMA0.65-co-MA0.35) was dissolved in 0.5 mL of phosphate buffer (pH ) 7). To this solution was added dropwise a solution of 2.5 mg of 5-(aminomethyl)fluorescein hydrochloride (in 250 µL DMF) under continuous stirring. The amount of fluorophore added corresponds to a maximum conversion of 3.25 mol % of the acid functions. After 15 min, 8 mg of EDC was added. After reacting for 2 h in the dark, the reaction mixture was dialyzed against 1 M NaCl to remove any unreacted 5-(aminomethyl)fluorescein hydrochloride. Residual 5-(aminomethyl)fluorescein hydrochloride in the dialysis water (outer part) was detected by UV absorbance at 492 nm. Finally, the product was dialyzed against water to remove traces of NaCl. The product was obtained by freeze-drying. The amount of fluorophore coupled was determined by UV (492 nm). 2.7. Complex Formation and DNA Condensation Studies. The charge ratio is defined as the number of cationic charges of the polymer to the number of anionic charges from DNA. For the calculation of the charge ratio, a mass per charge of 325 Da was used for DNA. The mass per charge of all cationic polymers was calculated assuming that, under the experimental conditions, only the amine functions

Figure 3. DNA condensation pattern of P(DMAEMA0.48-co-AEMA0.42co-HYMIMMA0.10) (left part of the gel) and P(DMAEMA0.96-coAEMA0.04) (right part of the gel). The polymer-DNA complexes were prepared at different charge ratios and applied on a 1% agarose gel. DNA staining was performed by incubating the gel in a 5 µg/mL EtBr solution.

of the side groups with the highest pKa (DMAEMA) are protonated. The acid groups (MA) are nonprotonated and thus negatively charged, whereas the imidazole groups are supposed to be nonprotonated and thus neutral. The values of the mass per charge for the different polymers are shown in Table 1. For PEI, a mass per charge of 208 Da was used. The DNA condensation properties of the polymers were evaluated by agarose gel electrophoresis. Polymer-DNA complexes were prepared in water at different charge ratios, ranging from 0.2/1 to 3/1 (() using linear calf thymus DNA, as described earlier.29-30 The results are shown in Table 2 and Figure 3. 2.8. Transfection and Toxicity Studies. The transfection efficiency of the polymers in the monkey kidney fibroblast Cos-1 cell line was studied using the pCMVbeta plasmid, encoding β-galactosidase controlled by the CMV promotor, as described earlier.30 The transfection mixtures, containing the polymer-DNA complexes, were prepared as follows. Three compounds were added and mixed in a precise order: DNA, water, and polymer. The final volume of the mixtures was 80 µL, containing 0.06 mg/mL plasmid DNA and the appropriate amount of polymer to obtain the desired charge ratio. The quantification of the β-gal mass was performed using a β-gal ELISA kit. Cellular protein determinations were performed with the BCA Protein Assay kit. The cytotoxicity of all polymer-DNA complexes was determined by means of the MTT assay as described earlier.30 All data (Table 3) are reported as mean ( standard deviation for triplicate samples. The results represent the toxicity of

Table 3. β-gal Mass and Amount of Cell Protein for the Transfection of Cos-1 Cells with Polymer-DNA Complexes at a 2/1 and a 4/1 Charge Ratio, n ) 3a 2/1 charge ratio polymer PDMAEMA P(DMAEMA0.96-co-AEMA0.04) P(DMAEMA0.48-co-AEMA0.42co-HYMIMMA0.10) P(DMAEMA0.65-co-MA0.35) polyethyleneimine

4/1 charge ratio

Mw (kDa)

β gal (ng/mL)

cell protein (µg/mL)

% cell viability

β gal (ng/mL)

cell protein (µg/mL)

% cell viability

201 273 47

5.78 ((0.63) 2.64 ((0.32) 0.01 ((0.03)

43.30 ((1.55) 47.20 ((2.01) 47.40 ((1.89)

108 ((3) 98 ((5) 107 ((1)

19.57 ((0.19) 6.48 ((0.21) 1.66 ((0.90)

40.04 ((2.38) 42.03 ((1.57) 39.52 ((2.62)

103 ((1) 108 ((5) 97 ((2)

316 25

0.02 ((0.02) 22.34 ((1.49)

57.84 ((1.53) 22.24 ((4.89)

106 ((2) 79 ((6)

0.01 ((0.01) 9.25 ((0.83)

48.70 ((3.80) 17.64 ((1.04)

102 ((7) 65 ((9)

a Control: DNA without polymer: β-gal mass 0.08 ng/mL (( 0.05); amount of cell protein 51.77 µg/mL (( 5.42). Lipofectamine: β-gal mass 60.10 ng/mL (( 22.54); amount of cell protein 19.56 µg/mL (( 1.83).

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the complexes compared to untreated cells (100% cell viability). Results were statistically analyzed with the student t-test. 2.9. Confocal Microscopy Studies. Cellular internalization studies were carried out with a Zeiss model LSM 410 invert on the basis of a Zeiss Axiovert 100 fluorescence microscope. The apparatus was equipped with an argon laser (488 nm) and a Helium-Neon laser (543 and 633 nm). Cos-1 cells were seeded at a density of 105 cells/0.25 cm2 and grown for 48 h in DMEM supplemented with 10% FBS and 1% Penicillin/Streptamycin (104 IU/mL). Cells were washed and preincubated in 200 µL DMEM. The complexes (7.5 µL complex prepared as in section 2.8) were added to each well. 30 min before measuring 50 nM Lysotracker Red was added. Prior to microscopy examination, cells were washed and placed in PBS. 3. Results and Discussion Previously, we described the synthesis and the physicochemical and biological evaluation of a series of cationic polymethacrylates as nonviral gene delivery systems.27,29-30 These studies showed that PDMAEMA, a polymer containing only tertiary amine functions, reaches 90% of the transfection efficiency of PEI, one of the golden standards in the field of polymeric gene delivery systems. Although DMAEMA-based copolymers containing imidazole groups (HYMIMMA) or acid functions (MA) possess similar buffering properties as PEI, these polymers were not able to transfect Cos-1 cells (monkey kidney fibroblasts). In the present work, we investigated the cellular internalization and the subcellular localization of the complexes based on these polymers. 3.1. Polymer Synthesis and Labeling. Since PDMAEMA and P(DMAEMAx-co-HYMIMMAy) do not contain primary amine functions, necessary for further functionalization of these polymers, we first synthesized a t-Boc protected amine containing methacrylate (t-Boc AEMA). In a subsequent step, this monomer was copolymerized with DMAEMA and/or t-Boc HYMIMMA yielding polymers containing primary amines, which could be used for the coupling of a fluorophore. t-Boc AEMA was synthesized in a two step procedure (Figure 1). First, the amine function of ethanolamine was protected by reaction with di-tert-butyl dicarbonate. This product was converted into the corresponding methacrylate by reaction with methacrylic anhydride. Starting from this monomer, two different types of polymers were synthesized: a copolymer containing DMAEMA and t-Boc AEMA and a terpolymer containing DMAEMA, t-Boc AEMA, and t-Boc HYMIMMA. The t-Boc protecting groups of both polymers were removed by acidolysis in trifluoro acetic acid. The chemical composition of the polymers as determined by 1H NMR spectroscopy and their molecular weight, as determined by GPC, are shown in Table 1. The refractive index increments (dn/dc) of the polymers needed for the calculation of the molecular weight are also given in Table 1. The chemical structure of the different types of polymers is shown in Figure 2. The synthetic pathway applied in this work is a simple procedure to obtain organic soluble methacrylate (or acry-

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late)-based polymers containing primary amine functions. Earlier, van Dijk-Wolthuis et al. proposed a method in which DMAEMA and AEMA were copolymerized in water.36 PEI, P(DMAEMA0.96-co-AEMA0.04) and P(DMAEMA0.48co-AEMA0.42-co-HYMIMMA0.10) were coupled via their amine groups to a spacer-linked succinimidyl ester of Oregon Green (see Figure 2).37 P(DMAEMA0.65-co-MA0.35) was modified with an amine containing fluorophore, 5-(aminomethyl)fluorescein hydrochloride (see Figure 2), using EDC as coupling agent. 3.2. DNA Condensation Studies. The ability of the different polymers to condense DNA was studied by agarose gel electrophoresis. The charge ratio at which DNA condensation occurs for the different polymers is shown in Table 2. As an example, the agarose gel of P(DMAEMA0.96-coAEMA0.04) and P(DMAEMA0.48-co-AEMA0.42-co-HYMIMMA0.10 is shown in Figure 3. The results show that all polymers are able to condense DNA, being a prerequisite for any polymer to be used as a DNA vector. All polymers, except PEI, are condensing DNA at a similar charge ratio (between 1.2/1 and 1.6/1). Most likely, the presence of backbone amine functions in PEI enhances the DNA condensation properties of this polymer, resulting in a lower charge ratio for DNA condensation (0.4/1). 3.3. Transfection and Toxicity Studies. The polymers synthesized in this work were evaluated for their biological activity, including their transfection efficiency (determination of β-gal mass) and their toxicity (determination of the amount of cell protein and MTT reduction). The results are shown in Table 3. The β-gal expression of cells grown for 48 h after 4 h exposure to the polymer-DNA complexes was comparable for PDMAEMA-DNA complexes prepared at a 4/1 charge ratio and PEI-DNA complexes prepared at a 2/1 charge ratio. Compared to PDMAEMA, copolymers containing tertiary amines (DMAEMA) and primary amine (AEMA) were significantly lower in the transfection efficiency, both at charge ratios of 2/1 and 4/1 (P < 0.002). This might be attributed to both differences in chemical composition and molecular weight. As shown previously, the molecular weight strongly influences the transfection efficiency of both PEI and polymethacrylates.30,38-39 Imidazole and carboxylic acid containing polymers were not able to transfect Cos-1 cells. The amount of cell protein (as determined by the BCA assay) of cells treated with polymethacrylate-DNA complexes is at least twice that of cells treated with PEI-DNA complexes. This is also reflected by the MTT assay in which cells treated with PEI-DNA complexes have a significantly (P < 0.02) lower cell viability compared to cells treated with polymethacrylate-DNA complexes (Table 3). Our results show that the determination of the total cell protein content is a simple and time-saving method for the toxicity determination of polymer-DNA complexes. This is in accordance with previous reports.30,40-41 3.4. Confocal Microscopy Studies. To find out whether the differences in transfection efficiency in Cos-1 cells are related to differences in cellular internalization and subcellular distribution, confocal microscopy experiments were performed on Cos-1 cells treated with the polymer-DNA



Figure 5. Cellular internalization of P(DMAEMA0.96-co-AEMA0.04)DNA complexes as studied by confocal microscopy. The complexes were prepared at a 2/1 charge ratio. The bars represent 10 µm.

Figure 4. Cellular internalization of PEI-DNA complexes as studied by confocal microscopy. The complexes were prepared at a 2/1 charge ratio. Part E represents endosomal/lysosomal staining of Cos-1 cells. The bars represent 10 µm.

complexes containing fluorescently labeled polymers. Cells were co-incubated with Lysotracker Red DND-99 probe, wich specifically stains acidic organelles (i.e., late endosomes and lysosomes).42 In Figures 4-7, the green fluorescence corresponds to polymer-DNA complexes, whereas red fluorescence arises from the Lysotracker Red probe. Colocalization of both signals is reflected by yellow fluorescence. The effect of the incubation time and the charge ratio of the complexes on the cellular internalization and subcellular localization were evaluated. 3.4.1. Effect of the Incubation Time. Figure 4 shows the results obtained for PEI-DNA complexes prepared at a 2/1 charge ratio. After 15 and 45 min incubation (Figure 4, parts A and B), most of the green fluorescently labeled polymer was colocalized with the LysoTracker Red probe, indicating endosomal and lysosomal localization of PEI-DNA complexes. It should also be noted that no membrane associated green fluorescence appears. These results are in very good agreement with those obtained earlier by Re´my-Kristensen et al. using synchronized L929 fibroblasts,43 showing that PEI-DNA complexes were efficiently taken up in the endosomes in less than 10 min.

At incubation times of 1.5 h or longer (Figure 4, parts C and D), all yellow fluorescence disappeared, indicating that co-localization decreased. PEI-DNA complexes are thus gradually released from the endosomes and lysosomes into the cytoplasm of the cell. It can be concluded that PEIDNA complexes are taken up rapidly and not continuously over the entire incubation period. These findings can be useful for fine-tuning the transfection protocols for PEIDNA complexes since shorter incubation times might decrease the toxicity of PEI-DNA complexes (or polymerDNA complexes in general). In the 4 h incubation period of PEI-DNA complexes with Cos-1 cells, we did not observe a clear uniform nuclear localization of labeled PEI. These results are in agreement with those obtained by Re´my-Kirstensen et al. in synchronized L929 fibroblasts43 and Bieber et al. in PaTu 8902 carcinoma cells,44 but they are in contrast with those obtained by Godbey et al. using EA.hy 926 cells,45-46 suggesting that PEI transfection may involve different mechanisms, depending on the cell type. After 15 min, P(DMAEMA0.96-co-AEMA0.04)-DNA complexes (2/1 charge ratio) are taken up by Cos-1 cells (Figure 5A). However, compared to PEI-DNA complexes (Figure 4A), a higher amount of red spots, indicative for endosomes without polymer-DNA complexes, is observed. After 30 min incubation, all of the Lysotracker Red probe is colocalized with P(DMAEMA0.96-co-AEMA0.04)-DNA complexes, indicated by an decrease in the amount of red spots and an increase in the amount of yellow spots (Figure 5B). After 4 h incubation (Figure 5 C), most P(DMAEMA0.96-coAEMA0.04)-DNA complexes are localized in the cytoplasm, whereas some complexes remain localized in the acidic cell organelles. These findings indicate a difference in the kinetics of cellular internalization and endosomal escape of PEI-

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Figure 7. Cellular internalization of P(DMAEMA0.65-co-MA0.35)-DNA complexes as studied by confocal microscopy. The complexes were prepared at a 2/1 charge ratio. The bars represent 10 µm.

Figure 6. Cellular internalization of P(DMAEMA0.48-co-AEMA0.42-coHYMIMMA0.1)-DNA complexes as studied by confocal microscopy. The complexes were prepared at a 2/1 charge ratio (parts A-C) or a 4/1 charge ratio (parts D and E). The bars represent 10 µm.

DNA complexes and P(DMAEMA0.96-co-AEMA0.04)-DNA complexes. Compared to P(DMAEMA0.96-co-AEMA0.04)DNA complexes, PEI-DNA complexes are taken up more rapidly into Cos-1 and are released more rapidly and to a greater extent into the cytosol. The cellular internalization of P(DMAEMA0.48-co-AEMA0.42-co-HYMIMMA0.10)-DNA complexes prepared at a 2/1 charge ratio is less efficient than for PEI- and P(DMAEMA0.96-co-AEMA0.04)-based complexes. After incubation for 15 min, no cellular accumulation of polymer-DNA complexes was observed (Figure 6A). At an incubation time of 2 h, no co-localization was observed (Figure 6B). After incubation for 4 h, some co-localization was observed, but most of the endosomes were only stained with the Lysotracker Red probe (Figure 6C). These results indicate that the low transfection efficiency of P(DMAEMA0.48-co-AEMA0.42-coHYMIMMA0.10)-DNA complexes at a 2/1 charge in Cos-1 might be attributed to the low cellular internalization of the complexes. After 15 min, the cellular internalization of P(DMAEMA0.65co-MA0.35) based complexes prepared at a 2/1 charge ratio

in Cos-1 cells was very low (Figure 7A). After 30 min, these complexes were localized at the plasma membrane but are not internalized in the cytosol (Figure 7B). After incubation for 1 h, co-localization of the complexes with the Lysotracker Red probe was observed, indicative for endosomal and lysosomal localization (Figure 7C). After incubation for 4 h, most complexes were still located in the endosomal compartment (data not shown), as no accumulation of the polymer in the cytosol occurred. Thus, the low transfection efficiency of this polymer compared to PEI can be attributed to a (s)lower uptake via endocytosis and an unefficient release of the complexes in the cytoplasm. 3.4.2. Effect of the Charge Ratio of the Complexes. Since P(DMAEMA0.48-co-AEMA0.42-co-HYMIMMA0.10)-DNA complexes were not able to transfect Cos-1 cells, neither at a 2/1 nor at a 4/1 charge ratio, we investigated if the low transfection activity at a 4/1 charge ratio is also related to a low cellular internalization of these complexes. At a 4/1 charge ratio, the amount of positive charges in the complexes is much higher compared to those prepared at a 2/1 charge ratio. It might thus be possible that the cellular internalization and the subcellular localization depend on the charge ratio of the complexes used. After incubation for 1 h, the polymer-DNA complexes prepared at a 4/1 charge ratio are localized at the cell membrane and in acidic cell organelles (Figure 6D). After incubation for 4 h, membrane staining decreased with some indication of complexes in the endosomes (Figure 6E). These results show that P(DMAEMA0.48-co-AEMA0.42-co-HYMIMMA0.10)-DNA complexes prepared at a 4/1 charge ratio are taken up faster than those at a 2/1 charge ratio. At both charge ratios, complexes are not able to escape from the acidic organelles in the 4 h incubation of Cos-1 cells, which might explain the low transfection efficiency.


It can be concluded that polymethacrylate based complexes with buffering properties similar to PEI are taken up in Cos-1 cells but are not released as efficiently as PEI from the endosomal compartment. Thus, the buffering properties of cationic polymethacrylates are not the only key to efficient escape from the endosomes. It should be considered that other factors such as the membrane interaction properties might limit the release of the complexes from the endosomes. Indeed, we demonstrated earlier that the membrane interactive properties of PEI-DNA complexes are higher compared to the polymethacrylate-DNA complexes (as shown earlier by haemolysis studies30). This may influence the release of the complexes from the endosomal compartment. Moreover PEI-DNA complexes swell when the pH is lowered from physiological to endosomal conditions, whereas the size of polymethacrylate-DNA complexes decreases when the pH is lowered.27 It can be anticipated that this will affect the release of the complexes into the cytoplasm of cells. To further explore both phenomena, we are presently studying the effect of varying pH on the particle size of PEIand PDMAEMA-DNA complexes in more detail, trying to mimmick the cellular internalization and endosomal release of polymer-DNA complexes. In addition, we are optimizing the membrane interactive properties of the polymethacrylates in an attempt to try to increase their transfection efficiency. 4. Conclusions In the present work, we synthesized a series of cationic polymethacrylates containing fluorophores. Transfection studies revealed that complexes based on imidazole and acid function containing polymethacrylates, which possess similar buffering properties as PEI, are not able to transfect Cos-1 cells. Confocal microscopy pointed out that these complexes are taken up into cells via endocytosis but are slowly released from acidic organelles (late endosomes and/or lysososmes). These findings clearly indicate that the buffering properties of polymethacrylates are not the only key to successful endosomal escape and gene delivery. Acknowledgment. The authors thank the Flemish institute for the promotion of Scientific-Technological Research in Industry (IWT), the Fund for Scientific Research-Flanders (FWO), the Belgian Ministry of Scientific Programming, IUAP/PAI-V, and the European Union’s Biotechnology Program Contract No. 97 2334 with support from INCO Contract No. IC 20 CT 970005. Abbreviations AEMA: aminoethyl methacrylate AIBN: 2,2′-azobis(2-methylpropionitrile) DMAEMA: 2-N,N-(dimethylaminoethyl) methacrylate MA: methacrylic acid HYMIMMA: 4-methyl-5-imidazoyl methyl methacrylate Mw: weight average molecular weight PEI: polyethyleneimine t-Boc: tert-butyloxycarbonyl GPC: gel permeation chromatography EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride DMEM: Dulbecco’s Modified Eagle medium


FBS: fetal bovine serum PBS: phosphate buffered saline NMR: nuclear magnetic resonance spectroscopy MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide AIBN: 2, 2′-azobis(2-methylpropiononitrile) DNA: deoxyribonucleic acid.

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BM034438D

Buffering Properties of Cationic Polymethacrylates Are Not the Only

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