Bioconjugate Chem. 2007, 18, 922−928
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Quaternized Poly(4-vinylpyridine)s as Model Gene Delivery Polycations: Structure-Function Study by Modification of Side Chain Hydrophobicity and Degree of Alkylation Aure´lie San Juan,† Didier Letourneur,† and Vladimir A. Izumrudov*,‡ Inserm, U698, Department of Bioengineering, X. Bichat Hospital, University Paris 7, Paris, F-75018, France, Institut Galilee, University Paris 13, Villetaneuse, F-93430, France, and M. V. Lomonosov Moscow State University, Department of Chemistry, 119992 Moscow, Russia. Received October 10, 2006; Revised Manuscript Received February 6, 2007
To optimize polycation-based gene delivery agents, the influence of molecular characteristics of the polycations on physicochemical properties of polycation/DNA complexes and their relationships to cellular gene transfer need to be understood. With this aim, we have synthesized a series of model polymers based on quaternized poly(4-vinylpyridine)s (CnPVP-β) with the same DP of 1600 but differing by the number n of methylene groups in N-alkyl ester substituents from 1 to 6 and/or by degree of alkylation β from 25% to 95%. For polycations CnPVP-95, the efficiency of transfection of a plasmid vector expressing a secreted form of alkaline phosphatase started to be detectable at n ) 4, noticeable at n ) 5, and again undetectable at n ) 6. A decrease in β of active C5PVP-95 from 95% to 65% resulted in a further noticeable increase of activity with a 9-fold increase for C5PVP65. This finding was attributed to the proton sponge mechanism due to protonation of non-alkylated pyridine moieties of CnPVP-β/DNA complexes in slightly acidic media that was supported by the fluorescence quenching assay. The data demonstrate the advantages of partial alkylation of tertiary polyamines with medium-length alkyl agents for preparation of efficient nonviral gene delivery vectors.
INTRODUCTION Gene therapy refers to the transfer of DNA encoding a therapeutic gene of interest into the target cells or organs with consequent expression of the transgene. In the past decade, gene therapy has received significant attention due to its potential application for treatment of acquired diseases (1, 2). The most effective vectors for gene transfection are those based on viruses and viral peptide sequences (3, 4). However, concerns with immunogenic and long-term oncogenic effects have prompted the concurrent development of synthetic gene delivery systems, such as liposomes, lipids, or polycations (5, 6). Although naked DNA has been found to transfect some cells like the skeletal muscle cells of the cardiac and diaphragm regions, the pronounced negative charge of the double helix tends to inhibit DNA from entering cell membranes. Moreover, the unprotected DNA is rapidly degraded by nucleases. Linear cationic polymers, such as poly(lysine) and its conjugates (7-9), poly(N-ethyl-4vinylpyridinium) bromide and its derivatives (10, 11), branched polymers such as poly(ethylene imine) (12), and poly(amidoimine) dendrimers (13, 14) effectively bind to the negatively charged DNA to form a polyelectrolyte complex that can be taken up by cells. The electrostatic binding of the polycation to the phosphate groups leads to DNA compaction. By being packed in the polyelectrolyte complex, DNA is shielded from contact with nucleases. It has been observed that in vitro transfection becomes efficient when complexes have a net positive charge to interact with negatively charged cell membranes. Numerous works on liposomes or polymers for gene therapy indicated that the modification of positively charged chains with hydrophobic residues enhances their binding to cell membranes * Corresponding author. Moscow State University, Department of Chemistry, Leninskiye Gori V-234, 119992 Moscow, Russia. Telephone 7-4959393117, fax 7-4959390174, E-mail:
[email protected]. † University Paris. ‡ M. V. Lomonosov Moscow State University.
and penetration into cells. For gene transfer with cationic oligomers, long alkyl sequences were commonly used. For instance, the transformation of various mammalian cells was performed by DNA complexes with lipospermines that were synthesized by chemical modification of natural oligocation spermine with two long C18 alkyl tails (15, 16). N-Terminal stearylation of oligoarginines consisting of 4-16 repeat units increased the transfection efficiency by 100 times with the maximal efficiency displayed by stearyl-Arg8 (17), but the decrease in the number of methylene groups from 16 in the stearyl-Arg8 to 10 in N-terminal lauryl-Arg8 significantly reduced the efficiency. Few detailed reports are available for higher molecular weight structures. Thus, the introduction of 3-4% of C16 N-alkyl moieties in poly(N-ethyl-4-vinylpyridinium) bromide noticeably enhanced its affinity with DNA to negatively charged phosphatidylcholine/cardiolipin liposomes used as models of biological membranes (18). However, the hydrophobized polycation exhibited less transfection efficacy than unmodified poly(N-ethyl-4-vinylpyridinium) bromide (19, 20). In another study, an increase of a number of methylene moieties in the side chain charge groups of cationic polyphosphoesters from 2 to 6 was accompanied by a 3-fold increase of the transfection efficacy (21). Similarly, an increase of the length of hydrophobic groups incorporated into the backbone of cationic polyvinyl ethers from 1 to 4 carbons was shown to significantly improve the transfection efficacy (22). In the present study, combinations of length of the hydrophobic side chains and their content were evaluated using a series of model cationic copolymers of 4-vinylpyridine and N-alkyl-4-vinylpyridinium obtained by quaternization with ω-bromoesters. The hydrophobicity of the copolymers was controlled by a number of methylene groups in the ω-bromoesters and the degree of the quaternization. The main advantages of such model copolymers are their controllable hydrophiliclipophilic balance and the presence of pH-induced ionizing 4-vinylpyridine units in their chains.
10.1021/bc060317+ CCC: $37.00 © 2007 American Chemical Society Published on Web 04/03/2007
Quaternized Poly(4-vinylpyridines) Scheme 1. Synthesis Route for Quaternization of Poly(4vinylpyridine) Yielding (1) Exhaustively Alkylated Polycations CnPVP (n ) 1 to 6) and (2) Polycarboxybetaines CnPB
EXPERIMENTAL SECTION Materials. NaOH, HCl, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), Tris, MES, and HEPES buffers were purchased from Sigma (St. Louis, MO). In all experiments, twice-distilled water was used and purified by Milli-Q (Millipore, U.S.A.). Dulbecco’s modified Eagle’s media (DMEM) and fetal calf serum were obtained from BioMedia, France. Ethidium bromide was purchased from Sigma (St. Louis, MO). Concentration of ethidium bromide in solution was determined spectrophotometrically assuming molar extinction coefficient 5600 L‚mol-1 cm-1 at 480 nm (23). Poly(ethylene imine) (PEI) was purchased from Aldrich (MW ) 25 000 g/mol) or from Euromedex (ExGen 500, MW ) 22 000 g/mol). Na salt of highly polymerized calf thymus DNA (∼10 000 base pairs) was purchased from Sigma (St. Louis, MO) and used without further treatment. Concentration of DNA phosphate groups in the solutions was determined by UV absorbance measurements at 260 nm assuming molar extinction coefficient 6500 L‚mol-1 cm-1 (24). pcDNA3-SEAP2 expression plasmid (pSEAP; 7443 base pairs) driven by the cytomegalovirus promoter was obtained by inserting the EcoRI-SalI fragment of the pSEAP2-control plasmid (Clontech), which contains the cDNA encoding for a secreted form of alkaline phosphatase (SEAP) preceded by the Kozak consensus translation initiation site and followed by the late polyadenylation signal of simian virus SV40, at the EcoR-SalI cloning sites of the pcDNA3 expression plasmid (Invitrogen). Characterization Techniques. 1H NMR spectra were recorded with a VARIAN Unity Inova 500 MHz spectrometer at 500.6 MHz using D2O as solvent. IR spectroscopy was performed using a ThermoNicolet AVATAR 370 FTIR spectrometer (Thermo Electron Corparation, Waltham, MA). Synthesis of Quaternized Poly(4-vinylpyridine)s (CnPVP) (1). Quaternized poly(4-vinylpyridine)s that possessed the same average degree of polymerization, DP 1600, but differed by the number n of methylene groups in N-alkyl ester substituents (n ) 1 to 6) were synthesized by alkylation of poly(4-vinylpyridine) sample (MW ) 168 000 g/mol; Aldrich, U.S.A.) with esters of corresponding ω-bromocarboxylic acid (Scheme 1) as described elsewhere (25). The values of degree of alkylation of CnPVP samples that were determined by FTIR measurements as described elsewhere (26) were 95 ( 3%.
Bioconjugate Chem., Vol. 18, No. 3, 2007 923 Scheme 2. Structural Formulas of Copolymers (3) of 4-Vinylpyridine and N-Alkyl-4-vinylpyridinium (CnPVP-β) with Different Content of N-Alkylpyridinium Moieties (β from 25% to 95%) and Copolymers (4) of N-Methyl-4-vinylpyridinium and N-Alkyl-4-vinylpyridinium (DMS-CnPVP-β) Obtained by Exhaustive Alkylation of 4-Vinylpyridine Units of CnPVP-β Samples with Dimethylsulfate
Synthesis of Polycarboxybetaines (CnPB) (2). Polycarboxybetaines were obtained from the corresponding CnPVP (n ) 4 and 5) (Scheme 1) by alkaline hydrolysis that was performed by dropwise addition of concentrated NaOH solution to the solution of CnPVP at 60 °C for several hours (25) according to the procedure described in ref 27. The hydrolysis was estimated from the potentiometric titration curves of aqueous solutions of the products that were conducted in the presence of sodium poly(styrene sulfonate) as described (25). The assay of aqueous solution of parent CnPVP, as well as CnPVP-β by the same approach, evidenced the absence of spontaneous hydrolysis in neutral media at room temperature even after storage of solutions for 2 weeks. Synthesis of Copolymers of 4-Vinylpyridine and N-Alkyl4-vinylpyridinium (CnPVP-β) (3). Copolymers of 4-vinylpyridine and N-alkyl-4-vinylpyridinium (CnPVP-β) with different content of N-alkylpyridinium moieties (β from 25% to 95%) were obtained by partial quaternization of poly(4vinylpyridine) with the esters (Scheme 2). Samples were successively withdrawn from the reaction mixture during the course of the alkylation, and the values of β were estimated by FTIR spectroscopy (26). The presence of N-alkylpyridinium moieties in the copolymers was evidenced by 1H NMR (e.g., the spectrum of C5PVP-65 in D2O with δ ) 8.60, 7.43, 4.16, 2.41, 1.96, 1.67, and 1.26 ppm is depicted in Figure 1). Synthesis of Copolymers of N-Methyl-4-vinylpyridinium and N-Alkyl-4-vinylpyridinium (DMS-CnPVP-β) (4). Copolymers of N-methyl-4-vinylpyridinium and N-alkyl-4-vinylpyridinium (DMS-CnPVP-β) were obtained by exhaustive
Figure 1. 1H NMR spectrum of C5PVP-65 copolymer. 500 MHz spectra were recorded using D2O as solvent.
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alkylation of 4-vinylpyridine units of CnPVP-β samples with dimethylsulfate in methanol at room temperature. Spectrophotometric Measurements were performed using Hitachi 150-20 Spectrometer (Japan) in water-thermostatic cell at 25 °C. Fluorescence Measurements. Fluorescence intensity of the solutions was measured using a Jobin Yvon-3CS spectrofluorimeter (France) with water-thermostatic stirred cell holder. The measurements were made in a capped quartz fluorescence cell upon permanent stirring at 25 °C. The excitation and emission wavelengths in experiments with ethidium bromide were set at 535 and 595 nm, respectively. Stability of CnPVP/DNA Complexes in Water-Salt Solutions. The DNA solution was directly mixed in the fluorescence cell with ethidium bromide at a molar ratio [ethidium bromide]/ [P] ) 0.25 where [P] is molar concentration of DNA phosphate groups. At this ratio corresponding to 1 molecule of ethidium bromide per 2 base pairs (4 nucleotides), the maximum of ethidium bromide fluorescence intensity was observed (28). The concentration of DNA phosphate groups was 4 × 10-5 mol‚L-1. The solution of DNA/ethidium bromide complex was titrated with CnPVP solution that was accompanied by quenching of ethidium bromide fluorescence due to displacement of the intercalated dye from DNA to the solution. The fluorimetric titration was conducted until the quenching was completed at a charge ratio [+]/[-] of 1.5. The mixtures were titrated with 4 M NaCl solution with a time interval between the titrant additions of 5 min. Destruction of the ion pairs in the CnPVP/ DNA polyelectrolyte complex by the added salt was monitored by the increase in fluorescence intensity I of the solution caused by re-intercalating of the dye into free sites of DNA. In parallel, a solution of DNA/ethidium bromide complex at the same concentration was titrated with 4 M NaCl solution, and the fluorescence intensity I0 was measured. The stability of CnPVP/ DNA complexes was estimated by values of the salt concentration, [NaCl]*, corresponding to I/I0 ) 1, i.e., when a complete dissociation of the complex CnPVP/DNA occurred (29). Note that the presence of the intercalated dye in the double helix weakens DNA binding to the polycations (28), but the contribution of this factor to the complex stability is relatively low and identical for all samples (29). pH Profiles of Polycations with DNA. The fluorimetric titrations of DNA/ethidium bromide complex with solutions of C5PVP-25, C5PVP-65, or PEI were conducted at different pH values in 0.01 M MES or HEPES buffer solutions. This approach particularly applies for polycations with a large content of the ionizable amino groups that provides high accuracy in the calculations of a degree of conversion Θ in the polyelectrolyte coupling reaction (where Θ is the ratio of a current number of the protonated amino groups to the ultimate one). In neutral and slightly acidic media, the fluorescence intensity decreased linearly with the increase in charge ratio from 0 to 1 (30). The degree of conversion was determined in an equimolar mixture ([+]/[-] ) 1) of polycations and DNA. The pHindependent contribution of (100 - β)% of N-alkylated units of C5PVP-β polymers to the interaction with DNA that was accompanied by a displacement of ethidium bromide was determined but not taken into account upon calculation of the pH profile. In Vitro Cell Transfection. Transfection experiments were carried out on rabbit vascular smooth muscle cells (SMCs; Rb-1 cell line (31)) cultured in DMEM supplemented with 1% penicillin, streptomycin, and amphotericin, 2% HEPES buffer, 1% glutamine, and 10% fetal calf serum. CnPVP-β polycation and pSEAP solutions were filtered onto 0.22 µm Millipore filters before use. Twenty-four well plates were seeded with 5 × 104 cells per well 24 h before transfection. Ten microliters of pSEAP
San Juan et al.
solution at 1 µg/µL was mixed with 10 µL of CnPVP-β polycation solution at a concentration ranging from 0.06 µg/ µL to 0.98 µg/µL, depending on the polycation, and corresponding to a charge ratio [+]/[-] of 1 or 5, assuming all amines were cationic. The resulting complex solution was diluted to a final volume of 50 µL with either serum-free or serumcontaining culture medium, vortexed for 10 s, and allowed to stand at room temperature for 30 min. Cells were washed, and 450 µL of serum-free or serum-containing culture medium per well was added. The complex solution (CnPVP-β/pSEAP) was then added to the wells, and cells were incubated at 37 °C in 95% air/5% CO2 for 24 h. Cells were cultured for 72 h in serumcontaining medium, and then the conditioned culture medium was removed and analyzed for SEAP activity. Phospha-Light kit (Tropix, Bedford, MA) was used for the measurement of SEAP activity, according to the manufacturer’s protocol. Light output was measured using a luminometer (LUMAT LB9501, Berthold, Wildbach, Germany), as described elsewhere (32). SEAP activity was expressed as relative light units (RLU) per 105 cells. Cells incubated with culture medium alone or with pSEAP alone were used as controls. Cytotoxicity was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (see below). For each condition, at least 3 experiments were performed. Cell Viability. Evaluation of cell viability was performed with the MTT assay. At the end of the transfection experiments, the culture medium was replaced with 500 µL of fresh Hanks’ Balanced Salt Solution and 50 µL of 5 mg/mL MTT. After 1 h incubation at 37 °C, MTT-containing medium was removed and 300 µL of DMSO was added to dissolve the formazan crystal formed by viable cells. Absorbance was measured at 540 nm. Cell viability was calculated according to the following equation: Cell viability (%) ) (OD540(sample)/OD540(control)) × 100, where the OD540(control) is measured with cells incubated in control medium. Size and ζ-Potential Measurements. For each measurement, 750 µL of pSEAP at 0.03 µg/µL was mixed with 750 µL of polycation at concentrations ranging from 0.15 µg/µL to 0.29 µg/µL corresponding to a charge ratio [+]/[-] of 5. All the solutions were prepared in Tris buffer (pH ) 7.4). The resulting mixtures were vortexed for 10 s and incubated for 30 min at room temperature before analysis. The size of polycation/DNA complexes was determined by quasi-elastic light scattering with a ZetaSizer 3000 instrument (λ ) 633 nm, Malvern Instruments, U.K.) at 25 °C, using a sample refractive index of 1.59 and a viscosity of 0.89 cP. Data were collected at a scattering angle of 90° and sizes calculated from the correlation function using an automated method. Three series of five measurements were performed. ζ potentials were measured on the same instrument. Complexes used for the size determination were recovered and injected into the cell. The system was calibrated with a -50 ( 5 mV ζ-potential polystyrene standard from Malvern. The analysis parameters were as follows: viscosity ) 0.89 cP, dielectric constant ) 79, temperature ) 25 °C, Smoluchowsky constant F (ka) ) 1.50, dispersant refractive index ) 1.33. Statistical Analysis. All results are expressed as mean ( SD. Statistical comparisons were performed with the use of ANOVA and post-hoc PLSD test. A value of p < 0.05 was considered significant.
RESULTS AND DISCUSSION A series of cationic copolymers based on quaternized poly(4-vinylpyridine) was synthesized. Schemes 1 and 2 indicate the synthesis conditions and the general formula of compounds from 1 to 4. The advantages of utilizing quaternized poly(4vinylpyridines) as model polycations were as follows: (i) All polycations possessed the same average degree of polymeriza-
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Table 1. Critical Salt Concentration, [NaCl]*, Corresponding to Complete Dissociation of Different CnPVP/DNA Complexes Measured by Ethidium Bromide Displacement Assay CnPVP [NaCl]*, M
Figure 2. Effect of the number n of methylene groups in N-alkyl side chains of CnPVP polycations on cell transfection efficacy. Plasmid pSEAP (1 µg) was used alone or with CnPVP polycation solutions at charge ratio [+]/[-] of 5 in serum-free culture medium. The degree of alkylation was 95% for all polymers. Gene expression was measured 3 days after transfection. Transfection efficacy is expressed as relative light units (RLU) per 105 cells. Results represent the mean and standard deviation of 3 experiments. *, p < 0.0001 vs pSEAP alone.
tion, DP ) 1600, that provided high stability of DNA/ polycations and allowed circumvention of the possible influence of DP on the transfection efficiency (33). (ii) The opportunity to terminate the N-alkyl side chains with carboxylic groups by hydrolysis of the ester moieties (Scheme 1) allowed comparison of the transfection efficiency of polycarboxybetaines (2) to their parent polycations (1). (iii) The polycations remained soluble in aqueous media upon variation of the number of methylene groups in N-alkyl side chains (3) from 1 to 6 and/or the degree of alkylation β g 25%. And, finally, (iv) non-alkylated pyridine units of all studied copolymers were accessible according to Scheme 2 to the alkylating agent dimethylsulfate, that allowed their quantitative quaternization (4) to establish the role of the pyridine groups in the transfection. Alkylated CnPVP Polycations (1). To elucidate the effect of hydrophobicity of the side chain on cell transfection, quaternized poly(4-vinylpyridine) polymers with the degree of quaternization of 95 ( 3% and an increasing number n of methylene groups from 1 to 6 in the N-alkyl substituents (Scheme 1) have been synthesized. The transfection efficiency was first estimated in the conditioned culture medium 3 days after incubation of cells for 24 h with mixtures of CnPVP and a plasmid DNA encoding for the secreted form of alkaline phosphatase (pSEAP) in DMEM. No toxicity was noticed after incubation of cells with pSEAP and CnPVP polycations (cell viability was 88.2 ( 9.9%). At a charge ratio [+]/[-] of 1, C1PVP and C5PVP were both inactive (1.2 ( 0.1 × 103 RLU/ 105 cells), whereas significant SEAP activity (1.2 ( 0.2 × 105 RLU/105 cells) was found with C5PVP at a charge ratio [+]/[] of 5. When all exhaustively alkylated poly(4-vinylpyridine) polymers were evaluated at a charge ratio [+]/[-] of 5, SEAP activity remained rather low for CnPVP polycations with short N-alkyl substituents containing 1 to 3 methylene groups (Figure 2). At n ) 4, there was noticeable SEAP activity that became pronounced at n ) 5. However, at n ) 6, the activity was rather low (Figure 2). The n-dependent increase of transfection capacity correlated with previously reported data on transfection performed by cationic polyvinyl ethers with C1-C4 side chains in the backbone (22) whose efficacy was low with methyl- and ethylcontaining side chains, noticeably higher in the case of propyl residues, and significant with butyl moieties. Interestingly, C6PVP was inactive as a gene transfer agent with cells (Figure 2). The reason is most likely the hydrophobic interactions between side chains that are strengthened upon lengthening of the side chains. In the case of C6 N-alkyl substituents, the
n)1 0.40
n)2 0.35
n)3 0.34
n)4 0.31
n)5 0.24
n)6 0.15
contribution of these intramolecular hydrophobic interactions could be large enough to hinder significantly the interaction of the C6PVP polycation with DNA in a physiological buffer (DMEM; pH ) 7, NaCl 0.15 M). This assumption is based on the n-dependent stability of CnPVP/DNA complexes against the added salt that we studied by ethidium bromide displacement assay as described in the Experimental Section. The values of critical salt concentration determined by this approach for all CnPVP polycations are listed in Table 1. It can be seen that the increase in n from 1 to 4 was accompanied by a progressive decrease in critical salt concentration [NaCl]* corresponding to complete dissociation of the CnPVP/DNA complex. In the case of C5PVP and most notably C6PVP, the critical salt concentration was reduced markedly, suggesting that the side chains induced a pronounced steric hindrance that encumbered the electrostatic interactions between the charged pyridinium moieties and DNA phosphate groups. Thus, the maximum transfection capacity displayed by CnPVP at n ) 5 could be the result of optimal structure and composition of the C5PVP chain that provided the balance between hydrophobic and electrostatic interactions needed for the transfection. Polycarboxybetaines CnPB (2). C4PVP and C5PVP polycations that exhibited significant transfection activity were transformed to the corresponding polycarboxybetaines, C4PB and C5PB (Scheme 1). The synthesis was performed by an exhaustive hydrolysis of ester moieties that terminated the N-alkyl substituents of polycations. Figure 3 shows the transfection efficacy determined upon incubation of cells with the polycarboxybetaine/DNA complexes. From the cell viability tests, it can be concluded that C4PB and C5PB polycarboxybetaines were nontoxic. Substitution in side chains of the ester moiety with a carboxylic group resulted in the complete loss of transfection activity for both polycarboxybetaines (Figure 3). For comparison, transfection activity for the parent polycations is also plotted in Figure 3. From a physicochemical point of view, the substitution is accompanied by a noticeable change of the hydrophilic-lipophilic balance of the side chain. Polycarboxybetaines that possess carboxylic groups are much more hydrophilic as compared to their precursors and, hence, less
Figure 3. Cell transfection with polycarboxybetaines C4PB and C5PB. Plasmid pSEAP (1 µg) was used alone or with CnPB polycation solutions at charge ratio [+]/[-] of 5 in serum-free culture medium. C4PVP and C5PVP (with similar methylene groups) are plotted for comparison. The degree of alkylation was 95% for all polymers. Gene expression was measured 3 days after transfection. Transfection efficacy is expressed as relative light units (RLU) per 105 cells. Results represent the mean and standard deviation of 3 experiments. *, p < 0.0001 vs pSEAP alone.
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Figure 4. Effect of the alkylation degree (β) for C5PVP-β polycations on cell transfection. Complex formation and incubation of cells were performed with either serum-free or serum-containing culture medium. Plasmid pSEAP (1 µg) was used alone or with C5PVP-β solutions at charge ratio [+]/[-] of 5. Gene expression was measured 3 days after transfection. The data are presented as the relative SEAP activity taking as 100% the activity displayed by the corresponding exhaustively alkylated C5PVP-95 in serum-free medium. Results represent the mean and standard deviation of 3 experiments. *, p < 0.0001 vs control (β ) 95% in serum-free medium).
active in transfection. Besides, the carboxylic group being ionized in neutral media forms an ion pair with the pyridinium group in the betaine moiety. This weakens the electrostatic binding with DNA. Thus, the C5PB/DNA complex was destroyed at physiological ionic strength (25). Partially Alkylated CnPVP-β Polycations (3) and Exhaustively Alkylated DMS-CnPVP-β Polycations (4). To ascertain combinations of the length of the hydrophobic side chains and their content in the macromolecule, we have synthesized several series of cationic copolymers CnPVP-β that possessed the same n but differed by degree of alkylation β. Note that the nonalkylated 4-vinylpyridine units are uncharged at physiological pH and their protonation occurred in acidic media according to the relatively low value of the apparent pKa ) 3.25 of poly(4vinylpyridine). So, the composition of mixtures of partially alkylated polycations with pSEAP defined as a charge ratio [+]/[-] of 5 corresponds to the ratio of molar concentration of the quaternized N-alkylpyridinium moieties to molar concentration of phosphate groups. Figure 4 shows the transfection results obtained with the use of different C5PVP-β copolymers. The data are presented as the relative SEAP activity taking the activity obtained with C5PVP-95 in DMEM to be 100%. It can be seen that a decrease in β enhances the transfection of C5PVP95, with a maximal 9-fold increase for C5PVP-65. For comparison, the relative SEAP activity obtained under the same conditions with ExGen 500, a commercial poly(ethylenimine) that is considered an excellent transfecting agent, was 6000%. It is remarkable that a further decrease in β for C5PVP-β copolymers reduced the transfection efficiency that became negligible at β ) 25%. Interestingly, cell transfection from inactive and highly hydrophobic C6PVP-95 increased by reducing the alkylation degree with an optimum of significant transfection activity (85-fold increase from background level) at β ) 40%. To characterize the complex particles formed by C5PVP-β with pSEAP, size and ζ-potential measurements were carried out at charge ratio [+]/[-] of 5 (Figure 5). It can be seen that all studied complexes including the complexes with exhaustively alkylated polycations did not differ both in size and in ζ potential being 150 ( 15 nm and 12 ( 2 mV, respectively. This suggests that the trends in transfection ability of C5PVP-β copolymers cannot be attributed to the change of these parameters. Contribution of N-alkyl substituents to intramolecular hydrophobic interactions may reduce flexibility of polycations and, hence, hinder their matching with DNA. To modulate polymer
Figure 5. Size and ζ potential of complexes obtained with C5PVP-β polycations and plasmid pSEAP. Sizes (circles) and ζ potentials (triangles) were measured in Tris buffer (pH 7.4) at a charge ratio [+]/[-] of 5 for all polycations.
flexibility, we performed quantitative alkylation of non-alkylated pyridine units of CnPVP-β with dimethylsulfate treatment (4). The alkylation did not change the content and distribution of hydrophobic side chains along the macromolecule but transformed hydrophobic 4-vinylpyridine units that are not charged at physiological pH (non-alkylated poly(4-vinylpyridine) is insoluble in water at pH > 3.5) to hydrophilic quaternized 4-vinylpyridinium moieties containing relatively small N-methyl substituents (Scheme 2). Despite the higher net mean positive charge of the hydrophilized DMS-CnPVP-β, the gene transfer activity of all these polycations was negligible (Figure 6). pH Profiles of DNA Interaction with Polycations. The plausible explanation of the transfection activity of partially alkylated CnPVP-β polycations is based on the ability of nonalkylated pyridine groups to be protonated in acidic media, according to a mechanism generally referred to as the “proton sponge hypothesis”. When applied to a cell, the polycation/ DNA complexes are thought to mediate transfection via a multistage process that includes hydrophobic and/or cationic binding to the cell membrane, internalization by endocytosis, and subsequent entry into the cytoplasm (12). The hypothesis asserts that polymers with high cationic charge potential, such as poly(ethylene imine) (PEI), act as a proton sponge upon acidification within endosomes or lysosomes. Under the acidic environment within lysosomes, every third nitrogen atom is protonated. The highly branched network of PEI absorbs a large amount of proton ions, like a sponge, upon lowering of the pH. The increased osmolarity resulted in endosomal or lysosomal swelling and subsequent escape of DNA into the cytosol. The proton sponge hypothesis has been built essentially around the transfecting activities of some cationic polymers such as PEI, PAMAM dendrimers, or polyhistidine (34) with apparent pKa values close to pH 5.5, in relation to the acidic environment of endosomes and lysosomes. Yet, partially alkylated CnPVP-β should be protonated in more acidic media, since aromatic
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Figure 6. Effect of the exhaustive quaternization of C5PVP-β polycations with DMS on cell transfection efficacy. Plasmid pSEAP (1 µg) was used alone or with the exhaustively quaternized DMSC5PVP-β polycation solutions at charge ratio [+]/[-] of 5 for the indicated alkylation degree (β). Gene expression was measured 3 days after transfection. Transfection efficacy is expressed as relative light units (RLU) per 105 cells. Results represent the mean and standard deviation of 3 experiments.
Figure 7. Protonation profiles of C5PVP-25 (1), C5PVP-65 (2), and PEI (3) determined by the ethidium bromide displacement assay. The degree of conversion (Θ) is plotted at the indicated pH for equimolar mixtures of polyamines and DNA (charge ratio [+]/[-] of 1).
tertiary amino groups of pyridine rings define relatively low apparent pKa of 3.25 determined for poly(4-vinylpyridine) (35). As documented in Figure 7 by fluorimetric titration of polycations in the mixtures with DNA, the DNA promotes the protonation of polyamines that could occur with a ∆pH shift up to 2-3 units in the alkaline media as compared to ionization of free polyamine (36). The experimental procedure of estimating the protonation degree Θ of amino groups of polyamine bound in the complex with DNA is based on the ethidium bromide displacement assay performed at different pH (29). Figure 7 (curve 3) shows the protonation profile of PEI determined in an equimolar ([+]/[-] ) 1) mixture of PEI and DNA. The inflection on curve 3 is in accordance with the basicity of the tertiary amino groups that noticeably differs from basicity of the primary and secondary amino groups of PEI. It is seen that the whole profile of PEI/DNA protonation is arranged in neutral, but not in slightly acidic, media, and hence, the transfection efficacy of PEI could not be attributed to protonation of the complex. Note that transfection with PEI or other polycations is commonly performed with a large excess of the polyamine. Consequently, apart from the ionizable amino group of PEI bound with DNA, a large number of amino groups that are not influenced by DNA are presented in endosomes and lysosomes and protonated in the acidic environment
according to their intrinsic basicity. The protonation of these unbound amino groups appears to be the main reason for the well-documented high transfection efficacy of branched PEI. We used the same titration procedure for C5PVP-65 (the most effective CnPVP-β transfection agent) and for C5PVP-25 that possessed the greatest amount of ionizable non-alkylated pyridine moieties in the series of C5PVP-β copolymers but still remained soluble in aqueous media. The protonation profiles determined in equimolar mixtures of DNA with C5PVP-25 (curve 1) or C5PVP-65 (curve 2) are depicted in Figure 7. The left position of the protonation profile of C5PVP-25 is in agreement with low pKa of the pyridine rings. Nevertheless, the profile is markedly shifted to the right as compared to the ionization of free poly(4-vinylpyridine) (35). The protonation profile of the C5PVP-65 copolymer (curve 2) practically coincided with the profile of C5PVP-25 (curve 1) and also arranged in slightly acidic media. These findings support the hypothesis that the transfection activity of partially alkylated C5PVP-β polycations could be related to the proton sponge mechanism that is accomplished due to protonation of nonalkylated pyridine moieties of CnPVP-β/DNA complexes in slightly acidic media. Complexation in Physiological Conditions. According to the literature, the transfection activity of polycations in the presence of serum could be noticeably reduced due to the interaction of the unbound polycation chains with serum proteins. In the present study, all studied CnPVP polycations were shown to form stable complexes with DNA in salt concentration above the physiological conditions (Table 1). Moreover, complexation and incubation of cells with C5PVP-β copolymers in serum-containing media resulted in transfection activity (Figure 4). No significant loss in SEAP activities was observed as compared to serum-free medium. The transfection capacity of C5PVP-65 and C5PVP-80 copolymers was even slightly higher in serum-containing medium. This finding also supports the explanation of their high activity by protonation of CnPVP-β/DNA complexes and evidenced a suitable chemical structure of these polycations to bind to DNA in physiological conditions (Table 1, Figure 4) and then transfer pSEAP inside the cell, providing successful gene expression.
CONCLUSION The data demonstrate the advantages of partial alkylation of tertiary polyamines with medium-length alkyl agents for the preparation of future efficient nonviral gene delivery vectors. With the DNA binding effect on protonation of polyamines in endosomes and lysosomes, this extends the range of polycations capable of improving the transfection, even in physiological conditions, by involvement of polyamines with tertiary amino groups that possess relatively low pKa.
ACKNOWLEDGMENT The authors are grateful to Laurent Ve´ron and Thierry Delair (CNRS-bioMe´rieux, UMR 2714, ENS Lyon, France) for size and ζ-potential measurements and to Erwann Gue´nin (CNRS, UMR 7033 University Paris 13, France) for NMR measurements. This work was supported by INSERM, University Paris 7, and University Paris 13. Vladimir A. Izumrudov thanks University Paris 13 for invitations as visiting professor that supported this work.
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