Interpolyelectrolyte Reactions in Solutions of Polycarboxybetaines, 2

Concentration of DNA phosphate groups in the solutions was determined by UV ..... The pH of cloud points slightly varied but always occurred in a rela...
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J. Phys. Chem. B 2005, 109, 17391-17399

17391

Interpolyelectrolyte Reactions in Solutions of Polycarboxybetaines, 2:† Influence of Alkyl Spacer in the Betaine Moieties on Complexing with Polyanions Vladimir A. Izumrudov,* Natalia I. Domashenko, Marina V. Zhiryakova, and Olga V. Davydova Department of Chemistry, Moscow State UniVersity, Leninskie gory, Moscow 119992, Russia ReceiVed: April 8, 2005; In Final Form: July 27, 2005

Series of polycarboxybetaines (PCB-n) of pyridiniocarboxylate structure with the same degree of polymerization but differing in the number, n, of methylene groups in the alkyl spacer between charges in the betaine moieties, n ) 1, 2, 3, 4, 5, and 8, were synthesized. The utility of PCB-n as positively charged components of polyelectrolyte complexes was elucidated by potentiometry, turbidimetry, and fluorescence spectroscopy. Affinity of PCB-n to the pyrenyl-tagged poly(methacrylic) acid (PMAA*) or DNA was judged from the stability of the corresponding polyelectrolyte complexes in water-salt solutions at different pH values as monitored by fluorescence quenching techniques. At pH ) 9.0, PCB-1 formed the least stable complexes due to strong interaction of charged groups positioned in close proximity in the betaine moieties. The increase in n resulted in the irregular change of the affinity. Thus, as expected, PCB-2 formed noticeably more stable complexes than PCB-1. However, PCB-3 and, in particular, PCB-4 revealed weaker affinity to PMAA* or DNA that is attributed to formation of stable ion pairs between charges in the betaine rings. At neutral and slightly acidic pH values binding of all PCB-n except PCB-1 was drastically enhanced due to protonation of PCB-n carboxylic groups that occurred with a ∆pH shift of 2-3 units to higher values as compared with the protonation of free PCB-n. The ability of added polyanion to compete with the betaine carboxylic groups in binding with the pyridinium groups was supported by potentiometric titration of PCB-n mixtures with sodium poly(styrenesulfonate): for n g 2, the binding of the polyanion-competitor also shifted protonation of carboxylic groups to higher values with ∆pH of more than 2 units. Practical ramifications of the revealed role of the alkyl spacer in polyelectrolyte complexation as well as the pH-induced stabilization of the complexes that occurs under enzyme-friendly conditions might extend to areas of biotechnology, specifically in bioseparation and gene delivery.

Introduction Polybetaines (or zwitterionic polymers) are polyampholytes that possess cationic and anionic moieties in the same repeat unit. Although polybetaines were synthesized almost 50 years ago,2 their properties in bulk and solution are less described3,4 as compared with polyampholytes that have been studied adequately (for review, see ref 5). Meanwhile, the properties of polybetaines that stem from the unique structure of their units could be of significant interest for different applications. Copolymers of betaines with acrylonitrile were used in staining of textiles.6 Grafting of sulfobetaine monomer onto a silicone surface improves the haemocompatibility because of the intrinsic antithrombogenity of polybetaines.7 Zwitterionic polymers have been proposed as perspective materials for nonlinear optics applications.8 Polybetaine based on imidazole was used to prepare a new type of polymer gel electrolyte that exhibited enhanced ionic conductance.9 The family of compounds that bears both anionic and cationic groups in the molecule takes its name from the compound betaine, (CH3)3N+-CH2-CO2-. Betaines could be considered as inner salts in which positive and negative charges are separated by an alkyl spacer that, as a rule, consists of one or more methylene groups. In most instances, the positive charge †

Part 1, see ref 1. * To whom correspondence should be addressed. Phone: 007-(095)9393117. Fax: 007-(095)-9390174. E-mail: [email protected].

is supplied by a quaternary ammonium moiety whereas the negatively charged group could be of a different chemical nature, usually a phosphate, sulfonate, and carboxylate group. Accordingly, polybetaines can be subdivided into polyphosphobetaines, polysulfobetaines, and polycarboxybetaines.4 The important role of phospholipids in design and function of biological membranes has motivated research on the synthesis and properties of polyphosphobetaines which are the polymeric analogues of phospholipids.10-12 The majority of studies of polybetaines have been performed on zwitterionic polymers with sulfobetaine functionalities.13-18 Both polyphosphobetaines and polysulfobetaines bear anionic moieties that are completely ionized in the pH range from 2 to 11, which is commonly used in studies of biopolymers. Inasmuch as a positive charge of the quaternary ammonium moiety is also pH independent, pH control over these polybetaines is minimized. In contrast, polycarboxybetaines (PCB) are pH-sensitive polymers as in acidic media these polyzwitterions are transformed to polycations due to protonation of the carboxylate groups. Studies of PCB were mainly aimed at elucidation of their solubility, viscosity, and ionization behavior of their solutions.19-23 Thus, inter- and intramolecular aggregation of PCB was shown to be dependent on alkyl spacer in the repeat units.21,24,25 Potentiometric titration of PCB revealed the significant influence of external salt on ionization of these polymers.26 Acid-base equilibrium and the viscosity of the

10.1021/jp0518207 CCC: $30.25 © 2005 American Chemical Society Published on Web 08/30/2005

17392 J. Phys. Chem. B, Vol. 109, No. 37, 2005 solutions were shown to be strongly affected by steric hindrance of the cationic moiety.27 The relationship between the structural differences of PCB and the pH-dependent behavior of their solutions was established recently by capillary electrophoresis.28,29 Reactions of polybetaines with positively or negatively charged polymers, i.e., interpolyelectrolyte reactions, are poorly described. Formation of polyelectrolyte complexes in solutions of poly-β-[N,N-dimethyl-N-(β-methacrylhydroxyethyl)]propiobetaine and synthetic polyacids in acidic media was experimentally proven more than 30 years ago.30 However, over the next 20 years there were no reports on their complexes with macromolecules. In succeeding years the interpolyelectrolyte complexes were incidentally found in water-salt solutions of polysulfobetaines.31-33 Stabilization of these complexes was attributed to ion-dipole interactions and hydrogen bonding. Recently the results of comprehensive studies of binding of poly(ammonium sulfopropylbetaine) with polycations and polyanions were reported by the group of Y. Osada.34,35 Of special interest is the established self-propagating association of the polysulfobetaines initiated by ionene polymers.35 To verify whether PCB also can readily form polyelectrolyte complexes, we synthesized PCB samples in which the betaine charges were isolated with one methylene group.1 All samples interacted rather poorly with poly(methacrylic) acid or DNA, and only sulfonate groups of strong polyanion poly(styrenesulfonate) were able to compete with carboxylate groups of PCB for binding with the amino groups. In this paper we present unambiguous evidence that complex formation between PCB and polyanions can be enhanced and efficiently controlled by the proper choice of alkyl spacer between charges in the betaine moieties. This finding shows the feasibility of designing polybetaine polyelectrolyte complexes that are able to form and dissociate at a desirable pH and ionic strength. The revealed pH-induced stabilization of DNA/PCB complexes that occurs under enzyme-friendly conditions is of specific importance for application in biotechnology, e.g., for selective binding and extraction of nucleic acids, and can be considered as a first step toward environmentally responsive gene delivery systems. Experimental Section Materials. KOH, HCl, TRIS, HEPES, and MES buffers were purchased from Sigma. In all experiments twice distilled and additionally purified by Milli-Q (Millipore) water was used. Ethidium Bromide. Ethidium bromide (EB) was purchased from Sigma. Concentration of EB in solution was determined spectrophotometrically assuming a molar extinction coefficient of 5600 L mol-1 cm-1 at 480 nm.36 Calf Thymus DNA. Na salt of highly polymerized calf thymus DNA (∼10 000 base pairs) was purchased from Sigma and used without further treatment. Concentration of DNA phosphate groups in the solutions was determined by UV absorbance measurements at 260 nm assuming a molar extinction coefficient of 6500 L mol-1 cm-1.37 Poly(acrylic) Acid. Poly(acrylic) acid (PAA) was prepared by radical polymerization of the monomer and fractionally precipitated in methanol/ethyl acetate mixture.38 The fraction with a weight-average molecular weight Mw ) 170 000 was used. Poly(methacrylic) Acid. Poly(methacrylic) acid (PMAA) was synthesized by radical polymerization and fractionally precipitated in methanol/ethanol mixture.39 The PMAA fraction with a weight-average molecular weight Mw ) 340 000 was used.

Izumrudov et al. SCHEME 1

SCHEME 2

PMAA tagged by fluorescent pyrenyl groups (PMAA*) was synthesized by interaction of the PMAA fraction with pyrenyldiazomethane as described elsewhere.39 PMAA* sample contained 1 fluorescence label per 320 monomer units as determined from the UV spectrum of PMAA* solution assuming a molar extinction coefficient of 5 × 104 L mol-1 cm-1 at 342 nm.39 Sodium Poly(styrenesulfonate). Sodium poly(styrenesulfonate) (PSS) samples with a weight-average molecular weight Mw ) 100 000 were purchased from Serva (Germany) and used without purification. Poly(N-ethyl-4-Vinylpyridinium) Bromide. Poly(N-ethyl-4vinylpyridinium) bromide (PEVP) with weight-average degree of polymerization Pw ) 1600 was synthesized by alkylation of poly-4-vinylpyridine (Aldrich), Mw ) 168 000, with ethyl bromide and characterized as described elsewhere.40 Polycarboxybetaines. Polycarboxybetaines (PCB-n) were synthesized by interaction of poly-4-vinylpyridine (Aldrich), Mw ) 168 000, with esters of the corresponding ω-bromocarboxylic acid, yielding a polymeric precursor, PE-n (n ) 1-5, Scheme 1). The precursors with n ) 1, 3, 4, 5 were converted in PCB-n by alkaline hydrolysis under heating29 (Scheme 1). Therefore, all utilized samples, PE-n and PCB-n, had an equal degree of polymerization, Pw ) 1600. Since the betaine moieties of PCB-2 are unstable under heating, PCB-2 was not prepared from precursor PE-2. In this particular case the same sample of poly(4-vinylpyridine) was alkylated with 3-bromopropionic acid (Scheme 2) in the sealed ampule at room temperature for 2 weeks as described elsewhere.40 PCB-8 was obtained by the same reaction of the poly4-vinylpyrydine sample with the corresponding ω-bromocarboxylic acid but at 60 °C. The utilized PCB-n possessed a pyridiniocarboxylate structure with a different length of the alkyl spacer (Schemes 1 and 2). The degrees of conversion in the polymer analogous reactions that are listed in Table 1 were estimated by IR spectroscopy.41

Reactions in Solutions of Polycarboxybetaines

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TABLE 1: Characteristics of Polycarboxybetaines and Polyesters-Precursors polymer

degree of substitution, %

Mw, 10-5, g mol-1

PCB-1 PE-1 PCB-2 PE-2 PCB-3 PE-3 PCB-4 PE-4 PCB-5 PE-5 PCB-8

>94

3.90 2.85 3.95 3.07 4.35 3.30 4.58 3.52 4.80 3.74 5.19

92 >94 >94 >94 >94 92

Methods. Spectrophotometric Measurements. Spectrophotometric measurements were performed using a Hitachi 150-20 Spectrometer (Japan) in a water-thermostatic cell under permanent stirring at 25 °C. Fluorescence Measurements. The fluorescence intensity of the solutions was measured using a Jobin Yvon-3CS spectrofluorimeter (France) with a water-thermostatic stirred cell holder. The measurements were made in a capped quartz fluorescence cell with permanent stirring at 25 °C. The excitation and emission wavelengths in experiments with EB were set at 535 and 595 nm, respectively, whereas in experiments with PMAA* the wavelengths were 342 and 395 nm, respectively. The DNA solution was directly mixed with EB in the fluorescence cell. The composition of the obtained complex DNA‚EB was [EB]/[P] ) 0.25, where [P] is the molar concentration of DNA phosphate groups. At this ratio, corresponding to one molecule of intercalated EB per two pairs of DNA bases (four nucleotides), the maximum EB fluorescence intensity was observed.42 The concentration of EB was 1 × 10-5 mol L-1; concentrations of DNA phosphate groups and PMAA* carboxylic groups, [P] and [PMAA*], were 4 × 10-5 mol L-1. Solutions of PMAA* or complex DNA‚EB were titrated with polycarboxybetaines or PEVP solutions. The stability of the complexes in water-salt media was checked by titration with 4 M NaCl solution. The time interval between titrant additions was 5 min. Potentiometric Titration. Potentiometric titration of aqueous solutions was conducted using a MultiLab 540 (WTW, Germany) potentiometer with a Metler Toledo glass calomel combination electrode. The measurements were performed in a water-thermostatic stirred cell holder under an argon atmosphere at 25 °C. A 14 mL amount of PCB-n solution with a concentration of repeat units [PCB-n] ) 1.5 × 10-3 mol L-1 containing one equivalent of HCl with respect to carboxylate groups of the polycarboxybetaines was prepared. A 7 mL amount of the solution was titrated with KOH solution using a Hamilton microsyringe. In parallel, 7 mL of the PCB-n solution was mixed with a portion of PSS solution, and the mixture was titrated with KOH solution. The portions of the titrant were added under vigorous stirring with 2-min time intervals, sufficient for achieving constancy in measured pH values. Potentiometric titration curves were presented as the pH dependence of a neutralization degree R of carboxylic groups. The apparent dissociation constant pKa was determined by modified Henderson-Hasselbalch equation, eq 1, with correction for the amount of free H+ 43,44

pKa ) pH - log

C R + [H+] C(1 - R) - [H+]

(1)

where C is the concentration of polybetaine carboxylic groups

Figure 1. Potentiometric titration curves of polybetaines solutions (PCB-1 (1), PCB-2 (2), PCB-3 (3), PCB-4 (4), PCB-5 (5), and PCB-8 (6)) and solutions of PMAA* (7) and PAA (8). Concentration of the solutions ) 1.5 × 10-3 mol L-1 (repeat units), 0.01 mol L-1 NaCl, 25 °C.

([PCB-n]). The intrinsic dissociation constant pK0 was estimated by extrapolation of pKa to R ) 0. Dynamic Light Scattering. Dynamic light scattering (DLS) measurements were conducted using a photon correlation spectrometer (PhotoCor Instruments Inc.). A Coherent 31-2082 He-Ne-Laser (632.8 nm, 10 mW) served as a light source. Signals were recorded with a photon counting system PhotoCorPC 3 and further processed by a digital flexible correlator PhotoCor-FC. Autocorrelation functions were automatically recorded on 288 channels, logarithmically spaced in time. The temperature was controlled by PhotoCor-TC to within (0.1 °C. For spherical particles the diffusion coefficient (D0) and hydrodynamic radius (R) are related by the Stokes-Einstein equation, eq 2

D0 )

kBT 6πηR

(2)

where kB is Boltzmann’s constant, T is the absolute temperature, and η is the solvent viscosity. Autocorrelation functions were analyzed using DynaLS software (Alango, Israel). Results and Discussion The zwitterionic structure of the PCB-n repeat units showed up most vividly when the acid-base equilibrium in their solutions was studied by potentiometry. Acid-Base Equilibrium. As mentioned above, quaternary amino groups of polybetaines are charged at any pH. When these groups strongly influence the ionization of the carboxylic groups, the titration curves should be similar to the curve of pure water; otherwise, PCB solutions should be titrated like poly(carboxylic) acids. Figure 1 shows the potentiometric titration curves of PCB-n solutions as well as the titration curves of poly(methacrylic) acid and poly(acrylic) acid. A pronounced kink in the PBC-n curves (Figure 1, curves 1-6) implies efficient suppression of protonation of the carboxylate groups of PCB-n from their protonation in neutral and weakly acidic media. The fact that the polycomplex titration curves are separated from both PMAA and PAA titration curves (curves 7 and 8, respectively) suggests that protonation of the carboxylate group of PCB-n is hindered by the adjacent quaternary

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TABLE 2: Intrinsic Dissociation Constants and Hansch Parameters PCB-n

pK0 of PCB-n

pK0 of PSS/PCB-n complexes

Hansch parameter, πx, of PCB-n alkyl substituent

PCB-1 PCB-2 PCB-3 PCB-4 PCB-5 PCB-8

1.9 2.5 2.4 2.4 2.4 2.4

3.1 6.1 6.5 6.6 6.7 7.2

+0.90 +1.43 +1.96 +2.49 +3.02 +4.61

amino group which forms a stable ion pair with the carboxylate group. In other words, pyridinium rings play the role of strong competitors for the protons. Protonation occurs only at pH < 4, as suggested by the relatively small slope of curves 1-6 of Figure 1 in acidic media. The values of the intrinsic dissociation constant pK0 obtained from the potentiometric titration data as described in the Experimental Section are summarized in Table 2. The pK0 values checked well with the reported data of capillary electrophoresis,28,29 which revealed the appearance and an increase of positive charge on PCB-1 in more acidic media compared to PCB-n with n g 2. Thus, quaternary amino group and carboxylate group located in the same repeat unit of polybetaines form a stable ionic pair and “inactivate” each other. It could be envisioned that this local binding results in significant inhibition of the electrostatic interaction of PCB with polycations or polyanions. An argument in favor of this assumption is the earlier reported weak affinity of PCB-1 to poly(methacrylic) acid or native DNA.1 Attempts to enhance binding by introducing hydrophobic alkyl fragments of different length in the betaine moiety of PCB-1 have not been successful. All studied polybetaine samples that possessed one methylene group between the charges formed unstable polyelectrolyte complexes over a wide pH range. Nevertheless, we succeeded in “activating” polybetaines by variation of the alkyl spacer between charges in the betaine moiety. Data presented in the next sections show that despite the similar potentiometric behavior of PCB-n samples (Figure 1, curves 1-6), these polybetaines could reveal noticeably different and pH-dependent affinity to the polyanions. Interaction of PCB-n with Poly(methacrylic) Acid. We performed fluorescence titration of PMAA* solution with solutions of PCB-n at different pH. The procedure is described in the Experimental Section. The successive addition of PCB-n to PMAA* solution at pH ) 9.0 (Figure 2, curves 1-6) resulted in a gradual decrease of the fluorescence intensity due to quenching of PMAA* fluorescence by pyridinium groups of the polybetaine. In most systems efficiency of quenching was higher than in the case of PCB-1 (curve 1), but it remained significantly lower as compared to fluorescence quenching of PMAA* by polycation poly(N-ethyl-4-vinylpyridinium) bromide (Figure 2, curve 7). This finding suggests that alkyl spacer in the betaine moiety enhances interpolyelectrolyte interaction between PCB and PMAA* chains. However, this effect did not correlate directly with the lengthening of the spacer (the increase in n) and was not able “to activate” PCB to the point where affinities of PCB and PEVP polycation to PMAA* would be comparable. The important role of the alkyl spacer was supported by data on the stability of the polyelectrolyte complexes in water-salt solutions (Figure 3). The dissociation of PMAA*/PCB-n complexes in sodium chloride solutions was monitored by the increase in fluorescence intensity of PMAA* which resulted from liberation of the tags from contact with the pyridinium quenchers of PCB-n. The complete dissociation of polyelec-

Figure 2. Dependencies of fluorescence intensity I on the ratio of concentrations of repeat units, Z ) [PCB-n]/[PMAA*] (or [PEVP]/ [PMAA*]), in solutions of PMAA* mixtures with polybetaines PCB-1 (1), PCB-2 (2), PCB-3 (3), PCB-4 (4), PCB-5 (5), and PCB-8 (6) and with polycation PEVP (7). [PMAA*] ) 4 × 10-5 mol L-1, 0.02 mol L-1 TRIS, pH ) 9.0, 25 °C.

Figure 3. Dependencies of relative fluorescence intensity I/I0 on NaCl concentration in solutions of PMAA*/PCB-n, n ) 1 (1), 2 (2), 3 (3), 4 (4), 5 (5), and 8 (6). The conditions are the same as in the legend to Figure 2.

trolyte complexes was accomplished at I/I0 ) 1.0, i.e., when the fluorescence intensity I of the mixture became equal to the intensity I0 of PMAA* solution. Figure 3 shows that at pH ) 9.0 the critical salt concentration, [NaCl]cr, required for complete dissociation of PMAA*/PCB-n complexes was dependent on parameter n, and the change of [NaCl]cr with an increase in n was also irregular. Furthermore, [NaCl]cr values for PCB-n samples were significantly lower than that obtained for PEVP polycation, [NaCl]crPEVP ) 0.52 mol L-1. The low stability of PMAA*/PCB-1 complex in the salt solution (Figure 3, curve 1) is readily explained by formation of stable ion pairs in the repeat units of PCB-1 due to the close proximity of the oppositely charged groups in the betaine moieties.1 As expected, the complex of PCB-2 was noticeably more stable (curve 2) due to weakening of the betaine ion pair with increased distance between the betaine charges. However, further lengthening of the spacer was not accompanied by stronger interaction and even led to considerable destabilization.

Reactions in Solutions of Polycarboxybetaines

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SCHEME 3

Thus, the [NaCl]cr values determined for PMAA*/PCB-3 complex (curve 3) and specifically for PMAA*/PCB-4 complex (curve 4) at I/Io ) 1.0 were significantly lower than the salt concentration corresponding to complete dissociation of PMAA*/ PCB-2 complex (curve 2). It is reasonable to assume that this reverse in the order of the curves reflects a tendency for the betaine charges to approach each other in ring-like structures which could form spontaneously in aqueous solutions of polybetaines.45 The six-membered betaine rings (Scheme 3) appear to be the most thermodynamically favorable structure. Accordingly, formation of the most stable ion pairs between the charges that are brought close together in the six-membered rings reduced the accessibility of PCB-4 amino group for the polyanion most significantly and, hence, resulted in a pronounced destabilization of the complex (Figure 3, curve 4). Argument in favor of this assumption is the highest value of [NaCl]cr ascertained for PCB-5 complex (curve 5). Formation of the seven-membered betaine ring in PCB-5 chain is thermodynamically unfavorable, and in this case complexing is enhanced due to the increased distance between charges in the betaine moieties. It is interesting that PCB-8 formed a rather unstable complex with PMAA* (Figure 3, curve 6). To explain this shift of the curve we are thus led to suggest formation of a 10-membered betaine ring; the latter is hardly the case. It is more realistic to suppose that the flexible spacer consisting of eight methylene groups is long enough to allow the terminal carboxylate group to locate in the close vicinity of the amino group of the neighboring betaine moiety. This cross-binding intramolecular electrostatic interaction could result in formation of stable ion pairs, “inactivating” the polybetaine and, therefore, destabilizing the interpolyelectrolyte complex. One can argue that destabilization of PCB-n complexes results from enhanced steric hindrance when an increase in the spacer length impedes accessibility of the polybetaine amino group for PMAA*. To verify this suggestion we checked the stability of complexes of PMAA* with polymeric precursors of PCB. The precursors PE-n, n ) 1, 2, 3, 4, and 5 (Scheme 1), were highly charged polycations that were synthesized by exhaustive alkylation of the poly(4-vinylpyridine) sample with the corresponding ω-bromoalkylesters. Fluorescence titration of solutions of PMAA*/PE-n complexes with sodium chloride was conducted in the same manner as for PMAA*/PCB-n complexes. Figure 4 shows that, as expected, complexes with highly charged polycation-precursors (curves 1-5) were much more stable than PCB-n complexes. There were no differences in the stability of all PMAA*/PE-n complexes (curves 1-5) except the moderate destabilization of the complex with the precursor that contained five methylene groups (curve 5). This finding suggests that the lengthening of the spacer does not affect significantly the accessibility of the amino group, at least in the studied range n ) 1-5. Consequently, the contribution of this factor to the

Figure 4. Dependencies of relative fluorescence intensity I/I0 on NaCl concentration in solutions of PMAA* with polycations-precursors PEn: n ) 1 (1, 1′), 2 (2, 2′), 3 (3, 3′), 4 (4, 4′), and 5 (5, 5′) in 0.02 mol L-1 TRIS, pH ) 9.0 (1-5, open symbols), and 0.02 mol L-1 MES, pH ) 6.0 (1′-5′, filled symbols). The dotted line corresponding to PMAA*/PCB-5 complex at pH ) 9.0 is taken from Figure 3 (curve 5). The other conditions are the same as in the legend to Figure 2.

Figure 5. pH dependencies of the normalized critical salt concentrations (see text) corresponding to complete destruction of PMAA*/PCB-n complexes, n ) 1 (1), 2 (2), 3 (3), 4 (4), 5 (5), and 8 (6). [PMAA*] ) 4 × 10-5 mol L-1, 25 °C.

shifts of curves 1-5 in Figure 3 can be ignored. The lack of the ester derivative of PCB-8 sample did not allow us to estimate the stability of PMAA* complex with the corresponding precursor. It is not inconceivable that in this particular case the relatively long spacer with eight methylene groups could hinder, at least partly, the interpolyelectrolyte interaction. This point will be studied in the future. We performed similar experiments with PMAA*/PCB-n complexes at different pH, and [NaCl]cr values were ascertained. In parallel, the PMAA*/PEVP complex was titrated with sodium chloride at the same pH, and the values of the critical salt concentration, [NaCl]crPEVP, were determined. The data obtained are presented in Figure 5 as pH dependencies of the normalized critical values, [NaCl]cr/[NaCl]crPEVP. It is seen that a decrease in pH from 9.0 to 7.0 resulted in moderate, if any, growth of the polybetaines affinity to PMAA*. At pH < 7.0, the stabilization profiles of all studied PCB-n except PCB-1 (curve

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Figure 6. Potentiometric titration curves of PSS/PCB-n mixtures: n ) 1 (1), 2 (2), 3 (3), 4 (4), 5 (5), and 8 (6). [PSS] ) [PCB-n] ) 1.5 × 10-3 mol L-1. Potentiometric titration curves of the PCB-n (open symbols) are taken from Figure 1. The conditions are the same as in the legend to Figure 1.

Figure 7. Potentiometric titration curves of PSS/PCB-4 mixtures of different ratios Z ) [PSS]/[PCB-4]: Z ) 0 (1), 0.25 (2), 0.5 (3), 1 (4), and 5 (5). The dotted lines mark the pH region corresponding to dissolution of the complexes. Concentration of PCB-4 solutions 1.5 × 10-3 mol L-1 (repeat units), 0.05 mol L-1 NaCl, 25 °C.

1) were markedly steeper. Note that the stability of complexes formed by PMAA* with the polycations-precursors PE-n remained virtually the same in the range 9.0 > pH > 7.0 (data not shown) and increased at pH ) 6.0 (Figure 4, curves 1′5′). This effect is probably due to additional stabilization of the polyelectrolyte complexes by H-bonds of PMAA* chains.46 Therefore, the sharp increase in the stability of PMAA*/PCB-n complexes at pH < 7.0 (Figure 5, curves 2-6) results from destruction of the betaine ion pairs due to protonation of the betaine carboxylate groups. The fact that the narrow pH region of this transition is markedly distinct from the protonation region of free PCB-n (without PMAA*) suggests that the destruction of betaine ion pairs is facilitated by the competitive binding of the polyacid with the pyridinium groups. The results of studies of PCB-n binding with poly(styrenesulfonate) given below further support this statement. Interaction of PCB-n with Sodium Poly(styrenesulfonate). In contrast to carboxylic groups of poly(carboxylic) acids, sulfonate groups of sodium poly(styrenesulfonate) do not change their charge with pH and do not interfere with potentiometric titration of carboxylic groups of PCB-n. Figure 6 shows that potentiometric titration curves of PSS mixtures with all PCB-n (curves 2-6) except PCB-1 (curve 1) differ markedly from the titration curves of free PCB-n (Figure 6, open symbols) and are much closer to the curves of the poly(carboxylic) acids (Figure 1, curves 7 and 8). This indicates binding of PSS with the pyridinium groups of PCB-n and reflects liberation of the carboxylic groups of PCB-n from the betaine ionic pairs and their protonation in the PSS/PCB-n complexes. The latter checks well with the stabilization of PMAA*/PCB-n (n g 2) complexes at pH < 7.0 revealed by fluorescence quenching (Figure 5, curves 2-6). In the case of PCB-1 when no significant stabilization was found (Figure 5, curve 1), protonation was small and occurred only at low pH values (Figure 6, curve 1). Thus, PSS/PCB-n complexes in acidic media may be viewed as a special type of poly(carboxylic) acids. Figure 6 shows that an increase in n at n g 2 was accompanied by a gradual shift of the potentiometric titration curves to neutral media. The values of pK0 determined from the titration curves in Figure 6 are listed in Table 2. It is seen that the acidity of these poly(carboxylic) acids was lower as the alkyl spacer in the betaine moieties was longer. This finding is in good agreement with

the reported data on dissociation of weak polyacids containing hydrophobic moieties, specifically copolymers of acrylic acid and 12-acryloyldodecanoic acid.47 Dissociation of the copolymers was also shifted to neutral media as the content of the hydrophobic residues increased. The latter reflected a growing contribution of hydrophobic interactions to ionization. To assess the hydrophobicity of the polymers, we used the approach introduced in ref 48. This approach uses the experimental values of the partition coefficient P, which is the ratio of the equilibrium concentration of a substance in two immiscible phasessorganic phase, most commonly 1-octanol, and water. For hydrophobic substances the concentration in the organic phase is larger than that in the aqueous phase, i.e., P > 1. The contribution of a substituent to the overall hydrophobicity of a molecule, πx, is calculated as

πx ) log Px - log PH

(3)

where Px and PH are the partition coefficients for the derivative and the parent molecule, respectively. For a series of alkyl substituents the choice of the parent molecule is not crucial. Taking into account the scale of the hydrophobicity of the substances, which is expressed in terms of parameter πx,48 we estimated the contribution of the alkyl spacer in the hydrophobicity of PCB-n betaine moieties. The calculated πx values are summarized in Table 2. A good correlation between πx and pK0 values (cf. the third and the second columns of Table 2) implies that the hydrophobic interactions are the main factor which hinders ionization of these polyacids. Note that this adverse action may not stem only from the side-by-side hydrophobic interactions of PCB-n alkyl moieties. The intramolecular hydrophobic interactions of the spacer with the hydrophobic fragments of the stoichiometric (1:1) complex formed by PSS sulfonate groups and PCB-n pyridinium groups are quite possible as well. The potentiometric and turbidimetric titration of PSS/PCB-n mixtures of different composition Z ) [sulfonate groups]/[amino groups] ) [PSS]/[PCB-n] also suggested formation of the stoichiometric complex. Figure 7 shows the results of potentiometric titration of PSS/PCB-4 mixture. It is seen that at Z e 1 (Figure 7, curves 2-4) there was one-to-one correspondence between the inflection points on the curves and Z values, i.e.,

Reactions in Solutions of Polycarboxybetaines for a higher relative content of sulfonate groups, more carboxylic groups were liberated from the betaine ion pairs. Moreover, at Z g 1 the titration curves did not depend on the composition Z, cf. curves 4 and 5 of Figure 7. These findings strongly suggest that the majority of the added sulfonate groups formed ionic pairs with the pyridinium groups until the latter are fully exhausted. In other words, in acidic media at Z < 1 the mixtures presumably consist of the interpolyelectrolyte PSS/PCB-4 complex and the unbound PCB-4 in a ratio dependent on the composition Z. There were two arguments in favor of this assumption. First, in acidic media the systems with n g 2 were turbid regardless of the composition Z. Since PCB-n samples were soluble in all studied pH ranges, the observed turbidity was due to formation of insoluble interpolyelectrolyte complex. Second, we revealed the coexistence of large particles and much smaller particles in PSS/PCB-4 mixtures with different composition Z < 1 by dynamic light scattering. The large particles with an average hydrodynamic radius of ca. 400 nm can be assigned to the aggregated insoluble stoichiometric complexes, whereas the smaller particles with a radius not exceeding 40 nm are reasonable to identify as the unbound PCB-n molecules. The large particles were formed immediately after mixing of the solutions, and their size increased only slightly during incubation of mixtures of all studied composition Z. Upon addition of potassium hydroxide, the turbidity decreased and completely disappeared in slightly acidic media. The pH of cloud points slightly varied but always occurred in a relatively narrow pH range that corresponded to ionization of the majority of the carboxylic groups. This implies that the complex solubility is controlled by a balance between charged carboxylic groups liberated from the betaine ion pairs due to PSS binding and hydrophobic sequences that consist of PSS sulfonate groups bound with the pyridinium groups. The insolubility of complexes in acidic media was due to the low solubilizing ability of the protonated noncharged carboxylic groups, which was not enough to retain complexes in solution. The charged carboxylic groups have a much higher solubilizing capacity and could ensure solubility of the complex particles. Treatment of heterogeneous mixtures of PCB-n with PMAA* or DNA with potassium hydroxide resulted in similar dissolution, with the only difference being that DNA/PCB-n mixtures became transparent at pH ) 6. In other words, in the latter case dissolution occurred with a ∆pH shift of one unit to acidic media. The reason for this peculiarity of DNA-containing complexes will be discussed in the next section. Note that solubilization of the complex particles by a relatively large amount of negatively charged betaine carboxylic groups might not be the only cause of complex dissolution. As it follows from low stability of PMAA*/PCB-n complexes in water-salt solutions at pH > 7.0 (Figure 5), these groups are able to bind efficiently with the pyridinium groups and displace part of the polyanion chains or their segments from the complex. It suggests destruction of hydrophobic sequences formed by the polyanion with PCB-n that should enhance the complex solubility. However, sodium poly(styrenesulfonate) is known as a strong binder with various polycations being an efficient competitor to polyanions with other acidic moieties.49,50 Accordingly, solubilization of PSS/PCB complex by the charged carboxylic groups is most likely the main factor for complex dissolution. In contrast, PMAA*/PCB-n complexes, as well as DNA/PCB-n complexes, seem to be dissolved mainly due to the competitive displacement of the polyanion from the complex. Dynamic light scattering and high-speed sedimentation experiments proving this hypothesis are underway.

J. Phys. Chem. B, Vol. 109, No. 37, 2005 17397

Figure 8. Dependencies of fluorescence intensity I on the ratio of concentrations of repeat units Z ) [PCB-n]/[P] (or [PEVP]/[P]) in solutions of DNA‚EB mixtures with polybetaines PCB-1 (1), PCB-2 (2), PCB-3 (3), PCB-4 (4), PCB-5 (5), and PCB-8 (6) and with polycation PEVP (7). [P] ) 4 × 10-5 mol L-1. The other conditions are the same as in the legend to Figure 2.

Interaction of PCB-n with DNA. The interaction was monitored by fluorescence quenching using the approach based on competitive displacement of intercalated cationic dye ethidium bromide by the added polycation.40 The addition of all studied polybetaines to the solution of complex DNA‚EB at pH ) 9 yielded in a decrease in fluorescence intensity (Figure 8, curves 1-6) which was markedly weaker than that caused by quenching by PEVP polycation (Figure 8, curve 7). In the latter case, the addition of one equivalent of positively charged pyridinium groups of PEVP per negatively charged phosphate groups of DNA was sufficient for almost complete transfer of the dye into solution where the fluorescence of ethidium bromide is fully quenched. Data on PCB-n binding with DNA (Figure 8) and PMAA* (Figure 2), though obtained by different fluorescence quenching methods, are in excellent agreement. This implies that the effect of the alkyl spacer on the affinity of PCB-n to polycarboxylic acids can be extended to DNAcontaining complexes. This statement was further confirmed by studies of the stability of DNA/PCB-n complexes in water-salt media. Dissociation of the complexes in sodium chloride solutions was inferred from an increase in the fluorescence intensity of ethidium bromide caused by re-intercalation of the dye to the unbound sites of DNA.40 A typical curve corresponding to titration of DNA/PCB-3 complex with sodium chloride at pH ) 5.0 is shown in Figure 9 (curve 2). The complete dissociation of the complex occurred at I/I0 ) 1.0, i.e., when the fluorescence intensity I of the mixture was equal to the intensity I0 of DNA‚ EB solution, marked by arrow in Figure 9. Figure 10 shows fluorescence titration curves of DNA/PCB-n complexes plotted as dependencies of the relative fluorescence intensity I/I0 on the salt concentration. Lengthening of the alkyl spacer in the betaine moieties resulted in similar changes of the complex stability in both DNA/PCB-n (Figure 10) and PMAA*/PCB-n (Figure 3) mixtures with the only difference being that DNAcontaining complexes are less stable, cf. the corresponding curves of Figures 10 and 3. Similar experiments were conducted with DNA/PCB-n complexes at different pH, and critical salt concentrations, [NaCl]cr, corresponding to I/I0 ) 1 were determined. In parallel, titration of DNA/PEVP complex was performed at the same

17398 J. Phys. Chem. B, Vol. 109, No. 37, 2005

Figure 9. Dependencies of fluorescence intensity I0 of DNA‚EB solution (1) and fluorescence intensity I of DNA‚EB/PCB-3 solution (2) on the salt concentration. [P] ) 4 × 10-5 mol L-1, 0.02 mol L-1 MES, pH ) 5.0, 25 °C.

Figure 10. Dependencies of relative fluorescence intensity I/I0 on NaCl concentration in solutions of DNA/PCB-n, n ) 1 (1), 2 (2), 3 (3), 4 (4), 5 (5), and 8 (6). [P] ) 4 × 10-5 mol L-1. The other conditions are the same as in the legend to Figure 3.

pH, and the value [NaCl]crPEVP ) 0.56 mol L-1 was ascertained. Figure 11 shows pH dependencies of the normalized critical values, [NaCl]cr/[NaCl]crPEVP. It is seen that the pH-dependent change of the stability of DNA/PCB-n complexes is only slightly different from that of PMAA*/PCB-n complexes; however, the noticeable stabilization of the DNA complexes occurred at pH < 6.0, i.e., with a ∆pH shift of one unit in more acidic media, cf. Figures 11 and 5. This finding suggests a weaker affinity of PCB-n to DNA that may be ascribed to the high rigidity of the double helix that hinders matching of DNA phosphate groups with pyridinium groups of the polybetaine. Nevertheless, even this relatively weak ability of DNA to “activate” PCB may have far-reaching implications. Demonstrated pH-controlled binding of DNA with PCB at enzyme-friendly pH and ionic strength holds much promise for applications in biotechnology and medicine. Concluding Remarks The reported study on the series of synthesized PCB-n with different number n of methylene groups, n ) 1, 2, 3, 4, 5, and

Izumrudov et al.

Figure 11. pH dependencies of the normalized critical salt concentration (see text) corresponding to complete destruction of DNA/PCB-n complexes, n ) 1 (1), 2 (2), 3 (3), 4 (4), 5 (5), and 8 (6). [P] ) 4 × 10-5 mol L-1, 25 °C.

8, and their complexation with the polyanions demonstrated the important role of the alkyl spacer in the interpolyelectrolyte interactions. As might be expected, the affinity of the polycarboxybetaine to the polyanion should be increased with an increase in n due to a larger distance between the oppositely charged groups in the betaine moiety. However, the revealed trend in PCB-n affinity to the synthetic polyanion PMAA* and DNA was more complicated. The interpolyelectrolyte interactions were characterized by the pronounced minima stemming from the irregular change in the intramolecular electrostatic interactions of the polyzwitterions. The propensity of PCB-n with n ) 3 and, in particular, 4 to form betaine rings spontaneously in aqueous solutions may be responsible for the first minimum, whereas the second minimum could be attributed to the ability of the relatively long spacer of PCB-8 for the crossbinding intramolecular electrostatic interaction of the charges within neighboring repeat units. Another important finding is the ascertained pH-dependent stabilization of PCB-n interpolyelectrolyte complexes at n g 2. The stabilization resulted from protonation of the betaines carboxylic groups, which was facilitated by polyanion binding and occurred with a ∆pH shift of 2-3 units to neutral media compared to the protonation of free polybetaines. The sharp increase in complex stability proceeded at pH < 7.0 in PMAA*/ PCB-n mixtures and at pH < 6.0 in DNA/PCB-n mixtures. The weaker affinity of PCB-n to DNA may be ascribed to the high rigidity of the double helix that hinders matching of DNA phosphate groups with pyridinium groups of the polybetaine. Nevertheless, even this relatively weak ability of DNA to “activate” PCB may have far-reaching implications. It is apparent that the established pH-controlled binding of DNA with PCB at enzyme-friendly pH and ionic strength holds much promise for applications in biotechnology and medicine. Thus, the ability of PCB-4 to combine high affinity to DNA with rather strong electrostatic interaction in the unbound betaine ion pairs is attractive for selective binding and extraction of nucleic acids; the selectivity could be rooted in the poor affinity of the mutually neutralized charges in the repeat units to the nontargeted substances, e.g., proteins. The same reasoning can be used as the basis for development of nonviral gene delivery systems.

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