Biomacromolecules 2005, 6, 1835-1838
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Water-Soluble Complexes through Coulombic Interactions between Bovine Serum Albumin and Anionic Polyelectrolytes Grafted with Hydrophilic Nonionic Side Chains Maria Sotiropoulou,† Georgios Bokias,‡ and Georgios Staikos*,†,§ Department of Chemical Engineering, University of Patras, GR-265 04 Patras, Greece, and Department of Chemistry, University of Patras, GR-265 04 Patras, Greece, Institute of Chemical Engineering and High-Temperature Chemical Processes, ICE/HT-FORTH, P.O. Box 1414, GR-265 04 Patras, Greece Received January 27, 2005; Revised Manuscript Received April 22, 2005
The interaction between bovine serum albumin (BSA) and the anionic graft copolymers poly(sodium acrylateco-sodium 2-acrylamido-2-methyl-1-propanesulfonate)-graft-poly(N,N-dimethylacrylamide) (P(NaA-coNaAMPS)-g-PDMAMx) was investigated within the acid pH region, 2 e pH e 7. The weight percentage, x, of the poly(N,N-dimethylacrylamide) (PDMAM) side chains varied from 0 up to 75% (w:w). When BSA and P(NaA-co-NaAMPS)-g-PDMAMx are oppositely charged, i.e., when pH is lower than the isoelectric point of BSA, the two macromolecules associate through Coulombic attractions. When the anionic graft copolymer is rich enough to the nonionic PDMAM side chains, x g 50% w:w, the associative phase separation is practically prevented, as revealed by the turbidimetric study of the BSA/P(NaA-co-NaAMPS)-g-PDMAMx mixtures in aqueous solution vs pH. In addition, the viscosity measurements support the formation through a charge neutralization process of a rather compact protein-polyelectrolyte complex stabilized by the hydrophilic PDMAM side chains grafted onto the anionic copolymer backbone. Introduction Biological macromolecules, such as proteins, interact with polyelectrolytes in water to form amorphous precipitates, complex coacervates or soluble complexes, depending on the ionic strength, the pH, and the concentration of the two components,1 similar to the polyelectrolyte complexes (PECs) formed between oppositely charged synthetic polyelectrolytes.2 What mostly happens is the formation of insoluble precipitates with polyanions at pH lower than the isoelectric point (IP) of the protein3-6 and with polycations at pH higher than the IP.3,4,7-10 This associative phase separation phenomenon has been exploited for practical applications, as the biochemical capacity of the protein is substantially retained in the resultant complex.11 For instance, protein precipitation with polyelectrolytes is very important for the separation and purification of proteins.12 Furthermore, coacervation of the bovine serum albumin (BSA)/acacia complex has been proposed for microencapsulation of drugs and biologically active substances.13 On the other hand, the formation of water-soluble complexes between proteins and polyelectrolytes seems to be rather rare, occurring within a relatively narrow pH range, just before phase separation.4,14 For example, the water-soluble complex formed between hemoglobin and dextran sulfate at pH values close to the IP of the protein3 could regulate the hemoglobin oxygen affinity.15 * To whom correspondence should be addressed. Fax: 30-2610-997266. E-mail:
[email protected]. † Department of Chemical Engineering, University of Patras. ‡ Department of Chemistry, University of Patras. § ICE/HT-FORTH.
The development of protein/polyelectrolyte complexes retaining water-solubility and stability over a wide pH range should be of importance, as it would enlarge their potential applications. As it regards PECs, a first approach to achieve such a goal was by using block ionogenic copolymers, namely synthetic copolymers where a polyelectrolyte is covalently attached to a nonionic hydrophilic block. Using such block ionogenic copolymers, instead of charged homopolymers, water-soluble stoichiometric PECs are formed. Covalent binding of both the polyanion and the polycation16,17 or of one of them18,19 to a nonionic hydrophilic block, such as poly (ethylene glycol) (PEG), results in the formation of a finally water-soluble product, comprised of a waterinsoluble core (the insoluble PEC) surrounded and stabilized by a hydrophilic PEG corona. Moreover, the formation of water-soluble complexes through Coulombic attractions between block ionomers and enzymes20,21 or oligonucleotides22-27 has also been proposed for potential applications in enzyme entrapment or gene therapy, respectively. Polyelectrolytes of a comb-type structure, provide an alternative, less-studied, flexible strategy to prepare watersoluble complexes through Coulombic interactions between polyelectrolytes and other oppositely charged (macro)molecules, like surfactants28 or DNA.29,30 In addition, we have recently synthesized the comb-type copolymers P(NaAco-NaAMPS)-g-PDMAMx, by grafting neutral poly (N,Ndimethylacrylamide) (PDMAM) chains onto an anionic poly(sodium acrylate-co-sodium 2-acrylamido-2-methyl-1propanesulfonate) backbone (P(NaA-co-NaAMPS))31 and demonstrated their ability to form water-soluble stoichio-
10.1021/bm050061v CCC: $30.25 © 2005 American Chemical Society Published on Web 05/07/2005
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°C for 6 h, using the redox couple APS/KBS. The product was obtained in its sodium salt form, poly(sodium acrylateco-sodium 2-acrylamido-2-methyl-1-propanesulfonate) (P(NaAco-NaAMPS)), after being fully neutralized with an excess of NaOH (pH ∼ 11), ultrafiltrated with a tangential flow filtration Pellicon System (Millipore) and freeze-dried. Its composition was determined by an acid-base titration and it was found to contain 21 mol % of acrylate units. The graft copolymers P(NaA-co-NaAMPS)-g-PDMAMx, shortly designated as Gx, where x is the weight percentage feed composition in PDMAM, were synthesized by means of a coupling reaction between the P(NaA-co-NaAMPS) copolymer and the amine-functionalized PDMAM. In a 5% aqueous solution of the polymer mixture, a 5-fold excess of the coupling agent, EDC, was added and let under stirring for 12 h at room temperature. Addition of EDC was repeated for a second time. The EDC excess was subsequently fully neutralized with a 5-fold quantity of NaOH and the products obtained were then purified by water with the Pellicon system and freeze-dried. The completion of the grafting reaction was confirmed by size exclusion chromatography, by means of a Waters system equipped with two Shodex OH-pak columns, B804 and B805. Table 1 summarizes the polyelectrolytes synthesized in the present study. The molar mass of all of the products was determined by static light scattering measurements of aqueous 0.1 M NaCl polymer solutions. The composition of the graft copolymers was determined by 1H NMR analysis in D2O. Turbidimetry. The change in transmittance at 490 nm of dilute aqueous BSA/polymer mixtures was monitored as a function of the mixture composition at room temperature by means of a Hitachi spectrophotometer model U 2001. Viscometry. Reduced viscosity studies were carried out with an automated viscosity measuring system (Schott-Gera¨te AVS 300, Germany) equipped with a micro-Ostwald-type viscometer, at 25 ( 0.02 °C. Preparation of the Solutions of the BSA/Polyelectrolyte Mixtures. Parent solutions of the desired concentration were prepared by dissolution of BSA or the anionic polyelectrolytes in 0.05 M sodium hydrogen phosphate-citric acid buffer solutions in 0.1 M NaCl under gentle agitation for 24 h. The BSA/polyelectrolyte mixtures solutions were prepared by dropwise addition of the solution of BSA into the polyelectrolyte solution under agitation.
Scheme 1
metric PECs with synthetic polycations, like poly(diallyldimethylammonium chloride).32 In the present work, we have broadened this concept to involve biological macromolecules, like proteins. As a model protein, we have used BSA, and we have studied its interactions with the above comb type copolymers, Scheme 1. It is shown that water-soluble stoichiometric protein/ polyelectrolyte complexes are formed, provided that the protein molecules are positively charged, i.e., pH is lower than 4.9, the IP of BSA. Experimental Section Materials. Bovine serum albumin (BSA) used was purchased from Sigma (A-7638) and used without further purification. The monomers, acrylic acid (AA), 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPSA), and N,N-dimethylacrylamide (DMAM), were purchased from Aldrich. Ammonium persulfate (APS, Serva), potassium metabisulfite (KBS, Aldrich), 2-aminoethanethiol hydrochloride (AET, Aldrich), and 1-(3-(dimethylamino) propyl)-3-ethyl-carbodiimide hydrochloride (EDC, Aldrich) were used for the synthesis of the graft copolymers. Water was purified by means of a Seralpur Pro 90C apparatus combined with an USF Elga water purification unit. Polymer Synthesis and Characterization. Amine functionalized PDMAM was synthesized by free radical polymerization of DMAM in water at 30 °C for 6 h using the redox couple APS and AET as initiator and chain transfer agent, respectively. The polymer was purified by dialysis against water in a tubing with MWCO 12000 (Sigma) and then freeze-dried. Its number average molecular weight was determined by an acid-base titration of the amine end groups and was found equal to 16 000. A copolymer of AA and AMPSA was prepared by free radical copolymerization of the two monomers dissolved in water in a 1:5 mol ratio respectively, after a partial neutralization (∼90% mol) with NaOH at pH ∼ 5-6, at 30
Results and Discussion Figure 1 shows the pH-dependence of the optical density of dilute aqueous solutions of mixtures of BSA with the anionic backbone, P(NaA-co-NaAMPS), and the three graft
Table 1. Polyelectrolytes Synthesized and Used in This Study polyelectrolyte
short designation
feed composition
chemical characterization
Mwx 105
P(NaA-co-NaAMPS) P(NaA-co-NaAMPS)-g-PDMAM25 P(NaA-co-NaAMPS)-g-PDMAM50 P(NaA-co-NaAMPS)-g-PDMAM75
G25 G50 G75
80 mol % NaAMPS 25 wt % PDMAM 50 wt % PDMAM 75 wt % PDMAM
79 mol % NaAMPSa 23 wt % PDMAMb 47 wt % PDMAMb 72 wt % PDMAMb
2.6c 3.5c 5.2c 10.1c
a
Acid-base titration.
b 1H
NMR analysis. c Static light scattering results.
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Figure 1. Variation of the optical density with pH of 0.1% w/v BSA mixtures with 0.02% w/v NaA-co-NaAMPS (b); 0.027% w/v G25 (1); 0.04% w/v G50 (9); and 0.08% w/v G75 (2).
copolymers, G25, G50, and G75. In all mixtures, the concentration of BSA is constant, whereas the concentration of the polyelectrolytes varies, depending on their composition, to keep always constant the concentration of the anionic groups. As seen, the solutions of the BSA/P(NaA-coNaAMPS) mixtures turn strongly turbid just as pH decreases at 4.6, a value that is slightly lower than 4.9, the IP of BSA. This strong turbidity indicates the formation of the waterinsoluble BSA/polyelectrolyte complex, separating out from water, as expected. Nevertheless, the mixtures of BSA with the graft copolymers present a different behavior. The system BSA/G25 becomes strongly turbid at pH ) 4, the system BSA/G50 presents a low turbidity in the critical pH range, i.e., at pH < 4.9, and the system BSA/G75 is fully transparent in the whole pH range examined. This behavior is a result of the beneficial presence of the neutral PDMAM chains in the comb type copolymers. Due to their hydrophilic character, they inhibit the further aggregation of the hydrophobic complexes formed between the positively charged BSA globules at pH < 4.9 and the negatively charged AMPS units of the backbone of the graft copolymers by charge neutralization. This function becomes more effective as the composition of the graft copolymer in PDMAM hydrophilic chains increases. It seems that the complex formed between BSA and G75, the graft copolymer containing 75 w% graft PDMAM chains, is completely soluble even for pH values as low as 2. A dilute solution reduced viscosity study was also performed, as it is expected that the reduced viscosity of the polyelectrolyte will be influenced by the proteinpolyelectrolyte complexation. The influence of the presence of BSA, at two different concentrations, on the reduced viscosity of a 0.2% w/v G75 solution is presented in Figure 2 as a function of pH, in terms of the reduced viscosity ratio rηred rηred ) ηred,BSA/ηred
(1)
where ηred,BSA and ηred are the reduced viscosities of G75 in the presence and in the absence of BSA, respectively. Any deviation of the ratio rηred from unity (dotted line) offers a good qualitative measure of the interactions between BSA and the polyelectrolyte.
Figure 2. Variation of the reduced viscosity ratio, rηred, of a 0.2% w/v G75 solution with pH, in the presence of 0.05% w/v BSA (9) and 0.2% w/v BSA (b).
We observe that at pH higher than 5 the obtained rηred values are close to unity, indicating that no important interaction occurs between BSA and G75 in this pH region. This is expected, as at pH higher than the IP of BSA, the protein and the polyelectrolyte are similarly charged. On the contrary, as pH decreases to values lower than the IP of BSA, the reduced viscosity ratio rηred becomes lower than unity, indicating that a complex of a compact structure is formed through charge neutralization between the positively charged BSA and the anionic graft copolymer G75. At the low BSA concentration, 0.05% w/v, the ratio rηred becomes about 0.7, while at the higher BSA concentration, 0.2% w/v, the ratio rηred becomes much smaller. This behavior should be explained by the higher neutralization degree of the anionic graft copolymer at the higher concentration of the positively charged protein. Moreover, at this BSA concentration, a minimum rηred value is obtained, rηred ) 0.45, at pH ) 3-3.5, whereas rηred increases smootlhy to 0.6 as pH decreases further to pH ) 2. Probably, at the minimum, BSA has acquired the necessary positive charge to neutralize the corresponding negative charge of the polyelectrolyte. As pH decreases further, the excess positive charge of the protein makes the complex positively charged, leading to its partial swelling and the observed rηred increase. Conclusions In this work, we have studied the interactions between BSA and the anionic graft copolymers P(NaA-co-NaAMPS)g-PDMAMx in order to demonstrate the formation of watersoluble complexes through Coulombic interactions at pH lower than the IP of BSA. Indeed, when the composition, x, of the graft copolymer in the hydrophilic PDMAM side chains is high enough, water-soluble complexes are formed, characterized by a relatively compact structure. Such colloidal particles, consisting in BSA/polyelectrolyte complexes stabilized by PDMAM neutral side chains, could be proved useful for protein solubilization or for drug delivery purposes. Acknowledgment. We thank European Social Fund (ESF), Operational Program for Educational and Vocational
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Training II (EPEAEK II) and particularly the Program IRAKLEITOS, for funding the present work. References and Notes (1) Xia, J.; Dubin, P. L. Protein-polyelectrolyte complexes. In Macromolecular Complexes in Chemistry and Biology; Dubin, P. L., Bock, J., Davies, R. M., Schulz, D. N., Thies, C., Eds.; Springer-Verlag: Berlin, 1994; pp 247-271, and references therein. (2) Philipp, B.; Dautzenberg, H.; Linow, K.-J.; Ko¨tz, J.; Dawydoff, W. Prog. Polym. Sci. 1989, 14, 91-172. (3) Nguyen, T. Q.; Makromol. Chem. 1986, 187, 2567-2578. (4) Park, J. M.; Muhoberac, B. B.; Dubin, P. L.; Xia, J. Macromolecules 1992, 25, 290-295. (5) Xia, J.; Dubin, P. L.; Morishima, Y.; Sato, T.; Muhoberac, B. B. Biopolymers 1995, 35, 411-418. (6) Tsuboi, A.; Ixumi, T.; Hirata, M.; Xia, J.; Dubin, P. L.; Kokufuta, E. Langmuir 1996, 12, 6295-6303. (7) Dubin, P. L.; Murrell, J. M. Macromolecules 1988, 21, 2291-2293. (8) Xia, J.; Dubin, P. L.; Dautzenberg, H. Langmuir 1993, 9, 20152019. (9) Ahmed, L. S.; Xia, J.; Dubin, P. L. J. M. S. - Pure Appl. Chem. A 1994, 31, 17-29. (10) Ball, V.; Winterhalter, M.; Schwinte, P.; Lavalle, Ph., Voegel, J.-C.; Scaaf, P. J. Phys. Chem. B 2002, 106, 2357-2364. (11) Kokufuta, E.; Complexation of proteins with polyelectrolytes in a salt-free system and biochemical characteristics of the resulting complexes. In Macromolecular Complexes in Chemistry and Biology; Dubin, P. L., Bock, J., Davies, R. M., Schulz, D. N., Thies, C., Eds.; Springer-Verlag: Berlin, 1994; pp 247-271, and references therein. (12) Shieh, J.-y.; Glatz, C. E. Precipitation of proteins with polyelectrolytes: Role of polymer molecular weight. In Macromolecular Complexes in Chemistry and Biology; Dubin, P. L., Bock, J., Davies, R. M., Schulz, D. N., Thies, C., Eds.; Springer-Verlag: Berlin, 1994; pp 273-284, and references therein. (13) Burgess, D. J. Complex coacervation: Microcapsule formation. In Macromolecular Complexes in Chemistry and Biology; Dubin, P. L., Bock, J., Davies, R. M., Schulz, D. N., Thies, C., Eds.; SpringerVerlag: Berlin, 1994; pp 285-300, and references therein.
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