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Thermosensitive magnetic latex particles for controlling protein adsorption and desorption
Journal of Magnetism and Magnetic Materials 225 (2001) 151}155
Thermosensitive magnetic latex particles for controlling protein adsorption and desorption A. ElamK ssari*, V. Bourrel UMR-103, CNRS-bioMe& rieux, ENS de Lyon, 46 alle& e d+Italie, 69364 Lyon cedex 07, France
Abstract Thermosensitive core-shell magnetic latex with a magnetic polystyrene core and a rich poly(N-isopropylacrylamide) shell layer was prepared via the seed precipitation polymerization process using magnetic polystyrene particles (as the seed). The adsorption and desorption of Human Serum Albumin (HSA) onto the prepared thermosensitive magnetic latex particles were examined as a function of temperature, incubation time, pH and ionic strength. 2001 Elsevier Science B.V. All rights reserved. Keywords: Thermosensitive particles; Magnetic latex; Poly(N-isopropylacrylamide) ("NIPAM); Protein; Adsorption; Desorption; Core-shell particles; Polystyrene particles; Precipitation polymerization; Lower critical solution temperature (LCST)
1. Introduction With the choice of latex particles as particle carriers in biomedical applications, much work has been devoted to the preparation of hydrophobic magnetic polymer particles [1,2]. When used in the diagnostic "eld as a solid-phase support, it has been shown that polystyrene particles exhibit undesirable phenomena such as protein denaturation and an irreversible adsorption process. The introduction of thermosensitive microgel particles with a lower critical solution temperature (LCST) in biomedical research is of great interest, since their physico-chemical properties and the adsorption} desorption of proteins can be controlled by the pH,
* Corresponding author. Tel.: #33-472-728-364; fax: #33472-728-533. E-mail address: [email protected] (A. ElamK ssari).
temperature and salinity of the medium [3,4]. In addition, such particles may confer a non-denaturing environment to the immobilized protein, which is particularly suitable for immunoassays and protein concentration and puri"cation. This paper aims to report the use of thermosensitive magnetic latex particles for protein concentration and puri"cation by controlling both the adsorption and desorption processes as illustrated in Fig. 1.
2. Experimental section 2.1. Latex preparation Functionalization of a seed polystyrene magnetic latex (EM100/20, 0.8 m average size from Prolabo, France) bearing carboxylic groups was performed by precipitation polymerization of N-isopropylacrylamide (NIPAM) as the main monomer,
0304-8853/01/$ - see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 8 8 5 3 ( 0 0 ) 0 1 2 4 4 - 0
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A. Elan( ssari, V. Bourrel / Journal of Magnetism and Magnetic Materials 225 (2001) 151}155
3. Results and discussion 3.1. Characterization of latex particles The polymerization conversion was found to be close to 100%, with 12% water-soluble polymer formation. The average particle diameter as measured using Quasi-Elastic Light Scattering (QELS) of the surface-modi"ed particles was equal to 1 m in broad polydispersity. The "nal latex was puri"ed by repetitive magnetic separations and redispersion.
3.2. Adsorption kinetics Fig. 1. Diagram illustrating the protein concentration principle using magnetic latex particles.
methylene bisacrylamide (MBA) as the cross-linker and potassium persulfate (KPS) as the initiator. Before encapsulation, the seed magnetic latex was "rst cleaned so as to eliminate any free electrolyte and adsorbed surfactant. A given amount of seed magnetic latex (1 g of EM100/20) was purged with nitrogen for 2 h at a temperature of 703C, then a mixture of NIPAM (0.3 g), MBA (0.03 g) and KPS (0.006 g) was added. Polymerization was carried out for 2 h. The polymerization conversion was gravimetrically determined and the "nal latex was washed at least three times before the adsorption study.
Due to low and negligible protein adsorption below the LCST of poly [NIPAM] shell (i.e. LCST&323C) (as will be discussed below), the HSA adsorption kinetics was performed at 403C, pH 4.6 and at 10\ ionic strength. The adsorption was found to be rapid and a plateau value of 45 mg HSA g\ of latex was reached within 30 min as reported in Fig. 2. The result obtained on the adsorption kinetics of HSA onto such magnetic latex particles correlated well with those reported by Kawaguchi et al. [3] by investigating HCG adsorption onto negatively charged poly [NIPAM] microspheres. In fact, they pointed out that protein adsorption was rapid (within only 20 min) when the adsorption was performed above the LCST.
2.2. Protein adsorption and desorption A given concentration of protein Human Serum Albumin (HSA) and the magnetic latex particles (1.6 mg/mL) were incubated below (or above) the LCST of Poly [NIPAM] (LCST+323C) in 10 mM phosphate bu!er solution at various pH levels. After 2 h incubation, the amount of protein adsorbed was determined from the supernatant analysis by UV at a wavelength of 595 nm using Bradford titration. For both adsorption and desorption studies, the adsorbed amount (or desorbed amount) was determined using the depletion method.
Fig. 2. Adsorption kinetics of HSA at pH 4.7, 0.01 ionic strength and above LCST at 403C.
A. Elan( ssari, V. Bourrel / Journal of Magnetism and Magnetic Materials 225 (2001) 151}155
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3.3. Adsorption isotherms Typical adsorption isotherms obtained at both temperatures are reported in Fig. 3. The adsorption isotherms of HSA onto polystyrene magnetic latex bearing poly [NIPAM] layer show a linear amount of protein adsorbed versus the initial protein concentration and well-de"ned plateaus for both temperatures (20 and 403C). As expected, the adsorption of HSA protein was temperaturedependent and the maximal adsorbed amount of HSA was observed above the LCST rather than below it. The adsorption behavior versus temperature re#ects that the hydrophobic interactions are the driving forces in the adsorption process above the LCST. Indeed, a linear relationship between the adsorbed amount of protein and temperature was reported by Fujimoto et al. [5] by performing HCG protein adsorption onto negatively charged poly [NIPAM] microspheres. The low adsorbed amount of HSA observed below the LCST can be attributed principally to the contribution of electrostatic interactions, since latex and HSA are oppositely charged at pH 4.7. 3.4. Ewect of pH and ionic strength on HSA adsorption Fig. 4 shows the adsorbed HSA amount as a function of initial protein concentration and pH at a constant salinity (10\ M) and at 403C. The
Fig. 3. Adsorbed amount of HSA versus initial HSA concentration (pH 4.7, 0.01 ionic strength).
Fig. 4. Adsorption isotherms of HSA as a function of pH at 0.01 ionic strength and at 403C.
observed adsorption behavior shows that the adsorption level of protein onto this thermosensitive magnetic latex is not only governed by hydrophobic interactions but also by electrostatic interactions. In fact, the adsorbed amount of HSA decreases when the attractive electrostatic forces are reduced by increasing the pH. Furthermore, protein adsorption is totally reduced even above the LCST by increasing principally the charge} charge repulsive interactions between protein and latex particles negatively charged above pH*6 and at low salinity ()10\ M). The e!ect of ionic strength on the amounts adsorbed at acidic and basic pH levels was investigated in order to determine the e!ect of ionic strength on electrostatic adsorption. The adsorbed amount at the acidic pH of &4.7 was found to decrease on increasing the ionic strength. A similar behavior has also been observed in the case of BSA adsorption onto cationic poly [NIPAM] particles [4]. This can be explained by the reduction of attractive electrostatic interactions between the protein and the oppositely charged latex support. However, the adsorbed amounts at the basic pH of &8.6 were found to be increased when the ionic strength increases. In fact, the increase in salinity leads to the reduction of repulsive electrostatic interactions between the protein and the latex particles (both negatively charged) as discussed above and reported in the case of polystyrene latexes [6].
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A. Elan( ssari, V. Bourrel / Journal of Magnetism and Magnetic Materials 225 (2001) 151}155
3.5. Ewect of pH and ionic strength on HSA desorption The pH e!ect on the desorbed HSA amount was investigated by "rst performing the adsorption at acidic pH&4.6, 0.01 ionic strength and at 403C. Firstly, the desorption at pH&8.6, 0.01 ionic strength and at 203C, reveals that desorption was not complete as reported in Fig. 5 and the desorbed amounts were found to be between 70% and 80% of the initially adsorbed HSA. Secondly, HSA desorption depends not only on the pH of the medium and the temperature, but also on the ionic strength and the incubation time as shown in Fig. 6. The maximal desorbed HSA amount was observed for 0.1 ionic strength, whereas, the desorbed HSA
Fig. 7. Adsorbed amount of HSA versus ionic strength and pH 8.6 and pH 4.7 and at 403C.
amount decreases on increasing the salinity before reaching a plateau, thereby corroborating the e!ect of ionic strength on the adsorption behavior discussed above and reported in Fig. 7. The maximum desorption observed versus salinity may be attributed to the e!ect of ionic strength on (i) electrostatic interactions between protein and latex particles at the investigated pH&8.6 and (ii) LCST variation versus salinity. We have no tangible explanation at present for this observation. Fig. 5. Residual adsorbed amount of HSA after desorption step.
4. Conclusion
Fig. 6. Desorbed amount of HSA as a function of ionic strength and incubation time (at pH 8.6).
Thermosensitive core-shell magnetic latexes can be used to control protein adsorption and desorption. The adsorption of HSA protein was principally governed by hydrophobic interactions above the LCST of the poly (NIPAM). The contribution of electrostatic interactions to the adsorption process was clearly evidenced versus pH and salinity. The desorbed amount of protein below the LCST was drastically a!ected by the experimental conditions such as incubation time, pH, salinity and temperature of the medium. Indeed, the maximal desorbed amount of protein was obtained at basic pH'IEP (isoelectric point of the protein), high salinity (&0.1 M NaCl) and at 203C (particles in a hydrophilic state). The obtained results indicate that such particles could serve as an alternative route for protein
A. Elan( ssari, V. Bourrel / Journal of Magnetism and Magnetic Materials 225 (2001) 151}155
concentration and puri"cation, thus supplementing other techniques such as precipitation using high salt-concentrated medium, Sepharose gel column system, and concentration via speci"c capture.
References [1] J.C. Olivier, M. Taverna, C. Vauthier et al., Electrophoresis 15 (1994) 234.
155
[2] P. Van Dulm, W. Norde, J. Colloid Interface Sci. 91 (1983) 248. [3] H. Kawaguchi, K. Fujimoto, Y. Mizuhara, Colloid Polym. Sci. 53 (1992) 270. [4] A. ElamK ssari, L.C. Holt, F. Voisset et al., J. Biomater. Sci. Polymer Edn. 10 (1999) 403. [5] K. Fujimoto, Y. Mizuhara, N. Tamura, H. Kawaguchi, J. Intelligent Material Systems Struct. 4 (1993) 184. [6] A.V. Elgersma, R.L. Zsom, W. Norde, J. Lyklema, J. Colloid Interface Sci. 138 (1990) 145.
Thermosensitive magnetic latex particles for controlling protein adsorption and desorption A. ElamK ssari*, V. Bourrel UMR-103, CNRS-bioMe& rieux, ENS de Lyon, 46 alle& e d+Italie, 69364 Lyon cedex 07, France
Abstract Thermosensitive core-shell magnetic latex with a magnetic polystyrene core and a rich poly(N-isopropylacrylamide) shell layer was prepared via the seed precipitation polymerization process using magnetic polystyrene particles (as the seed). The adsorption and desorption of Human Serum Albumin (HSA) onto the prepared thermosensitive magnetic latex particles were examined as a function of temperature, incubation time, pH and ionic strength. 2001 Elsevier Science B.V. All rights reserved. Keywords: Thermosensitive particles; Magnetic latex; Poly(N-isopropylacrylamide) ("NIPAM); Protein; Adsorption; Desorption; Core-shell particles; Polystyrene particles; Precipitation polymerization; Lower critical solution temperature (LCST)
1. Introduction With the choice of latex particles as particle carriers in biomedical applications, much work has been devoted to the preparation of hydrophobic magnetic polymer particles [1,2]. When used in the diagnostic "eld as a solid-phase support, it has been shown that polystyrene particles exhibit undesirable phenomena such as protein denaturation and an irreversible adsorption process. The introduction of thermosensitive microgel particles with a lower critical solution temperature (LCST) in biomedical research is of great interest, since their physico-chemical properties and the adsorption} desorption of proteins can be controlled by the pH,
* Corresponding author. Tel.: #33-472-728-364; fax: #33472-728-533. E-mail address: [email protected] (A. ElamK ssari).
temperature and salinity of the medium [3,4]. In addition, such particles may confer a non-denaturing environment to the immobilized protein, which is particularly suitable for immunoassays and protein concentration and puri"cation. This paper aims to report the use of thermosensitive magnetic latex particles for protein concentration and puri"cation by controlling both the adsorption and desorption processes as illustrated in Fig. 1.
2. Experimental section 2.1. Latex preparation Functionalization of a seed polystyrene magnetic latex (EM100/20, 0.8 m average size from Prolabo, France) bearing carboxylic groups was performed by precipitation polymerization of N-isopropylacrylamide (NIPAM) as the main monomer,
0304-8853/01/$ - see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 8 8 5 3 ( 0 0 ) 0 1 2 4 4 - 0
152
A. Elan( ssari, V. Bourrel / Journal of Magnetism and Magnetic Materials 225 (2001) 151}155
3. Results and discussion 3.1. Characterization of latex particles The polymerization conversion was found to be close to 100%, with 12% water-soluble polymer formation. The average particle diameter as measured using Quasi-Elastic Light Scattering (QELS) of the surface-modi"ed particles was equal to 1 m in broad polydispersity. The "nal latex was puri"ed by repetitive magnetic separations and redispersion.
3.2. Adsorption kinetics Fig. 1. Diagram illustrating the protein concentration principle using magnetic latex particles.
methylene bisacrylamide (MBA) as the cross-linker and potassium persulfate (KPS) as the initiator. Before encapsulation, the seed magnetic latex was "rst cleaned so as to eliminate any free electrolyte and adsorbed surfactant. A given amount of seed magnetic latex (1 g of EM100/20) was purged with nitrogen for 2 h at a temperature of 703C, then a mixture of NIPAM (0.3 g), MBA (0.03 g) and KPS (0.006 g) was added. Polymerization was carried out for 2 h. The polymerization conversion was gravimetrically determined and the "nal latex was washed at least three times before the adsorption study.
Due to low and negligible protein adsorption below the LCST of poly [NIPAM] shell (i.e. LCST&323C) (as will be discussed below), the HSA adsorption kinetics was performed at 403C, pH 4.6 and at 10\ ionic strength. The adsorption was found to be rapid and a plateau value of 45 mg HSA g\ of latex was reached within 30 min as reported in Fig. 2. The result obtained on the adsorption kinetics of HSA onto such magnetic latex particles correlated well with those reported by Kawaguchi et al. [3] by investigating HCG adsorption onto negatively charged poly [NIPAM] microspheres. In fact, they pointed out that protein adsorption was rapid (within only 20 min) when the adsorption was performed above the LCST.
2.2. Protein adsorption and desorption A given concentration of protein Human Serum Albumin (HSA) and the magnetic latex particles (1.6 mg/mL) were incubated below (or above) the LCST of Poly [NIPAM] (LCST+323C) in 10 mM phosphate bu!er solution at various pH levels. After 2 h incubation, the amount of protein adsorbed was determined from the supernatant analysis by UV at a wavelength of 595 nm using Bradford titration. For both adsorption and desorption studies, the adsorbed amount (or desorbed amount) was determined using the depletion method.
Fig. 2. Adsorption kinetics of HSA at pH 4.7, 0.01 ionic strength and above LCST at 403C.
A. Elan( ssari, V. Bourrel / Journal of Magnetism and Magnetic Materials 225 (2001) 151}155
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3.3. Adsorption isotherms Typical adsorption isotherms obtained at both temperatures are reported in Fig. 3. The adsorption isotherms of HSA onto polystyrene magnetic latex bearing poly [NIPAM] layer show a linear amount of protein adsorbed versus the initial protein concentration and well-de"ned plateaus for both temperatures (20 and 403C). As expected, the adsorption of HSA protein was temperaturedependent and the maximal adsorbed amount of HSA was observed above the LCST rather than below it. The adsorption behavior versus temperature re#ects that the hydrophobic interactions are the driving forces in the adsorption process above the LCST. Indeed, a linear relationship between the adsorbed amount of protein and temperature was reported by Fujimoto et al. [5] by performing HCG protein adsorption onto negatively charged poly [NIPAM] microspheres. The low adsorbed amount of HSA observed below the LCST can be attributed principally to the contribution of electrostatic interactions, since latex and HSA are oppositely charged at pH 4.7. 3.4. Ewect of pH and ionic strength on HSA adsorption Fig. 4 shows the adsorbed HSA amount as a function of initial protein concentration and pH at a constant salinity (10\ M) and at 403C. The
Fig. 3. Adsorbed amount of HSA versus initial HSA concentration (pH 4.7, 0.01 ionic strength).
Fig. 4. Adsorption isotherms of HSA as a function of pH at 0.01 ionic strength and at 403C.
observed adsorption behavior shows that the adsorption level of protein onto this thermosensitive magnetic latex is not only governed by hydrophobic interactions but also by electrostatic interactions. In fact, the adsorbed amount of HSA decreases when the attractive electrostatic forces are reduced by increasing the pH. Furthermore, protein adsorption is totally reduced even above the LCST by increasing principally the charge} charge repulsive interactions between protein and latex particles negatively charged above pH*6 and at low salinity ()10\ M). The e!ect of ionic strength on the amounts adsorbed at acidic and basic pH levels was investigated in order to determine the e!ect of ionic strength on electrostatic adsorption. The adsorbed amount at the acidic pH of &4.7 was found to decrease on increasing the ionic strength. A similar behavior has also been observed in the case of BSA adsorption onto cationic poly [NIPAM] particles [4]. This can be explained by the reduction of attractive electrostatic interactions between the protein and the oppositely charged latex support. However, the adsorbed amounts at the basic pH of &8.6 were found to be increased when the ionic strength increases. In fact, the increase in salinity leads to the reduction of repulsive electrostatic interactions between the protein and the latex particles (both negatively charged) as discussed above and reported in the case of polystyrene latexes [6].
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A. Elan( ssari, V. Bourrel / Journal of Magnetism and Magnetic Materials 225 (2001) 151}155
3.5. Ewect of pH and ionic strength on HSA desorption The pH e!ect on the desorbed HSA amount was investigated by "rst performing the adsorption at acidic pH&4.6, 0.01 ionic strength and at 403C. Firstly, the desorption at pH&8.6, 0.01 ionic strength and at 203C, reveals that desorption was not complete as reported in Fig. 5 and the desorbed amounts were found to be between 70% and 80% of the initially adsorbed HSA. Secondly, HSA desorption depends not only on the pH of the medium and the temperature, but also on the ionic strength and the incubation time as shown in Fig. 6. The maximal desorbed HSA amount was observed for 0.1 ionic strength, whereas, the desorbed HSA
Fig. 7. Adsorbed amount of HSA versus ionic strength and pH 8.6 and pH 4.7 and at 403C.
amount decreases on increasing the salinity before reaching a plateau, thereby corroborating the e!ect of ionic strength on the adsorption behavior discussed above and reported in Fig. 7. The maximum desorption observed versus salinity may be attributed to the e!ect of ionic strength on (i) electrostatic interactions between protein and latex particles at the investigated pH&8.6 and (ii) LCST variation versus salinity. We have no tangible explanation at present for this observation. Fig. 5. Residual adsorbed amount of HSA after desorption step.
4. Conclusion
Fig. 6. Desorbed amount of HSA as a function of ionic strength and incubation time (at pH 8.6).
Thermosensitive core-shell magnetic latexes can be used to control protein adsorption and desorption. The adsorption of HSA protein was principally governed by hydrophobic interactions above the LCST of the poly (NIPAM). The contribution of electrostatic interactions to the adsorption process was clearly evidenced versus pH and salinity. The desorbed amount of protein below the LCST was drastically a!ected by the experimental conditions such as incubation time, pH, salinity and temperature of the medium. Indeed, the maximal desorbed amount of protein was obtained at basic pH'IEP (isoelectric point of the protein), high salinity (&0.1 M NaCl) and at 203C (particles in a hydrophilic state). The obtained results indicate that such particles could serve as an alternative route for protein
A. Elan( ssari, V. Bourrel / Journal of Magnetism and Magnetic Materials 225 (2001) 151}155
concentration and puri"cation, thus supplementing other techniques such as precipitation using high salt-concentrated medium, Sepharose gel column system, and concentration via speci"c capture.
References [1] J.C. Olivier, M. Taverna, C. Vauthier et al., Electrophoresis 15 (1994) 234.
155
[2] P. Van Dulm, W. Norde, J. Colloid Interface Sci. 91 (1983) 248. [3] H. Kawaguchi, K. Fujimoto, Y. Mizuhara, Colloid Polym. Sci. 53 (1992) 270. [4] A. ElamK ssari, L.C. Holt, F. Voisset et al., J. Biomater. Sci. Polymer Edn. 10 (1999) 403. [5] K. Fujimoto, Y. Mizuhara, N. Tamura, H. Kawaguchi, J. Intelligent Material Systems Struct. 4 (1993) 184. [6] A.V. Elgersma, R.L. Zsom, W. Norde, J. Lyklema, J. Colloid Interface Sci. 138 (1990) 145.