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Thermosensitive N-isopropylacrylamide-vinylphenyl boronic acid copolymer latex particles for nucleotide isolation
Colloids and Surfaces A: Physicochem. Eng. Aspects 232 (2004) 253–259
Thermosensitive N-isopropylacrylamide-vinylphenyl boronic acid copolymer latex particles for nucleotide isolation a , A. Tuncel c,∗ B. Elmas a , M.A. Onur b , S. Senel ¸ a
Department of Chemistry, Hacettepe University, Ankara, Turkey Department of Biology, Hacettepe University, Ankara, Turkey Chemical Engineering Department, Hacettepe University, Ankara, Turkey b
c
Received 28 April 2003; accepted 4 November 2003
Abstract In this study, thermosensitive N-isopropylacrylamide-4-vinylphenylboronic acid copolymer latex, poly(NIPA-co-VPBA) particles were obtained by dispersion polymerization. A nucleotide isolation procedure was proposed by using the latex particles as the sorbent. As a model nucleotide, -nicotiamide adenine dinucleotide (-NAD) was adsorbed onto the latex particles at a low temperature (i.e. +4 ◦ C) via the interaction between boronic acid groups of particles and diol groups of -NAD. The equilibrium -NAD adsorption capacities up to 40 mg/g were obtained by the proposed sorbent. The equilibrium -NAD adsorption capacity of the latex particles significantly decreased with increasing temperature and nearly zero nucleotide adsorption was observed at the temperatures higher than 30 ◦ C. On the other hand, poly(NIPA-co-VPBA) latex particles forming a stable aqueous suspension in the temperature range of 4–25 ◦ C exhibited a thermoflocculation behavior at the temperatures higher than 30 ◦ C. The effects of temperature both on the -NAD adsorption capacity and the colloidal stability of the particles were used for -NAD recovery. After adsorption of -NAD onto the stable latex particles at +4 ◦ C, the particles were flocculated by elevating the temperature to 37 ◦ C and -NAD was desorbed from the flocculating particles simultaneously. Hence, -NAD was recovered in the clear supernatant. Therefore, the desorption of -NAD to a medium at any ionic strength and at any pH could be achieved by using temperature as an on-off switch controlling the adsorption/desorption behavior. © 2003 Elsevier B.V. All rights reserved. Keywords: -Nicotinamide adenine dinucleotide; Boronate affinity chromatography; N-Isopropylacrylamide; Thermosensitive polymer; Vinylphenyl boronic acid; Thermoresponsive chromatography
1. Introduction Thermosensitive polymers have been widely tried as carriers for the immobilization or isolation of different biological agents like enzymes, proteins, oligonucleotides and cells [1–14]. The interaction of boronic acid functionalized thermosensitive polymers with the diol-carrying biomolecules was investigated elsewhere [15–18]. A thermosensitive copolymer of N,N-dimethylacrylamide and 3-acrylamidophenylboronic acid (AcPBA) was synthesized by the radical copolymerization [15]. The thermosensitive copolymers of N-isopropylacrylamide (NIPA) and AcPBA and the terpolymers of NIPA-AcPBA-dimethylaminopropylacrylamide (DMAPA) showed a LCST change against diol carrying biomolecules [16]. The thermosensitive terpolymer ∗
Corresponding author. Fax: +90-312-299-21-24. E-mail address: [email protected] (A. Tuncel).
0927-7757/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfa.2003.11.007
gels in the form of NIPA-AcPBA-dimethylamino-propylmethacrylamide (DMAPM) were utilized in the endothelial cell differentiation as a cell substratum [18]. In the “boronate-affinity chromatography” applications, diol carrying biomolecules were selectively adsorbed onto the support material based on the complex formation between boronic acid and diol groups [19,20]. The selective adsorption allows the separation of diol carrying structure from other biological molecules in the aqueous medium. Then diol-carrying molecules were desorbed into an appropriate solution and obtained in the purified form after applying a dialysis process. Boronic acid-carrying agarose or acrylamide-based stationary phases have been commonly utilized in the chromatographic studies involving the separation of nucleotides, oligonucleotides, glycoproteins and glycoenzymes [21–29]. We also produced various sorbent materials suitable for the isolation of nucleotides and nucleic acids [30–37].
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On the other hand, the carriers based on the uniform latex particles were commonly examined in the biotechnological applications aiming the immobilization or specific isolation of biomolecules. Various emulsion or dispersion polymerization methods were used for the synthesis of uniform latex particles carrying desired functionality depending upon the intended use [37–42]. Recently, thermosensitive polymeric latexes in sub-micron or micron size range were utilized as support materials in the immobilization of different enzymes, oligonucleotides, nucleic acid fragments and antibodies [43–46]. In this study, bononic-acid carrying thermoresponsive latex particles prepared by the emulsion copolymerization of NIPA and a boronic acid carrying comonomer, 4-vinylphenylboronic acid (VPBA) were tried as sorbent for nucleotide isolation. In the proposed procedure, a model nucleotide (-nicotinamide adenine dinucleotide, -NAD) was adsorbed onto the poly(NIPA-co-VPBA) particles by the complex formation taking place at a low temperature (+4 ◦ C). Following this, -NAD adsorbed particles were thermoflocculated in a separate medium at 37 ◦ C. During the thermoflocculation, -NAD was desorbed from the particles to the solution. By the precipitation of flocculated particles, -NAD was recovered in the clear supernatant with the isolation yields up to 95% (w/w).
2. Experimental 2.1. Materials N-Isopropylacrylamide (NIPAM, Aldrich Chemical Co., Milwaukee, WI) was crystallized from hexane-acetone solution. The comonomer, 4-vinylphenylboronic acid was supplied from Aldrich Chemical Co. N,N-methylenebisacrylamide (MBA, BDH Chemicals Ltd., Poole, UK) and potassium persulfate (KPS, Analar grade, BDH Chemicals Ltd.). were used as the crosslinking agent and the initiator, respectively. -Nicotinamide adenine dinucleotide (-NAD, Cat No.: N1511, Sigma Chemical Co., St. Louis, USA) was selected as the model nucleotide. N-[2-Hydroxyethyl]piperazine-N -[2-ethane sulfonic acid] (HEPES, Sigma Chemical Co.) buffer solutions were used in the adsorption experiments. 2.2. Preparation of poly(NIPA-co-VPBA) copolymer latex The dispersion copolymerization is summarized as follows: NIPA (1 g), VPBA (0.05 g) and MBA (0.04 g) were dissolved in distilled water (40 ml) and the solution was transferred to a cylindrical pyrex reactor. The initiator, KPS (0.03 g) was dissolved in the solution. Following nitrogen purge for 10 min, the reactor was sealed. Emulsion copolymerization was performed at 70 ± 0.5 ◦ C for 24 h in a temperature-controlled water bath shaken at 150 cpm. At the end of polymerization, the latex was cooled down to room
temperature and cleaned by centrifugation-decantation. For this purpose, the latex suspension was centrifuged at 10.000 rpm for 10 min. The supernatant was discarded and the latex particles were redispersed in water (50 ml) by ultrasonication. This operation was repeated several times for the removal of any possible unreacted monomer from the latex dispersion. In these experiments, VPBA/NIPA feed ratio was varied for the synthesis of poly(NIPA-co-VPBA) particles with different boronic acid contents. 2.3. Characterization of latex particles The final latex yield was determined by a conventional gravimetric procedure. The average particle size was measured by a transmission electron microscope (TEM, JEOL, JEM 1200EX, Japan). The detailed procedures both for the preparation of the particle samples and electron microscopic examination were given elsewhere [46]. Boronic acid content of the latex particles was determined by Carmine method as described elsewhere [46]. Thermosensitive behavior of poly(NIPA-co-VPBA) latex was followed by a UV-Vis spectrophotometer equipped with a temperature-control system (Shimadzu, Japan). Typically, the cleaned latex suspension was diluted at an appropriate ratio for obtaining a suspension with a solid content of 0.1% (w/w). Then, the pH of latex was adjusted by aqueous NaOH solution to a prescribed value (i.e. 5, 7 and 9). The absorbance was measured at 500 nm by increasing the temperature at a heating rate of approximately 1 ◦ C/min, by starting from 15 ◦ C. Critical flocculation temperature (CFT) was determined according to the procedure described elsewhere [46]. 2.4. β-NAD adsorption experiments -NAD adsorption experiments were performed as follows: A certain volume of latex suspension including poly(NIPA-co-VPBA) particles (0.05 g based on dry weight) was centrifuged at 10.000 rpm and +4 ◦ C for 10 min and the supernatant was discarded. The particles were redispersed in HEPES buffer (5 ml, pH 8.5, ionic strength:0.1) containing -NAD at a certain concentration. The adsorption medium was magnetically stirred at 250 rpm for 1 h at +4 ◦ C. After completion of adsorption period, the entire mixture was centrifuged at 10,000 rpm for 10 min at +4 ◦ C and the supernatant was isolated. It should be noted that the centrifugation was conducted at the temperature identical to that of adsorption. -NAD concentration in the supernatant was determined by measuring the absorbance at 260 nm in a UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan). The adsorbed amount of -NAD onto poly(NIPA-co-VPBA) particles (Q-NAD , mg -NAD/g dry particles) was calculated according to Eq. (1). Where Co (mg/ml) and Cf (mg/ml) are the initial and final -NAD concentrations in the HEPES buffer, respectively. MG (g) and V (ml) are the amount of dry particles and the volume of HEPES buffer,
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respectively. Q−NAD = (Co − Cf )
V MG
(1)
2.5. β-NAD desorption experiments Following centrifugation at +4 ◦ C, -NAD adsorbed poly(NIPA-co-VPBA) particles were put into the desorption medium (usually HEPES buffer at pH 8.5, including 0.1 M MgCl2 ) at +4 ◦ C. The particles were redispersed in the solution at +4 ◦ C and the temperature was raised to 37 ◦ C. The mixture was magnetically stirred for 30 min at this temperature. At the end of this period, the stirring was stopped and the precipitation of poly(NIPA-co-VPBA) particles by the thermoflocculation was allowed for another 30 min. After completion of the precipitation, a certain volume of sample (2–3 ml) was withdrawn from the supernatant and the absorbance was measured at 260 nm in a UV-Vis spectrophotometer. The desorption yield was calculated as the ratio of desorbed amount of -NAD (mg) to the adsorbed amount of -NAD on the particles (mg).
3. Results and discussion 3.1. Characterization of thermosensitive latex particles The dispersion copolymerization conditions are given in Table 1. As seen here, a set of copolymerizations were performed based on the systematical change of VPBA feed concentration between 0 and 7.1 mol% based on total monomer. As seen in Table 1, VPBA content of the particles increased with increasing feed concentration of VPBA. On the other hand, the copolymer particles produced with different VPBA concentrations had approximately the same size (i.e. 0.6 m). The TEM photographs of poly(NIPA-co-VPBA) particles are exemplified in Fig. 1. As seen here, the latex particles obtained by the dispersion copolymerization of NIPA and VPBA were nearly uniform. This case was also observed for the other latexes synthesized in our study. For poly(NIPA-co-VPBA) latexes, the variation of absorbance with the temperature is given in Fig. 2. In this figure, the magnitude of absorbance difference between the lower and upper plateau values was evaluated as an indicator for the
Fig. 1. TEM photographs of poly(NIPA-co-VPBA) latex particles taken in the dry state. Magnification: 5000×, latex code: (A) IB3, (B) IB4.
Table 1 The production conditions of poly(NIPA-co-VPBA) particles Code
NIPA (mg)
VPBA (mg)
MBA (mg)
KPS (mg)
Water (g)
QVPBA (mg/g)
IB1 IB2 IB3 IB4
1000 1000 1000 1000
0.0 25.0 50.0 100.0
40 40 40 40
30 30 30 30
40 40 40 40
0.0 24.9 46.7 95.1
QVPBA : final VPBA content of particles (mg VPBA/g dry particles), polymerization conditions: KPS: 30 mg, water: 40 ml, temperature:70 ◦ C, shaking rate: 150 cpm, and time: 24 h.
Fig. 2. Thermosensitive behavior of poly(NIPA-co-VPBA) latex particles followed by UV-Vis spectrophotometry (pH 7.0).
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thermosensitivity of the latex particles [45,46]. Because the lower plateu observed at the low-temperature range corresponds to the latex suspension including swollen particles whereas the upper plateau in the high temperature region gives the absorbance of the latex suspension of shrunken particles. Hence, the latex providing the largest absorbance difference between fully shrunken and fully swollen states should be the most thermosensitive one among the produced materials. As seen in Fig. 2, this absorbance difference decreased with increasing VPBA content of the latex particles. In other words, the thermosensitivity of particles probably decreased with increasing boronic acid content. Among these latexes, poly(NIPA-co-VPBA) particles produced with the VPBA feed concentration of 3.7 mol% (i.e. the latex sample encoded as IB3 in Table 1) were mostly used as the sorbent in the -NAD adsorption experiments since it exhibited an appreciable thermosensitive behavior with a sufficiently high VPBA content (46.7 mg VPBA/g dry particles). The thermosensitive behavior of IB3 was also determined in the aqueous media prepared with different ionic strengths. The variation of absorbance with the temperature in the aqueous media containing NaCl at different concentrations at pH 7 is given in Fig. 3. As seen here, bell-shaped curves were obtained in the media containing NaCl at relatively high concentrations whereas an s-shaped curve was observed in the aqueous medium containing no salt. In the case of bell-shaped curves, the absorbance decrease after the peak point occurred due to the flocculation of latex particles induced by the temperature increase. In other words, thermoflocculation of poly(NIPA-co-VPBA) latex particles was more clearly observed at higher salt concentrations. Here, the temperature at which the absorbance exhibited a
Fig. 4. The variation of critical flocculation temperature with the salt concentration at pH 7.0 for the NIPA–VPBA copolymer latex encoded as IB3.
maximum was defined as the critical flocculation temperature. For the latex encoded as IB3, the variation of CFT by the salt concentration is given in Fig. 4. As seen here, CFT significantly decreased with increasing salt concentration. Note that the thermoflocculation behavior was utilized for the separation of the sorbent material from the aqueous medium in the -NAD desorption stage conducted at the temperatures higher than the CFT of the selected latex. 3.2. β-NAD adsorption onto thermosensitive latex particles
Fig. 3. Typical absorbance-temperature curves indicating the critical flocculation temperature of NIPA–VPBA copolymer latex encoded as IB3 (pH 7.0).
In the first group of experiments, the effect of pH on the -NAD adsorption behavior of poly(NIPA-co-VPBA) particles was investigated. In these experiments, the particles encoded as IB3 were utilized as the sorbent. The adsorption experiments were performed at +4 ◦ C in a HEPES buffer (5 ml) containing the selected sorbent (0.05 g based on dry weight). The initial -NAD concentration was fixed at 0.5 mg/ml. The effect of pH on the -NAD adsorption is given in Fig. 5. As seen here, -NAD adsorption increased with increasing pH and exhibited a plateau at the pHs higher than 8.5. In the literature, boronic acid functionalized supports (i.e. mostly agarose based gels) have been commonly utilized in the isolation of nucleotides by different chromatographic methods [21–25]. The binding of nucleotides onto the boronic acid functionalized supports occurred via the formation of cyclic borate ester by the reaction between the boronic acid groups in the hydrophilic tetrahedral anionic form and the diol groups of nucleotides. Note that pKa value of boronic acid was 8.86 [22]. Based on this value, most of the boronic acid groups on the poly(NIPA-co-VPBA) particles should be in the hydrophobic trigonal form at the pH
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Fig. 5. The effect of pH on the -NAD adsorption behavior of poly(NIPA-co-VPBA) particles (sorbent: IB3, -NAD concentration: 0.5 mg/ml, +4 ◦ C).
values lower than neutral pH. As described in the literature, the trigonal form of boronic acid is not reactive against the diol groups of the target biomolecules [15,17,46,47]. For this reason, -NAD adsorption onto the latex particles was low at the pHs lower than or equal to 7. However, cyclic borate ester formation should effectively occur by the interaction of tetrahedral boronic acid groups with the diol groups of -NAD at the pH values close to the pKa of phenylboronic acid [15,17,46,47]. Therefore, relatively higher -NAD adsorption was observed at the pHs higher than 8.5 (i.e. the pH region in which most of the boronic acid groups are in the hydrophilic tetrahedral anionic form). By considering the behavior in Fig. 5, pH 8.5 was selected as an appropriate value for the other adsorption experiments. The effect of temperature on the -NAD adsorption of poly(NIPA-co-VPBA) particles is given in Fig. 6. Here, the adsorption experiments were performed at pH 8.5 with the initial -NAD concentration of 0.5 mg/ml. The sorbent concentration was fixed at 10 mg/ml in a batch volume of 5 ml. As seen in Fig. 6, -NAD adsorption onto the poly(NIPA-co-VPBA) particles markedly decreased with increasing temperature. All particle types showed nearly the same behavior. Note that the latex particles are in the fully swollen form at +4 ◦ C. At the low temperatures, poly(NIPA-co-VPBA) particles probably had a typical tailor-made structure comprised of a swollen-core and relatively flexible, boronic acid carrying-copolymer chains linked to the surface of swollen core [46]. The boronic acid groups on the particles should be in the partly ionized form in the adsorption medium with slightly basic character (pH 8.5). For this reason, an increase in the flexibility of copolymer chains attached to the swollen core should be expected
257
Fig. 6. The effect of temperature on the -NAD adsorption behavior of poly(NIPA-co-VPBA) particles (sorbent concentration: 10 mg/ml, initial -NAD concentration:0.5 mg/ml, pH 8.5).
[46]. Hence, the complex formation between the diol groups of -NAD molecules and the boronic acid groups available on the flexible copolymer chains should occur more easily in the low-temperature range. This model involves higher -NAD adsorption onto poly(NIPA-co-VPBA) particles at the lower temperatures. By increasing temperature, the core shrinks and probably a stiff layer is formed on the surface of shrunken particles by folding of the tailored copolymer chains. In this case, the interaction of -NAD molecules with the boronic acid functionality should become more difficult. Hence, -NAD adsorption decreases with increasing temperature. By considering the behavior in Fig. 6, -NAD adsorption isotherms were derived at +4 ◦ C. On the other hand, by considering low -NAD adsorption capacity at relatively high temperatures, 37 ◦ C was selected as an appropriate value for the desorption of -NAD from the latex particles. Except for the particles with the highest VPBA content, the magnitude of the adsorbed amount of -NAD at the low-temperature range (i.e. 4–20 ◦ C) was approximately proportional to the VPBA content of the sorbent material. For the particles with the highest VPBA content, -NAD adsorption at low temperatures was lower relative to the other particles. This behavior may be attributted to the formation of a surface structure not so suitable for nucleotide adsorption. As discussed before, the particle-surface carrying flexible, tailored copolymer chains with thermosensitive character is particularly suitable for high -NAD adsorption at the low temperatures. The flexibility of tailored copolymer chains should be strongly related to their thermosensitive character. High VPBA feed concentration involves an increase in the VPBA content of these chains which in turn their thermosensivity decreases. In this case, the formation possibility of linear chains making all boronic acid groups available may decrease in the low-temperature range. The
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Fig. 7. The variation of -NAD adsorption onto the poly(NIPA-co-VPBA) particles by the initial -NAD concentration (sorbent: IB3, sorbent concentration: 10 mg/ml, pH 8.5, +4 ◦ C).
chains with low thermosensivity can be found in the folded form that makes difficult the interaction of boronic acid groups with the -NAD molecules in the solution. This case probably leads to low -NAD adsorption at the low temperatures. The variation of -NAD adsorption with the initial -NAD concentration is given in Fig. 7. Here, the poly(NIPA-co-VPBA) particles encoded as IB3 were used as the sorbent. The poly(NIPA) particles prepared in the absence of VPBA were selected as the control-material. The adsorption experiments were performed at +4 ◦ C and pH 8.5. The other conditions were the same with those of the set in which the effect of pH was tested. As seen here, the plateau value of -NAD adsorption onto the poly(NIPA-co-VPBA) particles was reasonably higher relative to that obtained with the poly(NIPA) particles. This result is probably explained by the formation of cyclic borate ester via the interaction between boronate and diol groups [15,17,46,47]. It should be noted that the plateau value of adsorbed -NAD was obtained for the initial -NAD concentrations higher than 0.5 mg/ml in both cases. 3.3. Desorption of β-NAD from thermosensitive latex particles The tendency in Fig. 6 clearly showed that -NAD adsorption onto the poly(NIPA-co-VPBA) particles was controlled by adjusting the temperature. This behavior also indicated that the temperature should be considered as an effective variable for controlling the desorption process. The desorption of -NAD from poly(NIPA-co-VPBA) particles encoded as IB3 was investigated at different temperatures. For this purpose, -NAD was adsorbed onto
Fig. 8. The variation of -NAD desorption yield with temperature. Desorption conditions: sorbent concentration: 10 mg/ml, 37 ◦ C, 0.1 M MgCl2 , 30 min.
the poly(NIPA-co-VPBA) particles with the -NAD initial concentration of 0.5 mg/ml at pH 8.5 and at +4 ◦ C. For the desorption, -NAD adsorbed particles were redispersed in another HEPES buffer solution (pH 8.5) at +4 ◦ C. Then the temperature was elevated to the desired value and the entire mixture was kept at this temperature for 30 min. The variation of desorption yield by the temperature is given in Fig. 8. As expected -NAD desorption yield significantly increased with increasing temperature at pH 8.5. The desorption yield obtained at 37 ◦ C (i.e. 95%, w/w) was satisfactory. The desorption experiments were performed in the temperature range of 4–37 ◦ C and the thermoflocculation of latex particles was only observed at 37 ◦ C. By the precipitation of thermoflocculated particles, the separation of the sorbent from the desorption medium was simultaneously achieved. So, the desorbed -NAD was recovered in the clear supernatant at 37 ◦ C. In the desorption experiments conducted at the temperatures lower than 37 ◦ C, the latex particles were separated from the desorption medium by centrifugation. On the other hand, the effect of pH on the desorption yield was also examined at 37 ◦ C. For this purpose, HEPES buffer solutions at different pH values ranging between 6 and 10 were tried. The same experimental procedure was also followed in this set. The effect of pH on the -NAD desorption yield is given in Fig. 9. As seen here, pH 8.5 was found as the most appropriate value. The desorption process is controlled by the decomposition behavior of cyclic borate ester formed at pH 8.5 in our study (i.e. the pH at which -NAD adsorption was performed). The low desorption observed at pH 6–7 indicated that the formed cyclic borate ester is relatively stable in this pH region. Stronger affinity of tetrahedral boronic acid groups to the diol groups of -NAD at the
B. Elmas et al. / Colloids and Surfaces A: Physicochem. Eng. Aspects 232 (2004) 253–259
Fig. 9. The effect of pH on the -NAD desorption yield (sorbent concentration: 10 mg/ml, 37 ◦ C, 0.1 M MgCl2 , 30 min, pH variable).
pHs higher than 9 probably led to lower -NAD desorption yields (i.e. Fig. 5).
4. Conclusion In the studies involving the isolation of nucleotides and nucleic acids by using the boronic acid functionalized support materials, the desorption process is usually conducted in basic media containing (pH 10–10.5) a salt at extremely high concentration (approximately 1 M). The presence of salt at such a high concentration usually involves extensive dialysis of desorption solution for the purification of desorbed biomolecule. The use of boronic acid carrying sorbent in the thermosensitive form allowed the desorption of -NAD in a relatively wide pH range without occurring a significant change in the yield.
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Thermosensitive N-isopropylacrylamide-vinylphenyl boronic acid copolymer latex particles for nucleotide isolation a , A. Tuncel c,∗ B. Elmas a , M.A. Onur b , S. Senel ¸ a
Department of Chemistry, Hacettepe University, Ankara, Turkey Department of Biology, Hacettepe University, Ankara, Turkey Chemical Engineering Department, Hacettepe University, Ankara, Turkey b
c
Received 28 April 2003; accepted 4 November 2003
Abstract In this study, thermosensitive N-isopropylacrylamide-4-vinylphenylboronic acid copolymer latex, poly(NIPA-co-VPBA) particles were obtained by dispersion polymerization. A nucleotide isolation procedure was proposed by using the latex particles as the sorbent. As a model nucleotide, -nicotiamide adenine dinucleotide (-NAD) was adsorbed onto the latex particles at a low temperature (i.e. +4 ◦ C) via the interaction between boronic acid groups of particles and diol groups of -NAD. The equilibrium -NAD adsorption capacities up to 40 mg/g were obtained by the proposed sorbent. The equilibrium -NAD adsorption capacity of the latex particles significantly decreased with increasing temperature and nearly zero nucleotide adsorption was observed at the temperatures higher than 30 ◦ C. On the other hand, poly(NIPA-co-VPBA) latex particles forming a stable aqueous suspension in the temperature range of 4–25 ◦ C exhibited a thermoflocculation behavior at the temperatures higher than 30 ◦ C. The effects of temperature both on the -NAD adsorption capacity and the colloidal stability of the particles were used for -NAD recovery. After adsorption of -NAD onto the stable latex particles at +4 ◦ C, the particles were flocculated by elevating the temperature to 37 ◦ C and -NAD was desorbed from the flocculating particles simultaneously. Hence, -NAD was recovered in the clear supernatant. Therefore, the desorption of -NAD to a medium at any ionic strength and at any pH could be achieved by using temperature as an on-off switch controlling the adsorption/desorption behavior. © 2003 Elsevier B.V. All rights reserved. Keywords: -Nicotinamide adenine dinucleotide; Boronate affinity chromatography; N-Isopropylacrylamide; Thermosensitive polymer; Vinylphenyl boronic acid; Thermoresponsive chromatography
1. Introduction Thermosensitive polymers have been widely tried as carriers for the immobilization or isolation of different biological agents like enzymes, proteins, oligonucleotides and cells [1–14]. The interaction of boronic acid functionalized thermosensitive polymers with the diol-carrying biomolecules was investigated elsewhere [15–18]. A thermosensitive copolymer of N,N-dimethylacrylamide and 3-acrylamidophenylboronic acid (AcPBA) was synthesized by the radical copolymerization [15]. The thermosensitive copolymers of N-isopropylacrylamide (NIPA) and AcPBA and the terpolymers of NIPA-AcPBA-dimethylaminopropylacrylamide (DMAPA) showed a LCST change against diol carrying biomolecules [16]. The thermosensitive terpolymer ∗
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gels in the form of NIPA-AcPBA-dimethylamino-propylmethacrylamide (DMAPM) were utilized in the endothelial cell differentiation as a cell substratum [18]. In the “boronate-affinity chromatography” applications, diol carrying biomolecules were selectively adsorbed onto the support material based on the complex formation between boronic acid and diol groups [19,20]. The selective adsorption allows the separation of diol carrying structure from other biological molecules in the aqueous medium. Then diol-carrying molecules were desorbed into an appropriate solution and obtained in the purified form after applying a dialysis process. Boronic acid-carrying agarose or acrylamide-based stationary phases have been commonly utilized in the chromatographic studies involving the separation of nucleotides, oligonucleotides, glycoproteins and glycoenzymes [21–29]. We also produced various sorbent materials suitable for the isolation of nucleotides and nucleic acids [30–37].
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On the other hand, the carriers based on the uniform latex particles were commonly examined in the biotechnological applications aiming the immobilization or specific isolation of biomolecules. Various emulsion or dispersion polymerization methods were used for the synthesis of uniform latex particles carrying desired functionality depending upon the intended use [37–42]. Recently, thermosensitive polymeric latexes in sub-micron or micron size range were utilized as support materials in the immobilization of different enzymes, oligonucleotides, nucleic acid fragments and antibodies [43–46]. In this study, bononic-acid carrying thermoresponsive latex particles prepared by the emulsion copolymerization of NIPA and a boronic acid carrying comonomer, 4-vinylphenylboronic acid (VPBA) were tried as sorbent for nucleotide isolation. In the proposed procedure, a model nucleotide (-nicotinamide adenine dinucleotide, -NAD) was adsorbed onto the poly(NIPA-co-VPBA) particles by the complex formation taking place at a low temperature (+4 ◦ C). Following this, -NAD adsorbed particles were thermoflocculated in a separate medium at 37 ◦ C. During the thermoflocculation, -NAD was desorbed from the particles to the solution. By the precipitation of flocculated particles, -NAD was recovered in the clear supernatant with the isolation yields up to 95% (w/w).
2. Experimental 2.1. Materials N-Isopropylacrylamide (NIPAM, Aldrich Chemical Co., Milwaukee, WI) was crystallized from hexane-acetone solution. The comonomer, 4-vinylphenylboronic acid was supplied from Aldrich Chemical Co. N,N-methylenebisacrylamide (MBA, BDH Chemicals Ltd., Poole, UK) and potassium persulfate (KPS, Analar grade, BDH Chemicals Ltd.). were used as the crosslinking agent and the initiator, respectively. -Nicotinamide adenine dinucleotide (-NAD, Cat No.: N1511, Sigma Chemical Co., St. Louis, USA) was selected as the model nucleotide. N-[2-Hydroxyethyl]piperazine-N -[2-ethane sulfonic acid] (HEPES, Sigma Chemical Co.) buffer solutions were used in the adsorption experiments. 2.2. Preparation of poly(NIPA-co-VPBA) copolymer latex The dispersion copolymerization is summarized as follows: NIPA (1 g), VPBA (0.05 g) and MBA (0.04 g) were dissolved in distilled water (40 ml) and the solution was transferred to a cylindrical pyrex reactor. The initiator, KPS (0.03 g) was dissolved in the solution. Following nitrogen purge for 10 min, the reactor was sealed. Emulsion copolymerization was performed at 70 ± 0.5 ◦ C for 24 h in a temperature-controlled water bath shaken at 150 cpm. At the end of polymerization, the latex was cooled down to room
temperature and cleaned by centrifugation-decantation. For this purpose, the latex suspension was centrifuged at 10.000 rpm for 10 min. The supernatant was discarded and the latex particles were redispersed in water (50 ml) by ultrasonication. This operation was repeated several times for the removal of any possible unreacted monomer from the latex dispersion. In these experiments, VPBA/NIPA feed ratio was varied for the synthesis of poly(NIPA-co-VPBA) particles with different boronic acid contents. 2.3. Characterization of latex particles The final latex yield was determined by a conventional gravimetric procedure. The average particle size was measured by a transmission electron microscope (TEM, JEOL, JEM 1200EX, Japan). The detailed procedures both for the preparation of the particle samples and electron microscopic examination were given elsewhere [46]. Boronic acid content of the latex particles was determined by Carmine method as described elsewhere [46]. Thermosensitive behavior of poly(NIPA-co-VPBA) latex was followed by a UV-Vis spectrophotometer equipped with a temperature-control system (Shimadzu, Japan). Typically, the cleaned latex suspension was diluted at an appropriate ratio for obtaining a suspension with a solid content of 0.1% (w/w). Then, the pH of latex was adjusted by aqueous NaOH solution to a prescribed value (i.e. 5, 7 and 9). The absorbance was measured at 500 nm by increasing the temperature at a heating rate of approximately 1 ◦ C/min, by starting from 15 ◦ C. Critical flocculation temperature (CFT) was determined according to the procedure described elsewhere [46]. 2.4. β-NAD adsorption experiments -NAD adsorption experiments were performed as follows: A certain volume of latex suspension including poly(NIPA-co-VPBA) particles (0.05 g based on dry weight) was centrifuged at 10.000 rpm and +4 ◦ C for 10 min and the supernatant was discarded. The particles were redispersed in HEPES buffer (5 ml, pH 8.5, ionic strength:0.1) containing -NAD at a certain concentration. The adsorption medium was magnetically stirred at 250 rpm for 1 h at +4 ◦ C. After completion of adsorption period, the entire mixture was centrifuged at 10,000 rpm for 10 min at +4 ◦ C and the supernatant was isolated. It should be noted that the centrifugation was conducted at the temperature identical to that of adsorption. -NAD concentration in the supernatant was determined by measuring the absorbance at 260 nm in a UV-Vis spectrophotometer (Shimadzu, Kyoto, Japan). The adsorbed amount of -NAD onto poly(NIPA-co-VPBA) particles (Q-NAD , mg -NAD/g dry particles) was calculated according to Eq. (1). Where Co (mg/ml) and Cf (mg/ml) are the initial and final -NAD concentrations in the HEPES buffer, respectively. MG (g) and V (ml) are the amount of dry particles and the volume of HEPES buffer,
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respectively. Q−NAD = (Co − Cf )
V MG
(1)
2.5. β-NAD desorption experiments Following centrifugation at +4 ◦ C, -NAD adsorbed poly(NIPA-co-VPBA) particles were put into the desorption medium (usually HEPES buffer at pH 8.5, including 0.1 M MgCl2 ) at +4 ◦ C. The particles were redispersed in the solution at +4 ◦ C and the temperature was raised to 37 ◦ C. The mixture was magnetically stirred for 30 min at this temperature. At the end of this period, the stirring was stopped and the precipitation of poly(NIPA-co-VPBA) particles by the thermoflocculation was allowed for another 30 min. After completion of the precipitation, a certain volume of sample (2–3 ml) was withdrawn from the supernatant and the absorbance was measured at 260 nm in a UV-Vis spectrophotometer. The desorption yield was calculated as the ratio of desorbed amount of -NAD (mg) to the adsorbed amount of -NAD on the particles (mg).
3. Results and discussion 3.1. Characterization of thermosensitive latex particles The dispersion copolymerization conditions are given in Table 1. As seen here, a set of copolymerizations were performed based on the systematical change of VPBA feed concentration between 0 and 7.1 mol% based on total monomer. As seen in Table 1, VPBA content of the particles increased with increasing feed concentration of VPBA. On the other hand, the copolymer particles produced with different VPBA concentrations had approximately the same size (i.e. 0.6 m). The TEM photographs of poly(NIPA-co-VPBA) particles are exemplified in Fig. 1. As seen here, the latex particles obtained by the dispersion copolymerization of NIPA and VPBA were nearly uniform. This case was also observed for the other latexes synthesized in our study. For poly(NIPA-co-VPBA) latexes, the variation of absorbance with the temperature is given in Fig. 2. In this figure, the magnitude of absorbance difference between the lower and upper plateau values was evaluated as an indicator for the
Fig. 1. TEM photographs of poly(NIPA-co-VPBA) latex particles taken in the dry state. Magnification: 5000×, latex code: (A) IB3, (B) IB4.
Table 1 The production conditions of poly(NIPA-co-VPBA) particles Code
NIPA (mg)
VPBA (mg)
MBA (mg)
KPS (mg)
Water (g)
QVPBA (mg/g)
IB1 IB2 IB3 IB4
1000 1000 1000 1000
0.0 25.0 50.0 100.0
40 40 40 40
30 30 30 30
40 40 40 40
0.0 24.9 46.7 95.1
QVPBA : final VPBA content of particles (mg VPBA/g dry particles), polymerization conditions: KPS: 30 mg, water: 40 ml, temperature:70 ◦ C, shaking rate: 150 cpm, and time: 24 h.
Fig. 2. Thermosensitive behavior of poly(NIPA-co-VPBA) latex particles followed by UV-Vis spectrophotometry (pH 7.0).
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thermosensitivity of the latex particles [45,46]. Because the lower plateu observed at the low-temperature range corresponds to the latex suspension including swollen particles whereas the upper plateau in the high temperature region gives the absorbance of the latex suspension of shrunken particles. Hence, the latex providing the largest absorbance difference between fully shrunken and fully swollen states should be the most thermosensitive one among the produced materials. As seen in Fig. 2, this absorbance difference decreased with increasing VPBA content of the latex particles. In other words, the thermosensitivity of particles probably decreased with increasing boronic acid content. Among these latexes, poly(NIPA-co-VPBA) particles produced with the VPBA feed concentration of 3.7 mol% (i.e. the latex sample encoded as IB3 in Table 1) were mostly used as the sorbent in the -NAD adsorption experiments since it exhibited an appreciable thermosensitive behavior with a sufficiently high VPBA content (46.7 mg VPBA/g dry particles). The thermosensitive behavior of IB3 was also determined in the aqueous media prepared with different ionic strengths. The variation of absorbance with the temperature in the aqueous media containing NaCl at different concentrations at pH 7 is given in Fig. 3. As seen here, bell-shaped curves were obtained in the media containing NaCl at relatively high concentrations whereas an s-shaped curve was observed in the aqueous medium containing no salt. In the case of bell-shaped curves, the absorbance decrease after the peak point occurred due to the flocculation of latex particles induced by the temperature increase. In other words, thermoflocculation of poly(NIPA-co-VPBA) latex particles was more clearly observed at higher salt concentrations. Here, the temperature at which the absorbance exhibited a
Fig. 4. The variation of critical flocculation temperature with the salt concentration at pH 7.0 for the NIPA–VPBA copolymer latex encoded as IB3.
maximum was defined as the critical flocculation temperature. For the latex encoded as IB3, the variation of CFT by the salt concentration is given in Fig. 4. As seen here, CFT significantly decreased with increasing salt concentration. Note that the thermoflocculation behavior was utilized for the separation of the sorbent material from the aqueous medium in the -NAD desorption stage conducted at the temperatures higher than the CFT of the selected latex. 3.2. β-NAD adsorption onto thermosensitive latex particles
Fig. 3. Typical absorbance-temperature curves indicating the critical flocculation temperature of NIPA–VPBA copolymer latex encoded as IB3 (pH 7.0).
In the first group of experiments, the effect of pH on the -NAD adsorption behavior of poly(NIPA-co-VPBA) particles was investigated. In these experiments, the particles encoded as IB3 were utilized as the sorbent. The adsorption experiments were performed at +4 ◦ C in a HEPES buffer (5 ml) containing the selected sorbent (0.05 g based on dry weight). The initial -NAD concentration was fixed at 0.5 mg/ml. The effect of pH on the -NAD adsorption is given in Fig. 5. As seen here, -NAD adsorption increased with increasing pH and exhibited a plateau at the pHs higher than 8.5. In the literature, boronic acid functionalized supports (i.e. mostly agarose based gels) have been commonly utilized in the isolation of nucleotides by different chromatographic methods [21–25]. The binding of nucleotides onto the boronic acid functionalized supports occurred via the formation of cyclic borate ester by the reaction between the boronic acid groups in the hydrophilic tetrahedral anionic form and the diol groups of nucleotides. Note that pKa value of boronic acid was 8.86 [22]. Based on this value, most of the boronic acid groups on the poly(NIPA-co-VPBA) particles should be in the hydrophobic trigonal form at the pH
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Fig. 5. The effect of pH on the -NAD adsorption behavior of poly(NIPA-co-VPBA) particles (sorbent: IB3, -NAD concentration: 0.5 mg/ml, +4 ◦ C).
values lower than neutral pH. As described in the literature, the trigonal form of boronic acid is not reactive against the diol groups of the target biomolecules [15,17,46,47]. For this reason, -NAD adsorption onto the latex particles was low at the pHs lower than or equal to 7. However, cyclic borate ester formation should effectively occur by the interaction of tetrahedral boronic acid groups with the diol groups of -NAD at the pH values close to the pKa of phenylboronic acid [15,17,46,47]. Therefore, relatively higher -NAD adsorption was observed at the pHs higher than 8.5 (i.e. the pH region in which most of the boronic acid groups are in the hydrophilic tetrahedral anionic form). By considering the behavior in Fig. 5, pH 8.5 was selected as an appropriate value for the other adsorption experiments. The effect of temperature on the -NAD adsorption of poly(NIPA-co-VPBA) particles is given in Fig. 6. Here, the adsorption experiments were performed at pH 8.5 with the initial -NAD concentration of 0.5 mg/ml. The sorbent concentration was fixed at 10 mg/ml in a batch volume of 5 ml. As seen in Fig. 6, -NAD adsorption onto the poly(NIPA-co-VPBA) particles markedly decreased with increasing temperature. All particle types showed nearly the same behavior. Note that the latex particles are in the fully swollen form at +4 ◦ C. At the low temperatures, poly(NIPA-co-VPBA) particles probably had a typical tailor-made structure comprised of a swollen-core and relatively flexible, boronic acid carrying-copolymer chains linked to the surface of swollen core [46]. The boronic acid groups on the particles should be in the partly ionized form in the adsorption medium with slightly basic character (pH 8.5). For this reason, an increase in the flexibility of copolymer chains attached to the swollen core should be expected
257
Fig. 6. The effect of temperature on the -NAD adsorption behavior of poly(NIPA-co-VPBA) particles (sorbent concentration: 10 mg/ml, initial -NAD concentration:0.5 mg/ml, pH 8.5).
[46]. Hence, the complex formation between the diol groups of -NAD molecules and the boronic acid groups available on the flexible copolymer chains should occur more easily in the low-temperature range. This model involves higher -NAD adsorption onto poly(NIPA-co-VPBA) particles at the lower temperatures. By increasing temperature, the core shrinks and probably a stiff layer is formed on the surface of shrunken particles by folding of the tailored copolymer chains. In this case, the interaction of -NAD molecules with the boronic acid functionality should become more difficult. Hence, -NAD adsorption decreases with increasing temperature. By considering the behavior in Fig. 6, -NAD adsorption isotherms were derived at +4 ◦ C. On the other hand, by considering low -NAD adsorption capacity at relatively high temperatures, 37 ◦ C was selected as an appropriate value for the desorption of -NAD from the latex particles. Except for the particles with the highest VPBA content, the magnitude of the adsorbed amount of -NAD at the low-temperature range (i.e. 4–20 ◦ C) was approximately proportional to the VPBA content of the sorbent material. For the particles with the highest VPBA content, -NAD adsorption at low temperatures was lower relative to the other particles. This behavior may be attributted to the formation of a surface structure not so suitable for nucleotide adsorption. As discussed before, the particle-surface carrying flexible, tailored copolymer chains with thermosensitive character is particularly suitable for high -NAD adsorption at the low temperatures. The flexibility of tailored copolymer chains should be strongly related to their thermosensitive character. High VPBA feed concentration involves an increase in the VPBA content of these chains which in turn their thermosensivity decreases. In this case, the formation possibility of linear chains making all boronic acid groups available may decrease in the low-temperature range. The
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Fig. 7. The variation of -NAD adsorption onto the poly(NIPA-co-VPBA) particles by the initial -NAD concentration (sorbent: IB3, sorbent concentration: 10 mg/ml, pH 8.5, +4 ◦ C).
chains with low thermosensivity can be found in the folded form that makes difficult the interaction of boronic acid groups with the -NAD molecules in the solution. This case probably leads to low -NAD adsorption at the low temperatures. The variation of -NAD adsorption with the initial -NAD concentration is given in Fig. 7. Here, the poly(NIPA-co-VPBA) particles encoded as IB3 were used as the sorbent. The poly(NIPA) particles prepared in the absence of VPBA were selected as the control-material. The adsorption experiments were performed at +4 ◦ C and pH 8.5. The other conditions were the same with those of the set in which the effect of pH was tested. As seen here, the plateau value of -NAD adsorption onto the poly(NIPA-co-VPBA) particles was reasonably higher relative to that obtained with the poly(NIPA) particles. This result is probably explained by the formation of cyclic borate ester via the interaction between boronate and diol groups [15,17,46,47]. It should be noted that the plateau value of adsorbed -NAD was obtained for the initial -NAD concentrations higher than 0.5 mg/ml in both cases. 3.3. Desorption of β-NAD from thermosensitive latex particles The tendency in Fig. 6 clearly showed that -NAD adsorption onto the poly(NIPA-co-VPBA) particles was controlled by adjusting the temperature. This behavior also indicated that the temperature should be considered as an effective variable for controlling the desorption process. The desorption of -NAD from poly(NIPA-co-VPBA) particles encoded as IB3 was investigated at different temperatures. For this purpose, -NAD was adsorbed onto
Fig. 8. The variation of -NAD desorption yield with temperature. Desorption conditions: sorbent concentration: 10 mg/ml, 37 ◦ C, 0.1 M MgCl2 , 30 min.
the poly(NIPA-co-VPBA) particles with the -NAD initial concentration of 0.5 mg/ml at pH 8.5 and at +4 ◦ C. For the desorption, -NAD adsorbed particles were redispersed in another HEPES buffer solution (pH 8.5) at +4 ◦ C. Then the temperature was elevated to the desired value and the entire mixture was kept at this temperature for 30 min. The variation of desorption yield by the temperature is given in Fig. 8. As expected -NAD desorption yield significantly increased with increasing temperature at pH 8.5. The desorption yield obtained at 37 ◦ C (i.e. 95%, w/w) was satisfactory. The desorption experiments were performed in the temperature range of 4–37 ◦ C and the thermoflocculation of latex particles was only observed at 37 ◦ C. By the precipitation of thermoflocculated particles, the separation of the sorbent from the desorption medium was simultaneously achieved. So, the desorbed -NAD was recovered in the clear supernatant at 37 ◦ C. In the desorption experiments conducted at the temperatures lower than 37 ◦ C, the latex particles were separated from the desorption medium by centrifugation. On the other hand, the effect of pH on the desorption yield was also examined at 37 ◦ C. For this purpose, HEPES buffer solutions at different pH values ranging between 6 and 10 were tried. The same experimental procedure was also followed in this set. The effect of pH on the -NAD desorption yield is given in Fig. 9. As seen here, pH 8.5 was found as the most appropriate value. The desorption process is controlled by the decomposition behavior of cyclic borate ester formed at pH 8.5 in our study (i.e. the pH at which -NAD adsorption was performed). The low desorption observed at pH 6–7 indicated that the formed cyclic borate ester is relatively stable in this pH region. Stronger affinity of tetrahedral boronic acid groups to the diol groups of -NAD at the
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Fig. 9. The effect of pH on the -NAD desorption yield (sorbent concentration: 10 mg/ml, 37 ◦ C, 0.1 M MgCl2 , 30 min, pH variable).
pHs higher than 9 probably led to lower -NAD desorption yields (i.e. Fig. 5).
4. Conclusion In the studies involving the isolation of nucleotides and nucleic acids by using the boronic acid functionalized support materials, the desorption process is usually conducted in basic media containing (pH 10–10.5) a salt at extremely high concentration (approximately 1 M). The presence of salt at such a high concentration usually involves extensive dialysis of desorption solution for the purification of desorbed biomolecule. The use of boronic acid carrying sorbent in the thermosensitive form allowed the desorption of -NAD in a relatively wide pH range without occurring a significant change in the yield.
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