Bioconlugate Chem. 1003, 4, 341-346
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Temperature-ResponsiveBioconjugates. 2. Molecular Design for Temperature-Modulated Bioseparationst Yoshiyuki G. Takei, Takashi Aoki, Kohei Sanui, Naoya Ogata, Teruo Okano,,',* and Yasuhisa Sakurait Department of Chemistry, Faculty of Science and Technology, Sophia University, 7-1 Kioi-cho, Chiyoda, Tokyo 102, Japan, and Institute of Biomedical Engineering, Tokyo Women's Medical College, 8-1 Kawada-cho, Shinjuku, Tokyo 162, Japan. Received March 15, 1993"
We have synthesized carboxyl semitelechelic oligo(Wisopropylacry1amide)(OIPAAm) using radical telomerization with 3-mercaptopropionic acid. This telomerization is also effective for the synthesis of carboxyl semitelechelic co-oligomers of IPAAm with butyl methacrylate (BMA) as hydrophobic or N,N-dimethylacrylamide (DMAAm) as hydrophilic comonomers. All co-oligomers are highly watersoluble a t lower temperatures and exhibit phase separation with increasing temperature. Pure OIPAAm exhibits a lower critical solution temperature (LCST) a t 32 OC, and the LCST for co-oligomers can be controlled to increase over 32 OC with increasing DMAAm composition and to decrease below 32 OC with increasing BMA composition. OIPAAm was grafted to bovine serum albumin (BSA) and bovine plasma fibrinogen (BPF) by activated ester-amine coupling. These OIPAAm-biomolecule conjugates maintain their temperature responses, are soluble in cold water, and precipitate over a range of temperatures related to oligomer content. Conjugates could be selectivelyprecipitated and independently separated from conjugate solution mixtures with increasing temperature. In this case, the number of OIPAAm molecules attached to a conjugate affects the aggregate sizes of precipitated conjugates in mixtures. Both conjugate mixture ratios and solution concentrations influence the contamination of oligo(1PAAm-co-DMAAm)-BSAconjugates in precipitated oligo(1PAAm-co-BMA)-BPF conjugates. Furthermore, precipitated conjugates separated using centrifugation and filtration redissolve in water and maintain their biofunctionality, indicating the potential of strategy in reversible bioreactors and protein separations.
INTRODUCTION
Poly(N-isopropylacrylamide) (PIPAAm)' is a watersoluble polymer exhibiting remarkable hydration-dehydration changes in aqueous media in response to changes in temperature, resulting in a lower critical solution temperature (LCST) (1). These networks have been investigated for molecular separation (2), sorption-desorption of solutes (3), control of enzyme activity ( 4 ) ,and release of solutes (5). Temperature-responsive PIPAAm hydrogels are capable of controlling drug release rates by temperature change, demonstrating a potential to achieve an intelligent drug-delivery system (6). Random copolymer hydrogels composed of IPAAm and alkyl methacrylates as comonomers are used to control the LCST and subsequent 'on-off" regulation of drug permeation (7-9). PIPAAm-grafted polystyrene is used as cell culture substrates and controls cell attachment-detachment using the hydration-dehydration phenomena of these polymer chains (10). Chen et al. have reported polymer-protein conjugates using PIPAAm (11,121. IPAAm copolymers with comone + For Part 1, see ref. 13.
* Author to whom correspondence should be addressed. Women's Medical College. * Abstract published in Advance ACS Abstracts, September 1 Tokyo
1, 1993. 1 Abbreviations used: IPAAm, N-isopropylacrylamide; PIPAAm, poly-IPAAm; OIPAAm, oligo-IPAAm; BMA, butyl methacrylak, DMAAm, Nfl-dimethylacrylamide; MPA, 3-mercaptopropionic acid; AIBN, NJV-azobisisobutylonitrile;DMF, Nfl-dimethylformamide; IBc, oligo(1PAAm-co-BMA) with carboxyl end group; IDc, oligo(1PAAm-co-DMAAm) with carboxyl end group; BSA, bovine serum albumin; BPF, bovine plasma fibrinogen; F-B3/30, BPF-IBc conjugate; A-D5/5, BSA-IDc conjugate. 1043-1802f93/2904-0341~04.00/0
mers containing reactive groups were synthesized and conjugated with proteins. It was unclear if protein is coupled with polymer chains by single or multiple attachments, which might significantly influence the activity and properties of the conjugated protein. Moreover, conjugation of copolymer with proteins may affect the bioactivity of proteins through the steric hindrance of polymers as well as promoting formation of protein aggregates. We previously reported the synthesis of oligoIPAAm (OIPAAm) with reactive end groups by the telomerization of IPAAm using 3-mercaptopropyonic acid as telogen (13). We also reported control of chain length, introduction of end-standing carboxyl groups, and temperature-responsive phase-transition phenomena of these oligomers. Oligomerswith reactive end groups allow facile synthesis not only of block and graft copolymers but also of oligomer-modified biomolecules. Semitelechelic oligomers would be expected not only to maintain high protein bioactivity by reduced steric hindrance arising from conjugated oligomers but also to import high temperature sensitivity due to the inherently high mobile nature of oligomer free end groups. These system avoids nonsepcific problems associated with the process of biomolecule conjugation because they posses one reactive end group per oligomer. OIPAAm-grafted collagen conjugates, a model for OIPAAm-biomolecule conjugates, dissolved in water regardless of isotonic conditions and demonstrated an LCST near 34 "C (13). As the phase transition of PIPAAm corresponds to the stability of hydrophobic groups in polymer chain in aqueous media, OIPAAms copolymerized with another hydrophobic or hydrophilic comonomers show different LCSTs compared to homogeneous PIPAAm. Moreover, bioconjugates coupled with OIPAAms of different LCSTs should maintain these different LCSTs attributable to 0 1993 American Chemical Society
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Table I. Preparation of IPAAm Oligomers. oligo(1PAAm-co-BMA) IPAAm BMA code (Wmol) (lo-" mol)
IBc-1 IBc-3 IBc-5
4.29 4.18 4.07
6.79 13.25 21.81
MPA (IO4 mol)
code
8.36 8.31 8.42
IDc-5 IDc-7 IDc-10 IDc-15
oligo(IPAAm-co-DMAAm) IPAAm DMAAm (10-2 mol) (lo-"mol) 4.10 4.00 3.85 3.62
2.24 3.07 4.35 6.43
MPA
(lo-"mol) 8.75 8.33 8.57 8.37
IPAAm, N-isopropylacrylamide; DMAAm, NJV-dimethylacrylamide; MPA, 3-mercaptopropionic acid, solvent, NJV-dimethylformamide (50 mL); initiator, NJV'-azobisisobutyronitrile(2 mmol/L); synthesized at 70 1 O C for 300 min; [ S I / [ M I b ~= 0.022; [SI, concentration of chain transfer agent; [ M l b ~total , monomer concentration.
*
the respective conjugated OIPAAms (11). These conjugates are expected to be bioactive in aqueous solution with an intrinsic ability to be conveniently separated from reactive products with small temperature increases. In bioseparation processes and clinical diagnosis, these compounds should prove valuable for assay and subsequent separation and recovery. This strategy introduces temperature-responsive activities into biomolecules, a first step toward the development of "intelligent bioconjugates". We now report the synthesis of semitelechelic OIPAAms with reactive carboxyl end groups which show different LCST values on the basis of radical telomerization. Moreover, we discuss temperature-modulated phaseseparation systems using OIPAAm-biomolecule conjugates. EXPERIMENTAL PROCEDURES
Materials. N-Isopropylacrylamide (IPAAm) and NJVdimethylacrylamide (DMAAm) were provided by Kojin (Tokyo, Japan). IPAAm was purified by recrystallization from toluenelpetroleum ether and dried at room temperature in vacuo (1). DMAAm was dried at room temperature in vacuo. Butyl methacrylate (BMA) was obtained from Wako Pure Chemicals (Tokyo, Japan). BMA was distilled under reduced pressure and the fraction of bp 61 "(214 mmHg was used. 3-Mercaptopropionic acid (MPA) was purchased from Aldrich Chemical Co. (Milwaukee, WI) and was distilled under reduced pressure and the fraction of bp 95 "C/5 mmHg was used. NJVazobisisobutyronitrile (AIBN),N,N'-dimethylformamide (DMF), and ethyl acetate were obtained from Wako Pure Chemicals and purified by conventional methods. U1trapure water used for sample solutions was provided by a commercial water-purification device (Toray, Model LV10T). Other chemicals used in the synthesis of oligo(IPAAm-o-BMA) and oliio(IPAAm-co-DMAAm) are the same as those described in previous work (13). Preparation of Biomolecules. Bovine serum albumin (BSA,A3350) and bovine plasma fibrinogen (BPF, F8630) were obtained from Sigma Chemical Co. (St.Louis, MO), and were dialyzed against 0.15 M phosphate buffer solution (PBS, pH 7.3) and then lyophilized. Albumin concentration was determined by formation of a color complex (A,,= = 630 nm) with bromocresol green (Merck) (14). Fibrinogen concentration was determined by clotting with thrombin (Sigma, T4648) digestion of the clot and biuret analysis of the resulting solution (15). Co-OligomerizationProcedure. Semitelechelic IPAAm co-oligomers (IBc-X; X denotes the mole fraction of comonomer in feed) were prepared by radical oligomerization of IPAAm and BMA with MPA using AIBN as an initiator in DMF. The reaction mixture was subjected to freezethaw cycles, in order to remove oxygen. The ampules were degassed and sealed under reduced pressure. The reaction was performed in sealed ampules at 70 f 1 "C for 5 h (16). The preparation of co-oligomers is
summarized in Table I. OIPAAms of molecular weight near 6000 g/mol were prepared in this study. After evaporation, the products were dissolved in DMF and poured into diethyl ether to precipitate the oligomers. Oligomerswere further purified by repeated precipitation from DMF into diethyl ether. Radical oligomerization of IPAAm with DMAAm (IDc-X) was carried out by the same method. Characterization of Co-Oligomers. Proton NMR spectra were recorded for CDC13 solutions of the oligomers at ambient temperature on a spectrometer operating at 270-MHz frequency (JEOL, NJ-270E, Tokyo, Japan). Oligomer molecular weights were determined by gelpermeation chromatography (GPC, Shimadzu; C-R4AX, Shodex; KF-80M X 2) in tetrahydrofran at 40 "C (polystyrene standard; Polyscience Inc., Warrington, PA). The molecular weight of oligomerswas also determined by endgroup titration using 0.01 M NaOH and phenolphthalein at 4 "C, which detects the carboxyl group at the end of oligomer molecules. Transmittance Measurements. Optical transmittance of oligomer solutions (10 mg/mL) at various temperatures was carried out by monitoring transmittance at 500 nm using a spectrophotometer (Shimadzu, UV-240). The observation cell was thermostatted by a circular water jacket (Iuchi, HT-OlC). Modification of Biomolecules with Co-Oligomers. Oligomers were activated by N-hydroxysuccinimide with dicyclohexylcarbodiimidein dry ethyl acetate at 4 "C for 16 h in a molecular ratio of 1:1.2:1.2, respectively. After evaporation, the residue was dissolved in ethyl acetate and poured into diethyl ether to precipitate activated OIPAAm. Activated ester groups were confirmed by infrared and ultraviolet spectroscopy (17). Bioconjugates of OIPAAm with protein were synthesized by following the protocol of Btickman et al. with a weight feed ratio of OIPAAm to biomolecule of 10 and 50 (18).The reaction solution was adjusted to and maintained at pH 8.5 during the course of the reaction and all steps were carried out at 4 "C. Activated OIPAAm was dissolved in 4 mL of dry DMF and added to 100 mg of biomolecule in 50 mL of PBS. This process was repeated four more times a t 30min intervals. The total reaction time was 8 h with gentle stirring at 4 "C. The solution was dialyzed (cellulose, 10 000 molecular weight cutoff) against PBS, pH 7.4, and lyophilized. The resulting biomolecule-OIPAAm conjugate molecular weights were measured by high-performance size-exclusionchromatography (HPSEC; HLC-802, equipped with TSK-G4000SW, Toso, Tokyo, Japan). A calibration curve for globular proteins was determined by the retention of standard proteins, obtained from Boehringer Mannheim Biochemica, including @-galactosidase (Mr = 465 000), IgG (Mr = 150 OW),IgG Fab fragment (Mr = 50 OOO), myoglobin (Mr = 17 000) and Gly-Tyr (Mr = 238). Transmittance changes of purified conjugates in
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Table 11. Analysis of Oligo(IPAAm-co-BMA)a
code IBc- 1 IBc-3 IBc-5
molecular weight from analysis of end groupd 6200 6300 5900
mole fraction of BMA (mol % ) in feed observedb 1 0.92 2.81 3 4.80 5
a Oligo(1PAAm-co-BMA), in DMF (1.0 mol/L) at 70 dAverage (n = 3).
-
-
Mn'
Mw' 6300 6500 5900
5800
6050 5800
number of COOH in one moleculesd 0.97 1.05 1.07
* 1OC, AIBN, 2 mmol/L; reaction time, 300 min. * 'H NMR.
LCST (OC) 31.0 28.0 26.0
GPC in THF at 40 OC.
Table 111. Analysis of Oligo(IPAAm-co-DMAAm)a
code IDc-5 IDc-7 IDc-10 IDc-15
mole fraction of DMAAm (mol % ) in feed observedb 5 7 10 15
6.44 8.12
11.93 17.21
molecular weight from analysis of end groupd 5900 6200 6300 5900
number
-
Mw'
one moleculesd
LCST ("C)
5600 5700 6600 5500
6200 5900 7100 6100
1.02 0.97 1.09 1.02
35.0 36.0 38.0
Mn'
of COOH in
42.0
Olieo(1PAAm-co-DMAAm), in DMF (1.0 mol/L) at 70 f 1 OC;AIBN, 2 mmol/L; reaction time, 300 min. 1H NMR. e GPC in THF at 40 OC. ;Average (n = 3).
aqueous solution (10 mg/mL) were also recorded as a function of temperature as described above. Separation by Temperature-Modulated Precipitation. Optical transmittance for the mixtures of bioconjugate (10 mg/mL total) was measured by spectrophotometry a t various temperatures. Contamination by BSA conjugates in aggregated BPF conjugates was calculated from the data of BSA solution concentration after BPF precipitation as a function of BSA conjugate ratios. Furthermore, effects of solution concentration on contamination by BSA conjugate in aggregated BPF conjugates were calculated.
RESULTS AND DISCUSSION Synthesis of IPAAm Co-Oligomers. Carboxyl semitelechelic co-oligomers of IPAAm with BMA or DMAAm were synthesized by telomerization using MPA as telogen at 70 "C in DMF. Compositions of oligo(IPAAm-co-BMA) and oligo(1PAAm-co-DMAAm) were determined, from an analysis of the 'H NMR spectra in CDCl3 solutions. The mole fraction of BMA in oligo(1PAAm-co-BMA) was calculated from the area of the singlet at 0.9 ppm due to the terminal methyl protons of the alkyl chains and the area of the singlet at 4.01 ppm, attributed to the resonance of the C-2 proton of the isopropyl groups. The mole fraction of DMAAm in oligo(1PAAm-co-DMAAm) was also calculated from the area of the triplet centered at 2.79 ppm attributed to the methyl protons of the N-substituted methyl groups and the area of the singlet at 4.01 ppm, attributed to the resonance of the C-2 proton of the isopropyl groups. Results obtained in the co-oligomerization of IPAAm with DMAAm or BMA are summarized in Table I1and 111,respectively. NMR confirms copolymer formation as shown by these data. Yields of co-oligomers were 20-30 ?6 for 300-min reaction time. Considering the small amounts of comonomer fed in these reactions, the copolymerizability of DMAAm with IPAAm is better than that of BMA with IPAAm. Monomer reactivity ratios evaluated according to the Fineman-Ross method (19) were r l = 0.341 and F Z = 1.937 (MI = BMA, MZ= IPAAm) and ~1 = 0.977 and ~2 = 0.820 (Mi = DMAAm, Mz = IPAAm), respectively. A value of r1rz reported previously for copolymerization of N-alkyl-substituted acrylamides (20)is consistent with formation of random copolymers. Our evidence supports this. As mentioned in the introduction, control of OIPAAm molecular weight is possible by adjusting the relative
20
24
28
32
36
40
44
Temperature ("C) Figure 1. Temperature dependence of optical transmittance for aqueous solutions (0,IBc-5, A;IBc-3, 0;IBc-1, 0 ;IDc-5, A; IDc-7, . ;IDc-10, *; IDc-15, 10 mg/mL solution, X = 500 nm).
concentration of monomers to MPA in the starting mixture for the telomerization. OIPAAm molecular weights near 6000 are readily achieved by this strategy, and copolymerization of IPAAm with BMA or DMAAm using MPA via telomerization is confirmed. Molecular weights calculated from the carboxy group analysis assuming one carboxyl group per OIPAAm molecular were approximately equivalent to those determined from GPC data. All data were consistent with one OIPAAm molecule averaging one carboxyl end group per polymer chain. Phase Transitions of IPAAm Co-Oligomers. All oligomers were soluble in water at room temperature. The series of OIPAAm samples in aqueous solution was examined for optical transmittance changes at various temperatures as shown in Figure 1. All OIPAAms obtained by the copolymerization demonstrated their LCST in the range 26-42 "C. Taylor and Cerankowski proposed as a general rule that the LCST should decrease with increasing polymer hydrophobicity (21).The LCST change of IBc and IDc aqueous solutions is shown in Figure 2. IBc oligomers exhibited LCST values below 32 "C and transmittance of IBc oligomer solutions collapsed sharply corresponding to dehydration. Upon increasing the mole fraction of BMA as hydrophobic comonomer, the LCSTs shift to lower temperatures. On the other hand, as hydrophilic components in polymer chains demand larger dehydration energy, IDc oligomers exhibit LCST values
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24 0
4
8
12
16
20
Mol% of comonomer (%)
Figure 2. LCST changes of co-oligomer aqueous solutions as a function of mole fraction of comonomers in co-oligomers (0;IBc, A; IDc, 10 mg/mL, X = 500 nm).
above 32 OC and transmittance changes of IDc oligomer solutions are broader. Increasing the mole fraction of DMAAm as hydrophilic comonomer, shifts LCSTs to higher temperatures. Phase-transition behavior of OIPAAm depends on OIPAAm molecular weight and solution concentration. For low molecular weight OIPAAm precipitated from low-concentration solution, microaggregates of precipitated OIPAAm disperse in aqueous media and are unable to be recovered by centrifugation (6,22). We have recovered higher molecular weight OIPAAms (Mw = 6100) from precipitated OIPAAm solutions (10 mg/mL). From these results, we show the regulation of LCST values in OIPAAms with carboxyl end groups by telomer mediated copolymerization with hydrophobic or hydrophilic comonomers. Temperature-ResponsiveBioconjugates. Table IV describes the preparation and analysis of these bioconjugates. OIPAAm-biomolecule conjugates were prepared by coupling activated esters of OIPAAm with free amines in biomolecules. Two biomolecules were modified by different temperature-responsive oligomers. BPF was conjugated with IBc oligomer exhibiting an LCST below 32 OC. BSA was conjugated with IDc oligomer exhibiting a higher LCST above 32 "C. OIPAAm-biomolecule conjugates are described by a three sequence code, such as F-B3/7, where F refers to BPF, B3 to IBc-3 oligomer, and 7 to the number of IBc-3 molecules grafted per conjugate. The number of grafted OIPAAm molecules was estimated by the molecular weight of conjugates from HPSEC data. The obtained conjugates are soluble in water at room temperature regardless of isotonic conditions. Also, these conjugates become soluble in organic solvents such as ethanol and DMF. Transmittance changes in aqueous solutions of OIPAAm-biomolecule conjugates are shown in Figure 3. Unconjugated biomolecule solutions were transparent at temperatures up to and over 50 OC. OIPAAm-biomolecule conjugates, however, precipitated with increasing temperature. Grafting of semitelechelic OIPAAms to biomolecules in this experiment yields the advantages of high polymer chain mobility and rapid response to small temperature changes compared to multiple-point conjugation. Precipitates grafted by increasing temperature were identified as either BPF-IBc conjugates or BSA-IDc conjugates by their molecular weight from HPSEC data. The A-D5/29 bioconjugate exhibited an LCST near 37 "C, the obvious physiologically relevant temperature. Although IBc-3 oligomers showed an LCST at 28 OC in aqueous systems, the F-B3/7 conjugates exhibited a
temperature response at 29.4 "C. The LCST for the conjugate system is shifted to a higher temperature range than the LCST of original temperature-responsive polymers. The conjugate constructed by water-soluble biomolecules and temperature-responsive polymers would disturb the OIPAAm dehydration and increase the energy needed for precipitation. The LCST of the F-B3/30 conjugate at 28.8 OC is slightly lower than that of F-B3/7, at 29.4 "C. Also, the LCST of A-D5/29 at 37.2 OC is obviously higher than that for A-D5/5, a t 36.2 OC. Increasing the hydrophobic component in IPAAm copolymer chains facilitates polymer chain dehydration. As F-B3/30 conjugates contain more grafted IBc-3 molecules than F-B3/7 conjugates, less thermal energy is required for the precipitation of the F-B3/30 conjugates, providing a lower LCST than that for F-B3/7. By contrast, as A-D5/ 29 conjugates contain more grafted IDc-5 molecules than the A-D5/5 conjugate, A-D5/29 conjugates require a significantly higher hydrophobic driving force. Therefore, the LCST of A-D5/29 is shifted to a higher temperature range than A-D5/5, reflecting the energy change required for sufficient dehydration of the former conjugate. We have also estimated the biofunction of conjugated biomolecules by the dye binding for albumin and the fibrin clot formation for finrinogen as described in the Experimental Procedures. It was apparant that all the bioconjugates exhibited the similar biofunctionality as the original proteins, with the exception of A-D5/29, in the assay. Although A-D5/29 conjugates indicate phasetransition phenomena, A-D5/29 conjugates may be deprived of the properties of BSA through conjugation.A-D5/ 29 contained an average of 29 molecules of IDc-5 oligomer per protein, and the amount of OIPAAm was 72.5 w t 5% of conjugate as shown in Table IV. The F-B3/30 conjugate included 30 molecules of IBc oligomer per protein molecule. By contrast with A-D5/29,the amount of OIPAAm in F-B3/ 30 was, however, only 27.5 wt % of the conjugate as shown in Table IV. We propose that expanded and hydrated polymer chains with highly mobile free end groups contribute not only to effective steric hindrance of the biomolecule active site but also import large structural hydration-dehydration changes with small temperature changes. Temperature-ModulatedBioseparations. Selective precipitation of bioconjugates from mixtures was investigated to assess the ability to separate active proteins. F-B3/30 conjugates (50 mg) and A-D5/5 conjugates (50 mg) were dissolved in water a t 4 OC. The mixture was heated from 27 OC, and transmittance changes in the mixture were measured a t 0.2 O C intervals. Transmittance changes of mixtures are shown in Figure 4. Precipitation was fiist observed at 30.4 OC. The mixture was then fitered at 32 OC. When the supernatant was subsequently heated from 32 "C to higher temperatures, another precipitation was observed at 36.4 OC. The first precipitate was redissolved in PBS and confirmed to consist of BPF, as the F-B3/30 conjugate. The second precipitate was redissolved in PBS and determined to be BSA, as the A-D5/5 conjugate. We conclude that different bioconjugates having different LCST values precipitate in the sequential order of LCST temperatures from mixtures with temperature increases. Purity of the precipitated F-B3/30 a t lower temperatures was also investigated, particularly with regard to contamination byA-D5/5 conjugates in the precipitate (ehown in Figure 5). When A-D5/5 conjugate ratios become higher than 0.5, A-D5/5 content is generally very low in the F-B3/ 30 precipitate. At a conjugate ratio of 0.2, contamination
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Table IV. Preparation and Analysis of Biomolecule-OIPAAm Conjugates
o*pAAm (Mw")
biomolecule
(ax 103)
code
activated OIPAAm in feed (mol/mol biomolecule)
molecular weight of conjugatesb (X103)
F-0 0 340 F-B3/7 BPF IBc-3 10 380 F-B3/30 (340) (6500) 50 524 A-0 0 66 A-D5/5 BSA IDc-5 10 99 A-D5/29 (66) (6200) 50 240 a GPC in THF at 40 OC. * Determined by HPSEC. (wt OIPAAm/wt conjugates).
amount of OIPAAm coupled moVmol biomolecule w t %' LCST (OC) 0
0
6.6 30.2
10.5 27.5 0 33.3 72.5
0
5.4 28.6
29.4 28.8 36.2 37.2
100 80
8
v
8 l2
'E
s
60
40
20
0 22 24 26 28 30 32 34 36 38 40 Temperature ("C)
Figure 3. Temperature dependence of optical transmittance for OIPAAm-biomolecule conjugate aqueous solutions (0; F-B3/ 30, A;F-B3/7, 0;A-D5/5, 0 ;A-D5/29, 10 mg/mL solution, h = 500 nm). 100
.
80
.
20
*
h
3 8
60-
2 E I-
40
2 E
20
15:
.
-
0 26
28
30
32
34
36
38
Temperature ( O C )
Figure 4. Temperature dependence of optical transmittance F-B3/ for OIPAAm-biomolecule conjugate solution mixtures (0; 30, A; A-D5/5, 10 mg/mL solution, bioconjugates ratio = 1:1, h = 500 nm). reached a maximum (8.7wt 5% 1. Alternatively,when F-B3/
30 conjugate ratios are higher than 0.5, F-B3/30 contamination of A-D5/5 precipitates is barely detectable. At low A-D5/5 conjugate ratios, F-B3/30 is proposed to enfold A-D5/5 molecules in mixtures, resulting in A-D.515 entrainment into the resulting F-B3/30 precipitate. As the F-B3/30 conjugate had 5 times greater molecular weight than the A-D5/5 conjugate, F-B3/30 molecules would readily distribute around A-D5/5 molecules at lower A-D5/5 conjugate ratios. Molecules of each conjugate would be phase-separated in mixtures at higher conjugate ratios. The concentration of A-D5/5 entrapped in precipitated F-B3/30 would be minimized, therefore, at A-D5/5 conjugate ratios higher than 0.5, where phase separation would inhibit interaction. The influence of solution concentration on A-D5/5 contamination into F-B3/30 precipitates is shown in Figure
6. Mixtures of equivalent conjugate ratios were used. At concentrations greater than 100 mg/mL, A-D5/5 contamination in F-B3/30 precipitates ranges from 5 to 15 wt 5%. Since mixtures had high viscosities at concentrations higher than 100mg/mL, we believe that F-B3/30 molecules interact and enfold molecules of A-D5/5 and then precipitate. Altering mixture concentration can be used to optimize the contamination of A-D5/5 conjugates into precipitated F-B3/30 conjugates. From these results, both the conjugate ratios and mixture concentrations optimize the contamination of the solute in the precipitate in temperature-modulated phase separation. Polymer mixtures usually exhibit simple coacervation. As bioconjugate water-solubility corresponds to the phase-transition behavior of the OIPAAms, the chain mobility and hydration of grafted OIPAAm mediates the temperature-modulated
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phase separation and the biofunction of conjugated biomolecules. CONCLUSIONS
(i) Temperature-responsive semitelechelic co-oligomers with carboxyl end-groups were synthesized by the radical telomerization of IPAAm with BMA or DMAAm using MPA as a telogen. (ii) Oligo(1PAAm-co-BMA) demonstrated an LCST below 32 OC corresponding to an increase in polymer chain hydrophobicity. Oligo(IPAAm-co-DMAAm)demonstrated an LCST above 32 OC, attributed to increases in the hydrophilic nature of the polymer chain. (iii) OIPAAm-biomolecule conjugates demonstrate LCST values ranging from 28 to 42 OC, depending upon the chemistry and molecular weight of the grafted OIPAAms. (iv) Large, highly temperature sensitive structural hydration-dehydration changes for bioconjugates are demonstrated due to the inherently high mobile nature of oligomer free end groups. (v) OIPAAm-biomolecule conjugates can be separated from mixtures by temperature-modulated precipitation with temperature increases. (vi) Both the conjugate mixture ratios and mixture concentration can modulate the entrapment and contamination of solute into conjugate precipitate in temperaturemodulated phase separation. ACKNOWLEDGMENT
The authors are grateful to Dr. David W. Grainger, Oregon Graduate Institute of Science and Technology, and Dr. Glen S. Kwon, International Center for Biomaterial Science, Tokyo Women’s Medical College, for their valuable comments and discussions. LITERATURE CITED (1) Heskins, M., Guillet, J. E., and James, E. (1968) Solution properties of poly(N-isopropylacrylamide). J . Macromol. Sci. Chem. A2,1441-1455. (2) Freitas, R. F. S., and Cussler, E. L. (1987) Temperature sensitive gels as extraction solvents. Chem. Eng. Sci. 42,97103. (3) Hoffman, A. S., Afrassiabi, A., and Dong, L. C. (1986) Thermally reversible hydrogels; 11. Delivery and selective removal of substances from aqueous solutions. J . Controlled Release 4 , 213-222. (4) Dong, L. C. and Hoffman, A. S. (1986) Thermally reversible hydrogels; 111. Immobilization of enzymes for feedback reaction control. J. Controlled Release 4 , 223-227. (5) Bae, Y. H., Okano, T., Hsu, R, and Kim, S. W. (1987)Thermosensitive polymer as on-off switches for drug release. Makromol. Chem., Rapid Commun. 8,481-485. (6) Bae, Y. H., Okano, T., and Kim, S. W. (1990) Temperature dependence of swelling of crosslinked poly(N,”-alkyl substituted acrylamides) in water. J . Polym. Sci. Polym. Phys. 28, 923-936.
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