Thermally Reversible Hydrogels - ACS Symposium Series (ACS

Chapter 16

Thermally Reversible Hydrogels Swelling Characteristics and Activities of Copoly(N-isopropylacrylamide-acrylamide) Gels Containing Immobilized Asparaginase

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on April 13, 2016 | http://pubs.acs.org Publication Date: September 14, 1987 | doi: 10.1021/bk-1987-0350.ch016

Liang Chang Dong and Allan S. Hoffman Chemical Engineering Department, Center for Bioengineering, FL-20, University of Washington, Seattle, WA 98195 Hydrogels synthesized from polymers and copolymers of N-isopropyl acrylamide (NIPAAm) shrink or swell as the temperature is raised or lowered through their lower critical solution temperature (LCST). We show here that when an enzyme is immobilized within such gels it may be "switched" on and off reversibly as the temperature is cycled. Such catalytic hydrogels may be used to control reactions by a thermal feedback mechanism. Hydrogels may be synthesized by polymerizing or copolymerizing a variety of mainly hydrophilic monomers with minor amounts of crosslinking agents (1). If the monomers chosen are precursors of water soluble polymer or copolymer backbones which exhibit a phenomenon known as a lower critical solution temperature (LCST), (2) then the gel made from them will shrink significantly over a relatively narrow temperature range as the temperature is raised to the LCST and above (36). One can change the LCST of the gel, the rates of shrinking and swelling, and the permeation rates of substances within these hydrogels by copolymerizing the LCST monomer with more hydrophilic or more hydrophobic monomers (7-9). Since the deswelling and shrinking occur over a relatively narrow temperature range, one may release or "deliver" at these specific temperatures, substances which have been previously absorbed into the gel. The reverse process is also possible, wherein substances in the solution are absorbed into the gel when it is cooled and reswells. In such a manner, thermally reversible gels have been applied for delivery (4) or separation (4,6) of substances to andfromthe surrounding aqueous medium. Drug delivery from uncrosslinked thermally reversible copolymers has also been studied, where the copolymer contains hydrophobic segments which act as "crosslinkers" and prevent it from dissolving in aqueous solutions (10). Specific binding pair ligands, such as antibodies, antigens and haptens may be immobilized on the polymer backbones used to prepare the gels (4,11-14). Then one can selectively bind and remove from the surrounding medium, substances which are specifically bound to the immobilized ligand. An immunodiagnostic assay has been developed on this principle (12,14). One can subsequently deliver these same substances to a surrounding medium when it contains an eluting solute (4,13). In this paper we have immobilized an enzyme within a thermally reversible hydrogel. Immobilized enzymes have been used in a variety of applications, ranging from treatment of diseases to sensors, assays, and industrial processes (15-20). When an enzyme is immobilized within a gel which exhibits reversible shrinking and swelling as the temperature is raised and lowered through the LCST of the gel matrix polymer, the enzyme may be switched off and on as the substrate diffusion rate is regulated by the gel pore size (5). In addition to enzymes, a variety 0097-6156/87/0350-0236$06.00/0 © 1987 American Chemical Society

Russo; Reversible Polymeric Gels and Related Systems ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

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of catalysts, cocatalysts or reactants may be immobilized within LCST hydrogels. We propose that such catalytic, thermally reversible gels may be used to control reactions rates and temperatures by a thermal feedback mechanism. We report here on copolymer hydrogels of N-isopropyl acrylamide (NIPAAm) and acrylamide (AAm) which are crosslinked with methylene-bis-acrylamide (MBAAm) and which contain the enzyme asparaginase immobilized within the gel. Materials and Methods


Materials N-isopropyl acrylamide (NIPAAm) was obtainedfromEastman Kodak. Acrylamide (AAm, electrophoresis grade), methylene-bis-acrylamide (MBAAm, electrophoresis grade), tetramethylethylenediamine (TEMED), and ammonium persulfate were obtained from Aldrich. L-asparaginase amidohydrolase (asparaginase) derived from Escherichia Coli was obtained as ELSPAR from Merck & Co., Inc. Asparagine, Trizma base (tris(hydroxymethyl)aminomethane) and Sigma Ammonia Color Reagent (Nessler reagent) were obtained from Sigma, and Triton X-100 was procuredfromPackard Instrument Company. All chemicals were used as received, and solvents used were reagent grade. PD-10 Sephadex columns were purchasedfromPharmacia. Methods The methods of gel synthesis, immobilization of monomer conjugated enzyme, assay of enzyme activity, and determination of gel water content have been published elsewhere (4,5). A schematic of the synthesis is shown in Fig. 1. The gel compositions are identified as "NA-100" (100% NIPAAm), "NA-95" (95% NIPAAm, 5% AAm), "NA-90" (90% NIPAAm, 10% AAm) and "NA85" (85% NIPAAm, 15% AAm); all are based on mole percents of monomers. Total monomer concentration was always 1.75 M. The experiment to determine the temperature dependence of enzyme activity was carried out after the enzyme reversibility experiment. Results and Discussion Poly(NIPAAm) has been previously shown to have an LCST ca. 31-33° (21) while copolymers of NIPAAm and AAm have LCST*s which rise as the AAm content increases (5, 7-9). At a sufficiently high content of AAm, the LCST phenomenon is no longer observed. Figure 2 illustrates the temperature dependence of relative gel water contents for copolymer gels of NIPAAm and AAm. The sharpest drop in water content with temperature is seen for the 100% NIPAAm gel. As the AAm content increases, the drop becomesflatter,and occurs at higher temperatures. Figures 3 and 4 show the kinetics of water deswelling of the NA-100 and NA-95 gels. It can be seen that as little as 5% AAm has a significant effect on both the rate and extent of deswelling at temperatures between ca. 34° and 40°. This is the region where collapse of the poly(NIPAAm) gel would be occurring due to the 31-33° LCST of poly(NIPAAm). The LCST of a copolymer of 95% NIPAAm/5% AAm has an LCST around 34-38°, as estimated from Fig. 2. Relatively rapid collapse of this gel would be expected only above 40°, as seen in Fig. 4. The specific enzyme activities of the gels are shown in Fig. 5 as a function of increasing temperature. It can be seen that the enzyme activities parallel the water contents of the gels seen in Fig. 2. All enzyme gel activities rise with temperature, as expected, until the gel LCST region is reached. These data indicate that the activity of an immobilized catalyst (enzyme) may be "shut off by raising the temperature above a critical temperature. This is happening because shrinkage of the gel with loss of pore water will both retard or eliminate diffusion of reactants (substrates) into the gel and products out of the gel, as well as change the microenvironment of the enzyme. A rise in temperature in some reaction systems (including the body) could be undesirable and the ability of such a solid phase enzyme gel to shut off a reaction over a narrow temperature range may be useful for control of reaction rates and temperatures by this feedback mechanism.


REVERSIBLE POLYMERIC GELS AND RELATED SYSTEMS

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CL *y — ι


CH«

... CH 3

©ν >V— ι

a

CH,= C - C —OH • HO>

MAAC

CH,= C - C - O - N

* NH,-{

NSMA

CH =C—C—NH-4 2

1

Λ

Ο NHS

NSMA

Ε )

CHt=C-C-NH-(

Enzyme

Ε )*

Monomerized Enzyme

/·

Ε )

Monomerized Enzyme

jC-N—CH NIPAAm

AAm

Ο CH2=CH-C-NH >CH

2

2

II ο MBAAffl Immobilized enzyme

Figure 1. Schematic of synthesis of enzyme immobilized within hydrogel exhibiting thermally reversible shrinking and swelling.


DONG AND HOFFMAN



16.

Time ( nr. ) Figure 3. Water deswelling kinetics of NA-100 gel.


239



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Temperature ( ° C ) Figure 5. Temperature dependence of immobilized enzyme a c t i v i t y i n LCST hydrogels. (Reproduced with permission from Ref. 5. Copyright 1986 Elsevier Science.)



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For a catalytic gel to be most useful for such reaction control, it must act reversibly. We have also studied the specific enzyme activities of the gels by cycling them between 30°C and 40°C. Figure 6 is a schematic of the protocol used. The results are shown in Figs. 7-10. It is evident that the enzyme activity is reversible in all of these gels. In addition, there is an espe cially dramatic drop in gel enzyme activity in goingfrom30° to 40° for the NA-100 and NA-95 gels in all three cycles. This is in sharp contrast to the NA-90 and NA-85 gels which show a rise in activity for the same temperature changes, again in all three cycles. This is expected, because the NA-90 and NA-85 gels are still below their LCST's at 40°. The data in Fig. 8 suggest that the level of enzyme activity at 40° for all of the Ν A gels may be in proportion to the "free pore water". The data also suggest that as much as 30-35% water is "bound" by the polymer chains within the gel matrix and not available as "pore" water. Thus the enzyme may be difficult to reach or even unavailable to substrate in the NA-100 and NA-95 gels at 40°. In addition, diffusion of the aspartate product molecules out of the gel would also be retarded. It can also be noted that all of the gelsrisein enzyme activity at 30° as the number of 30°40° cycles increases. (Compare Fig. 7 to Fig. 9 to Fig. 10) This may be due to scission of some crosslinks as the gels swell and shrink during the temperature cycling and/or to relative move ments of the enzyme and polymer segments within the gel which provide more rapid access of substrate (asparagine) to the enzyme as well as more rapid diffusion of product (aspartate) away from the enzyme, with increasing number of cycles.

30° C Buffer 15 mins.

Stored at 4° C in Buffer

30° C Substrate in Buffer 10 mins

8 - O r S

s s s ζ: s s

s

R.T. Buffer ca. 30 mins.

40° C Buffer 30 mins.

40° C Substrate in Buffer 10 mins.

R.T. Buffer ca. 30 mins.

Repeat cycle from steps 3 to 8 two more times Figure 6. Schematic of temperature cycle protocol used to measure r e v e r s i b i l i t y of immobilized enzyme a c t i v i t y i n LCST hydrogels. (Reproduced with permission from Ref. 5. Copyright 1986 Elsevier Science.)



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% AAm ( NiPAAm+ AAm= 100) Figure 7. Dependence of immobilized enzyme activity on composition of LCST hydrogels at 30° and 40°:firsttemperature cycle.

Water Content at 40° C ( %) Figure 8. The change of enzyme activity at 40°C in the NA series of gels (first cycle) as a function of the gel water contents at 40°C.



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% AAm( NiPAAm+ AAm= 100) Figure 9. Dependence of immobilized enzyme activity on composition of LCST hydrogels at 30° and 40°: second temperature cycle.

% AAm ( NiPAAm-f AAm= 100) Figure 10. Dependence of immobilized enzyme activity on composition of LCST hydrogels at 30° and 40°: third temperature cycle.


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In summary, we have shown for thefirsttime that catalysts such as enzymes may be immo bilized within LCST hydrogels. These gels may be warmed above their LCST's, significantly reducing the enzyme activity of the gel. We have also shown that this phenomenon is reversible. We propose that such gels may be used to control reaction rates and temperatures by a feedback mechanism


Acknowledgments We would like to acknowledge the support of Genetic Systems Corporation (GSC). In addition, one of the authors (ASH) is grateful for many stimulating discussions he has had with GSC per sonnel, especially Nobuo Monji, Carole Ann Cole, John Priest, John Plastino and Karen AuditoreHargreaves. We also want to thank Ali Afrassiabi, Sara Shoemaker and Cheryl Kruesel for their help on the manuscript, and Wu-Xiao-Ying for her help in the laboratory. Literature Cited 1.

Ratner, B. D.; Hoffman, A. S.; in Hydrogels; Andrade, J. D., Ed.; ACS Symposium Series, 1976; No. 31, p 1.

2.

Franks, F.; Chemistry and Technology of Water Soluble Polymers, Finch, C. Α., Ed.; Ple num Press: New York, 1983; Chapter 9.

3.

Tanaka, T.; Scient. Amer., 1981, 224, 124-138.

4.

Hoffman, A. S.; Afrassiabi, Α.; Dong, L. C.; J. Contr. Release, 1986, 4, 213-222.

5.

Dong, L. C.; Hoffman, A. S.; J. Contr. Release, 1986, 4, 223-227.

6.

Freitas, R. F. S.; Cussler, E. L.; Chem. Eng. Sci., (in press).

7.

Chiklis, C. K.; Grasshoff, J. M.; J. Polym. Sci., 1970, A2, 1617-1626.

8.

Taylor, L. D.; Cerankowski, L. D.; J. Polym. Sci (Polymer Chem.), 1975, 13, 2551-2570.

9.

Priest, J. H.; Murray, S. L.; Nelson, R. J.; Hoffman, A. S.; in Reversible Gelation of Poly mers, (Published in this Symposium volume.)

10.

Okano, T.; Bae, Y. H.; Kim, S. W.; Presentation at Amer. Chem. Soc. Mtg.: New York, NY, April 14-18, 1986.

11.

Cole, C. Α.; Schreiner, S. M.; Priest, J. H.; Monji, N.; Hoffman, A. S.; in Reversible Gela tion of Polymers, (Published in this Symposium volume.)

12.

Monji, N.; Hoffman, A.S.; Appl. Biochem. Biotech., 1987 (to appear in April, 1987 issue.)

13.

Hoffman, A.S.; J. Contr. Rel., 1987 (in press).

14.

Auditore-Hargreaves, K.; Houghton, R. L.; Monji, N.; Priest, J. H.; Hoffman, A. S.; Nowinski, R.C.;Clin. Chem., 1987 (in press).

15.

Guilbault, G. G.; Enzymatic Methods of Analysis, Pergamon Press: New York, 1970.

16.

Hoffman, A. S.; Schmer, G.; Kraft, W.; Harris, W., Trans. Amer. Soc. Artif. Internal Organs, 1972, 18, 10.

17.

Zaborsky, O.R.; Immobilized Enzymes, CRS Press: Cleveland, OH, 1973.

18.

Messing, R. Α., Ed.; Immobilized Enzymes for Industrial Reactors, Academic Press: New York, 1975.

19.

Maugh, T. H.; Science, 1984, 223, 474-486.

20.

Dong, L. C.; Hoffman, A. S.; Radiation Phys. and Chem., 1986, 28, 177-182.

21.

Heskins, M.; Guillet, J.E.; J. Macromol. Sci. - Chem., 1968, A2, 8, 1441-1455.

RECEIVED June 15, 1987


Thermally Reversible Hydrogels - ACS Symposium Series (ACS

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