Temperature-Modulated Interaction Changes with Adenosine Nucleotides on Intelligent Cationic,Thermoresponsive Surfaces1

Abstract

Thin layer poly(N-isopropylacrylamide-co-n-butyl-methacrylate-co-N,N-dimethylaminopropylacrylamide) (IBD) copolymer gels are covalently introduced to initiator immobilized silica bead surfaces to create thermally sensitive intelligent cationic surfaces. The surface shows thermoresponsive changes in charge density as well as hydrophilic/hydrophobic character. The polymer chains dehydrate and inter-/intra-molecular aggregation occurs due to weakly deprotonated cationic amino groups in the hydrophobized circumstances, resulting in the surface charge density decrease. This was corroborated by the thermoresponsive pKa shift of dimethylamino side groups in the copolymers as well as the surface potential changes at elevated temperature. The unique characteristics of the IBD copolymer-immobilized surfaces were applied to regulate adenosine nucleotides retention in high-performance liquid chromatography using aqueous mobile phase by thermal stimulus. At lower temperature, adenosine nucleotides showed higher retention which was primarily driven by ionic interaction with positively charged surfaces. With increasing temperatures, their retention was shortened and a drastic change was observed above the polymer transition temperatures. This is strong evidence that the solute interaction is being regulated by the thermoresponsive surface charge density changes and hydrophobic alterations. Furthermore, we confirmed the modulation of nucleotide retention by step-temperature gradient without changing mobile phase composition. These findings should be beneficial in utilizing this stimuli responsive surface for the separation of bioactive compounds in aqueous system and environmental impact.

Keywords

thermoresponsive polymer poly(n-isopropylacrylamide)adenosine nucleotides separation electrostatic interaction hydrophobic interaction.

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References

Heskins, M. , Guillet, J.E. and James, E. (1968). Solution Properties of poly (N-isopropylacrylamide). Journal of Macromolecular Science, Chemistry, A2(8): 1441—1445.

Okano, T. , Yamada, N. , Sakai, H. and Sakurai, Y. (1993). A Novel Recovery System for Cultured Cells using Plasma-treated Polystyrene Dishes Grafted with poly(N-isopropylacrylamide) , Journal of Biomedical Materials Research, 27(10): 1243—1251.

Okano, T. , Yamada, N. , Okuhara, M. , Sakai, H. and Sakurai, Y. (1995). Mechanism of Cell Detachment from Temperature-modulated, Hydrophilichydrophobic Polymer Surfaces, Biomaterials, 16(4): 297—303.

Kikuchi, A. , Okuhara, M. , Karikusa, F. , Sakurai, Y. and Okano, T. (1998). Two-dimensional Manipulation of Confluently Cultured Vascular Endothelial Cells using Temperature-responsive poly(N-Isopropylacrylamide)-Grafted Surfaces, Journal of Biomaterials Science, Polymer Edition , 9(12): 1331— 1348.

von Recum, H. , Kikuchi, A. , Okuhara , M. , Sakurai, Y. , Okano, T. and Kim , S.W. (1998). Retinal Pigmented Epithelium Cultures on Thermally Responsive Polymer Porous Substrates, Journal of Biomaterials Science, Polymer Edition , 9(11): 1241—1253.

Yamato, M. , Kushida, A. , Konno, C. , Kikuchi, A. , Sakurai, Y. and Okano, T. (1999). A Novel Tool of Temperature-responsive Cell Culture Surfaces and its Application to Matrix Biology, Connective Tissue, 31(1): 13—6.

Kanazawa, H. , Yamamoto, K. , Matsushima , Y. , Takai, N. , Kikuchi, A. , Sakurai, Y. and Okano, T. (1996). Temperature-responsive Chromatography using poly(N-isopropylacrylamide)-modified Silica, Analytical Chemistry, 68(1): 100—105.

Kanazawa, H. , Kashiwase, Y. , Yamamoto, K. , Matsushima, Y. , Kikuchi, A. , Sakurai, Y. and Okano, T. (1997). Temperature-Responsive Liquid Chromatography. 2. Effect of Hydrophobic Groups in N-Isopropylacrylamide Copolymer-Modified Silica, Analytical Chemistry, 69(5): 823—830.

Yakushiji, T. , Sakai, K. , Kikuchi , A. , Aoyagi, T. , Sakurai, Y. , and Okano, T. (1999). Effects of Cross-Linked Structure on Temperature-Responsive Hydrophobic Interaction of Pipaam Hydrogel Modified Surfaces with Steroids, Analytical Chemistry, 71(6): 1125—1130.

10.

Kobayashi, J. , Kikuchi, A. , Sakai , K. and Okano, T. (2003). Aqueous Chromatography Utilizing Ph/Temperature-Responsive Polymer Stationary Phases to Separate Ionic Bioactive Compounds, Analytical Chemistry , 73(9): 2027—2033.

11.

Idota, N. , Kikuchi, A. , Kobayashi, J. , Akiyama, Y. , Sakai, K. and Okano, T. (2006). Thermally Modulated Interaction of Aqueous Steroids Using Polymer-Grafted Capillaries, Langmuir, 22(1): 425—430.

12.

Bae, Y.H. , Okano, T. , Kim , S.W. (1991). ``On-off'' Thermocontrol of Solute Transport. I. Temperature Dependence of Swelling of N-Isopropylacrylamide Networks Modified with Hydrophobic Components in Water, Pharmaceutical Research, 8(4): 531—537.

13.

Bae, Y.H. ,. Okano, T. and Kim , S.W. (1991). ``On—off '' Thermocontrol of Solute Transport. II. Solute Release from Thermosensitive Hydrogels, Pharmaceutical Research, 8(5): 624—628.

14.

Yoshida, R. , Uchida, K. , Kaneko , Y. , Sakai, K. , Kikuchi, A. , Sakurai. Y. and Okano, T. (1995). Comb-type Grafted Hydrogels with Rapid Temperature Responses, Nature, 374(6519): 240—242.

15.

Takei, Y.G. , Aoki, T. , Sanui , K. , Ogata, N. , Sakurai, Y. and Okano, T. (1994). Dynamic Contact Angle Measurement of Temperature-Responsive Surface, Properties for Poly(N-Isopropylacrylamide) Grafted Surfaces. Macromolecules, 27(21): 6163—6166.

16.

Yakushiji, T. , Sakai, K. , Kikuchi , A. , Aoyagi, T. , Sakurai, Y. and Okano, T. (1998). Graft Architectural Effects on Thermo-Responsive Wettability Changes of poly(N-isopropylacrylamide)-modified surfaces, Langmuir, 14(16): 4657—4662.

17.

Yamada, N. , Okano, T. , Sakai, H. , Karikusa, F. , Sawasaki, Y. and Sakurai, Y. (1990). Thermo-responsive Polymeric Surfaces; Control of Attachment and Detachment of Cultured Cells, Makromolekulare Chemie, Rapid Communications, 11(11): 571—576.

18.

Hirose, M. , Kwon, O.H. , Yamato, M. , Kikuchi, A. and Okano, T. (2000). Creation of Designed Shape Cell Sheets that are Noninvasively Harvested and Moved onto Another Surface, Biomacromolecules , 1(3): 377—381.

19.

Yamato, M. and Okano, T. (2004). Cell Sheet Engineering, Materials Today, 7(5): 42—47.

20.

Nishida, K. , Yamato, M. , Hayashida, Y. , Watanabe, K. , Yamamoto, K. , Adachi, E. , Nagai, S. , Kikuchi, A. , Maeda, N. , Watanabe, H. , Okano, T. and Tano, Y. (2004). Corneal Reconstruction with Tissue-engineered Cell Sheets Composed of Autologous Oral Mucosal Epithelium, The New England Journal of Medicine, 351(12): 1187—1196.

21.

Gaur, R.K. and Gupta, K.C. (1989). A Spectrophotometric Method for the Estimation of Amino Groups on Polymer Supports, Analytical Biochemistry , 180(2): 253—258.

22.

Feil, H. , Bae, Y.H. , Feijen, J. and Kim, S.W. (1992). Mutual Influence of Ph and Temperature on the Swelling of Ionizable and Thermosensitive Hydrogels, Macromolecules, 25(20): 5528—5530.

23.

Urry, D.W. (1992). Free Energy Transduction in Polypeptides and Proteins Based on Inverse Temperature Transitions, Progress in Biophysics and Molecular Biolology, 57(1): 23—57.

24.

Leoncini, G. , Buzzi, E. , Maresca , M. , Mazzei, M. and Balbi, A. (1987). Alkaline Extraction and Reverse-Phase High-Performance Liquid Chromatography of Adenine and Pyridine Nucleotides in Human Platelets, Analytical Biochemistry, 165(2): 379—383.

25.

Teerlink, T. , Hennekes, M. , Bussemaker , J. and Groeneveld, J. (1993). Simultaneous Determination of Creatine Compounds and Adenine Nucleotides in Myocardial Tissue by High-Performance Liquid Chromatography, Analytical Biochemistry, 214(1): 278—283.

26.

Tekkanat, K.K. and Fox, I.H. (1988). Isocratic Separation of ATP and its Degradation Products from Biological Fluids by Automated Liquid Chromatography, Clinical Chemistry, 34(5): 925—932.

27.

Schobert, B. (1995). Enzymatic Synthesis of ATP Analogs and their Purification by Reversed-Phase High-Performance Liquid Chromatography, Analytical Biochemistry, 226(2): 288—292.

28.

Blanco, J. , Canela, E.I. , Sayos, J. , Mallol, H. , Lluis, C. and Franco, R. (1993). Adenine Nucleotides and Adenosine Metabolism in Pig Kidney Proximal Tubule Membranes, Journal of Cellular Physiology, 157(1): 77—83.

29.

Fujimori, H. , Sasaki, T. , Hibi , K. , Senda, M. and Yoshioka, M. (1990). Direct Injection of Blood Samples into a High-Performance Liquid Chromatographic Adenine Analyser to Measure Adenine, Adenosine, and the Adenine Nucleotides with Fluorescence , Journal of Chromatography, 515: 363—373.