Surface Plasma Polymerization of Polyethylene Glycol) Heterotelechelic Macromonomers for Construction of Cell Specific Surface

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Surface Plasma Polymerization of Polyethylene glycol) Heterotelechelic Macromonomers for Construction of Cell Specific Surface

Yukio Nagasaki''*, Yoshikazu Nakashima', Ryutaro Ogawa', Masao Kato1, Hidenori Ohtsuka2, Kazunori Kataoka2

1.Department of Materials Science , Science University of Tokyo,Noda 278-8510, Japan 2.Department of Materials Science , Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo113-8656, Japan

Keywords: Polyethylene glycol) Heterotelechelics,Macromonomers, Hyperbranched PEG Tethered Chain Surface, Surface Plasma Polymerization, Cell Specific Surface

1.Introduction ionic interactions to form PEG brush. Recently, end-reactive poly(ethylene Semitelechelic PEGs (including macromonomers) glycol)s (PEG) have become more and more have been utilized for the preparation of these important in a variety of fields such as biology, block or graft copolymers. In general, biomedical science and surface chemistry, due to commercially available methoxy-ended PEGs their unique properties such as solubility and having a hydroxyl group at the other terminal is flexibility of the chains and basicity of the ether utilized as the starting material for the oxygens in the main chain"2. One of the most semitelechelic PEG preparations. Thus, these important utilizations of PEG is the construction of PEG surface brushes possess inert free end groups. polymer brushes3, a densely packed layer of If certain reactive groups can be introduced to the tethered polymers anchored on the surface utilizing free ends of the brush, an opportunity of the PEG the end-functionality of the polymer chain. Such brush will be expanded. For example, the a PEG brush significantly changes the surface introduction of an affinity ligand to the brush free properties. For example, a PEGylated surface, end changes the surface to be utilized for affinity which means that the poly(ethylene glycol) chains separation, keeping a low non-specific adsorption. are densely packed on a surface and attached by One of the other issues for construction of the end of the polymer chain, shows effective PEG tethered chain was a rather lower PEG rejection of protein adsorption resulting in a good density on the surface. To construct an ideal blood compatibility"4s,6 A PEGylated surface is surface tethered chains, a conventional PEGylation also utilized as a capillary for high performance by semitelechelic PEG is not enough. capillary electrophoresis'°8. To solve above 2 problems at the same time, To construct polymer brushes on the surface, we would like to propose "Surface Plasma two major approaches are available. One is direct Polymerization of Heterotelechelic PEG chemical bonding by the end of the polymer Macromonomer Method". Heterotelechelics chain1'9. The other is physical adsorption of the mean oligomer possessing a functional group at block and/or graft copolymers1'1° In the latter one end and another functional group at the other case, a segment other than PEG adsorbs on the end. Thus, heterotelechelic PEG macromonomer surface by certain forces such as hydrophobic and is a PEG having a polymerizable end along with a functional group at the other end. Surface plasma

* T o whom all correspondence should be addressed.

Received April 7, 1999 Accepted May 25, 1999 59 J. Photopolym. SC1. Technol., Vol.12, No.1, 1999

polymerization of heteroPEG macromonomer 2.3Acid hydrolysis of the acetal end group: A would give tree-like tethered chain on the surface 1.5 g sample of the acetal-ended PEG was which may improve their surface density. In dissolved in a 90% acetic acid aqueous solution addition, each branch should have a reactive end. and allowed to react for 5 h at 30 °C. After the (Scheme 1) hydrolyzed polymer was extracted into chloroform, So far, we have synthesized several types of the polymer was precipitated in ether. The heterotelechelic PEGs such as N}{2-PEG-OH, polymer thus obtained was dried by freeze-drying CHO-PEG-OH, etc. 11'11'12,13 This synthetic with benzene. technique can be applicable to new heteroPEG 2.4PEG macromonomers having phenylboronic macromonomer synthesis. This paper acid end group: 0.50 g (0.25 mmol) of CHO-PEG communicates the synthesis of heteroPEG macromonomer in phosphate buffer (30 mL, pH = derivatives and plasma surface modification by the 6.5, I = 0.15M) and 0.116g (0.625 mmol) of m- macromonomers. Physicochemical aminophenylboronic acid were mixed in 100 mL characteristics of the surface are also described. glass reactor and stirred for 2h in ice bath. 0.392g (6025 mmol) of sodium cyanoboro hydride was 2.Experimental Part added to the mixture and reacted for 4d at r.t. 2.1Materials: Commercial tetrahydrofuran (Wako, After the polymer was recovered by chloroform TIF), methanol (Wako), 3,3-diethoxy-1-propanol extraction, the purification was carried out in a (Aldrich), methacryloyl anhydride (Aldrich) and 3- way similar to the above description. aminophenyl boromc acid (Aldrich) were purified 2.SSurface plasma polymerization of by conventional methods.14 EO (Saisan) was heteroPEG macromonomers: After poly(vinyl dried over calcium hydride and distilled under an chloride) sample was treated by oxygen plasma argon atmosphere. Potassium naphthalene was (0.15 Ton; Fwd =95W; Ref=20-16W; 1 min), the prepared according to a previous paper15 and the sample was soaked in hydrochloric acid (0.1 mM) concentration determined by titration. Other for 15 mm at ambient temperature, then dried in reagents were used as received. vacuo for id to introduce peroxide on the surface. 2.2Polymerization procedure: A glass vessel After 1 wt. % of heteroPEG macromonomer equipped with a three-way stop cock was degassed solution was degassed by argon bubbling, the and argon gas introduced. This cycle was peroxidized PVC was soaked and reacted at 80 °C repeated three times, then 45 mL of TIF, 0.47 mL for 5h. After the surface polymerization, the of 3,3-diethoxy-1-propanol (0.44 g; 3.0 mmol) and sample was washed by water and dried under 8.0 mL of potassium naphthalene (3 mmol; 0.38 vacuum for id. mol I L) were added via a syringe. After a few 2.6Analysis: GPC measurements were carried out minutes agitation to form potassium 3,3-diethoxy- using a Shimadzu 6A Liquid Chromatograph 1-propanoxide, 11.8 mL of EO (10.5 g; 238 mmol) equipped with a Shodex gel permeation column was added via a cooled syringe. After the (Shodex KD-806M • 2) and an internal RI mixture was allowed to react for 2d at r.t., 2.2 mL detector (RID-6A). DMF containing 10 mol L-1 of methaclyloyl anhydride (2.3 g; 15 mmol) was of lithium bromide was used as the eluent at a flow added and stirred for a further 24 h. The obtained rate of 1.0 mL min 1 at 40 LIC. 1H NMR spectra polymer was precipitated in cooled 2-propanol and were obtained using chloroform-d solutions (1.0 separated by centrifugation (5,000 rpm; 40 min, - wt%) with a JEOL EX400 spectrometer at 400 4 °C). The polymer was finally dried by freeze- MHz . Chemical shifts relative to CHCl3 (1H: d = drying with benzene. 7.26) were employed. MALDI-TOF-MS spectra

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were recorded using Brucker Reflex II. 2,5- As stated above, since an aldehyde group dihydroxybenzoic acid(DHB) was used as the can be easily react with primary amino group via a matrix for the ionization and operated in the reductive amination reaction, derivatization of the reflection mode. Cytochrom C was used for the aldehyde group to cell specific group was carried calibration of the detected ions. out. m-aminophenylboronic acid was utilized for the derivatization because of its high affinity to 3.Results and Discussion specific sugar moiety. Figure 1 shows 1H NMR 3.1HeteroPEG macromonomer synthesis: After spectrum of the polymer obtained. As can be construction of PEG brushes on the surface, seen in the figure, phenylboronic acid could be reactive groups at the free ends must be usually introduced at the end of PEG macromonomer. utilized in aqueous media for conjugation with The quantitative derivatization was confirmed by specific compounds such as proteins. A formyl both the NMR spectrum and MALDI-TOF MS group is very useful for conjugation with protein spectroscopy. due to its stability in water and its rapid reactivity 3.2Surface Plasma Polymerization of Hetero- with primary amino groups. In addition, no PEG Macromonomers: Heterotelechelic charge variation takes place by the modification macromonomers thus synthesized were utilized for because the resulting Schiff base can be easily the surface plasma polymerization. After the converted to a sec-amino group by reduction. No surface plasma polymerization, wettability change in charge distribution is considered to keep increased significantly. Actually, cos9 of the the modified protein much more native than other CHO-PEG macromonomer modified surface was techniques. Therefore, an aldehyde group was close to unity, while the initial PVC surface was selected for the introduction as a reactive group at less than 0.5. Thus, effective surface the PEG chain end. For introduction of an modification could be done by this method. aldehyde group at the a-terminal of a PEG chain, potassium 3,3-diethoxypropoxide was used as the initiator. We have already reported that a potassium 3,3-diethoxypropanoxide initiated the anionic ring opening polymerization of EO to form uniform size PEG having acetal group at a-terminus.16 After the polymerization co-end group can be converted to methacryloyl group by the addition of methacrylic anhydride into the mixture. The functionality of both terminal was almost quantitative. The acetal end group can be conveted to aldehyde group by an acid treatment. Under the appropriate conditions, the conversion attained more than 90%. Figure 2. Change in c-Potential of the Hyperbranched PEG Tethered Chain Possessing Boronic Acid Group at Each Free End as a Function of an Environmental pH

Figure 2 shows change in c-potential of the boronic acid-PEG macromonomer modified surface as a function of pH. With increasing pH of the environment, c-potential decreased, which is due to the formation of B- in the alkaline media. Thus, by the heteroPEG macromonomer surface modification method gave functional boronic acid moiety easily on the surface. Hyperbranched tethered PEG chain possessing functional group at each free end thus prepared is Figure 1. 'NMR Spectrum of HeteroPEG promising as high performance biointerface such Macromonomer possessing Boronic Acid End as cell separation surfaces. An interaction

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between hyperbranched PEG brushes and living 8. Nashabeh, W., Rassi, Z. E., J Chromatography , cells such as lymphocytes will be published 1991, 559, 367 elsewhere. 0. Linden, C. C. van der, Leemakers, F. A. M., Fleer, G. J. Macromolecules, 1996, 29,1000 References 1. Bailey, F. E. Jr., Koleske, J. V. Ed. "Alkylene 10. Jeong, B. J., Lee, J. H., Lee, H. B. J. Coll. Int. Oxide and Their Polymers" Marcel Dekker, Sci., 1996, 178, 757 New York, Vol. 35, 1991 11. Kim, Y. J., Nagasaki, Y., Kataoka, K., Kato, M., 2. Harris, J. M. Ed. "Poly(ethylene glycol) Yokoyama, M., Okano, T., Sakurai, Y. Chemistry, Biotechnical and Biomedical Polymer Bull. ,1994, 33, 1 Applications " Plenum Press, New York ,1993 3.Taunton, H. J., Toprakocioglu, C., Fettersh, L. J., 12.Nagasaki, Y., Kutsuna, T., Iijima, M., Kato, M., Kataoka, K., Bioconjugate Chem., 1995, 6, 231 Klein, L. J. Nature, 1988, 332, 712 4. Amiji, M., Park, K. J. Biomat. Sci. Polym. Ed., 13.Nagasaki, Y., Iijima, M., Kato, M., Kataoka, K., 1993, 4, 217 Bioconjugate Chem., 1995, 6, 702 5. Llanos, G. R., Sefton, M. V. J Biomater. Sci., 14. Perrin, D. D., Armarego, W. L. F., Moor, F. W. "Purification of Laboratory Chemicals Polym. Ed., 1993, 4, 381 , 2nd 6. Bergstom, K., Osterberg, E., Holmberg, K., Ed. " Pergamom Press: Oxford, 1980. Hoffman, A. S., Schuman, T. P., Kozlowski, A., Harris, J. M. J Biomat. Sc., Polym. Ed., 15: Szwarc, M., Levy, M., Milkovich, R. J. Am. 1994, 6,123 Chem. Soc., 1956, 78, 2656 7.Herren, B. J., Shafer, S. G., Alstine, J. V., Marris, 16. Nagasaki, Y., Ogawa, R., Yamamoto, S., Kato, J. M., Snyder, R. S. J. Coll. Int. Sci., 1987, M., Kataoka, K., Macromolecules, 1997, 30, 115, 46 6489