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Graft Copolymer Having Hydrophobic Backbone and Hydrophilic Branches. 33.† Interaction of Hepatocytes and Polystyrene Nanospheres Having Lactose-Immobilized Hydrophilic Polymers on Their Surfaces Toshiro Uchida,‡ Takeshi Serizawa,‡ Hirohiko Ise,§ Toshihiro Akaike,§ and Mitsuru Akashi*,‡ Department of Applied Chemistry and Chemical Engineering, Faculty of Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065, Japan; and Department of Biomolecular Engineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuda, Midori-ku, Yokohama 226-8501, Japan Received February 26, 2001 Revised Manuscript Received June 28, 2001
Introduction There is currently much interest in the preparation of polymer particles with diameters of less than a micrometer, not only owing to their unique physical and chemical properties but also for wide applications such as biomedical use.1 Polymer particles prepared by emulsion or suspension polymerization are well-known.2 However, it is difficult to accumulate multiple functional groups on their surfaces. Our research group has developed the technology for the preparation of hydrophobic polymer core nanospheres coated with a hydrophilic polymer chain corona with functional groups (core-corona nanospheres).3-20 The nanospheres are monomerically well dispersed in aqueous phases and are very useful for immobilization of biomolecules. The surfacemodified polymeric nanospheres are synthesized by using free radical dispersion copolymerization of hydrophilic macromonomers and hydrophobic comonomers in a polar solvent. Various kinds of polymeric particles consisting of polystyrene (polySt) or poly(methyl methacrylate) cores and functional coronas have been prepared by this macromonomer method. Consequently, the nanospheres are useful as immunolatex, when antibodies, for example, are introduced to them. The peptide drug calcitonin showed pharmaceutical activity with oral administration when it was physically absorbed onto polySt-core nanospheres.10 Lectins were covalently immobilized to a poly(methacrylic acid) corona of the polySt nanosphere, and the nanospheres were then used to capture HIV-1 gp120 and virions by the interaction between lectins on the nanosphere and the mannose of gp120.11,12 These surface-modified polySt nanospheres have various biomaterial applications and are also suitable for various technological applications as the polySt core is very stable. Moreover, their surface area is large enough for immobilization of biomolecules. † Part 32 of the series: Serizawa, T.; Yasunaga, S.; Akashi, M. Biomacromolecules 2001, 2, 469. * To whom all correspondence should be addressed. ‡ Kagoshima University. § Tokyo Institute of Technology.
Figure 1. Schematic representation of a lactose-immobilized nanosphere and subsequent hepatocyte binding.
Carbohydrates on cell surfaces play critical roles in a variety of biological functions such as cell growth, regulation, differentiation, cancer metastasis, and infection.21 Hepatocytes have surface asialoglycoprotein receptors (ASGPR), which interact with galactose. There is 0.7 × 105 of lectin protein on a hepatocyte surface. But this number represents only 5% of a whole hepatocyte.22 There have been several studies of the interaction between hepatocytes and galactose. The importance of multiple interactions for binding between ligands and ASGPR was reported by Lee and co-workers using neoglycoproteins, i.e., galactosylated proteins.23 Adachi et al. investigated the interaction between hepatocytes and galactose-immobilized polystyrene derivatives, which were physically adsorbed onto polystyrene latex.24 In this study, we investigated the interaction of polystyrene nanospheres having lactose-immobilized hydrophilic polymers on their surfaces and hepatocytes (Figure 1) as a model system for drug delivery system, using flow cytometry and confocal microscopy. Preparation of the nanospheres, in which lactose is covalently bound to a poly(vinylamine) (PVAm) corona on the polySt core, has been described previously.18,19 Experimental Section Materials. PolySt nanospheres having lactose-immobilized hydrophilic polymers on their surface were prepared from PVAm nanospheres.18,19 The particle size of the nanospheres was measured by a submicron particle analyzer (Coulter model N4SD). The amount of lactose that was conjugated to PVAm was quantified using the sulfuric anthrone test25 after the lactose had been removed from the nanospheres
10.1021/bm0100413 CCC: $20.00 © 2001 American Chemical Society Published on Web 09/22/2001
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Figure 2. Fluorescence histograms of hepatocytes incubated with Lac-NS.
by acid hydrolysis (2 N HCl) at 100 °C for 2 h. Bovine serum albumin (BSA) was obtained from Sigma Chemical Co. (Steinheim, Germany). Fluorescein isothiocyanate (FITC) was purchased from Nacalai Tesque Co. (Kyoto, Japan). N,NDimethylformamide (DMF) and water were distilled before use. Williams’ E medium (GIBCO BRL) was prepared according to the manufacturers instructions and the pH was adjusted to 7.0. Poly(N-p-vinylbenzyl-D-lactonamide) (PVLA) was synthesized by free radical polymerization of the corresponding lactose-substituted styrene monomer (N-pvinylbenzyl-[o-β- D -galactopyranosyl-(1,4)]- D -gluconamide) as described by Kobayashi et al.26 We used TCSNT (Leica Co. Ltd.) as a confocal micrograph. The confocal micrograph was used in the usual way. FITC Labeling of Lactose-Immobilized Nanospheres. FITC dissolved in DMF solution 200 µL (4 mg/mL) was added to 1.4 mL of lactose-immobilized nanosphere (LacNS) suspension (Lac-NS concentration; 2.1 mg/mL). After 30 min of stirring in the dark, the solution was dialyzed for 1 day against water. FITC seemed to be physically incorporated into the Lac-NS. The resulting FITC loaded LacNS was analyzed by fluorescent spectrometry (MTP-32, Corona Electric Co., Ltd.). Isolation of Mouse Hepatocytes. Hepatocytes were prepared by noncirculating perfusion of male ICR mouse (6-9 weeks old) (SLC, Shizuoka, Japan) livers using a twostep collagenase perfusion technique of Seaglen.27 The preparation was enriched with viable cells by Percoll density centrifugation. The resulting cell suspensions were routinely >95% viable and consisted of predominantly single cells. Flow Cytometry. Hepatocytes (5.5 × 106 cells/mL) were incubated in Williams’E medium (0.2% BSA-10%FCS) with the FITC-labeled Lac-NS. After 1 h of incubation at 4 or 37 °C, the hepatocyte suspension was washed three times with PBS containing Ca2+ by centrifugation for 2 min at 600 rpm. The fluorescence intensity of the hepatocyte suspension was measured with a flow cytometer (EpicsXLMCL, Beckman-Coulter Co.). Confocal Laser Microscopy. Lab-Tec Chamber Slide TM (Nalge Nunc International) was precoated with 0.05% collagen (0.2 mL/well) for 1 day at room temperature. Hepatocytes (2 × 103 cells/0.2 mL/well) were preincubated in Williams’ E medium (10%FCS) for 3 h at 37 °C. Subsequently, the hepatocytes were incubated with the FITClabeled Lac-NS (0.21 g/L). After incubation several times at 37 °C, for 1, 3, and 24 h, wells were washed three times
Notes
Figure 3. Inhibition of nanosphere association with hepatocytes by PVLA.
with PBS containing Ca2+, fixed in 70% ethanol for 30 min, and washed with PBS containing Ca2+ again. The slides were moved to a chamber, covered by a coverslip, and observed by confocal laser microscopy. Results and Discussion Lac-NS was prepared by the reaction of lactose-lactone with PVAm nanospheres in methanol under alkaline conditions. The size of the nanospheres was 415 ( 100 nm (SD), as analyzed by a particle analyzer. The amount of lactoseimmobilized per amount of Lac-NS, which was analyzed by the anthrone test, was 170 µg mg-1 (the amount per unit surface area of Lac-NS corresponded to 1.2 µg cm-2). LacNS was labeled with FITC in order to measure the interaction between the hepatocytes and the Lac-NS by flowcytometry and confocal microscopy. The resulting amount of FITC loaded onto the Lac-NS, which was determined by fluorescent spectrometer, was 18 µg mg-1, which was sufficient to conduct the following experiments. Flow cytometry was used to evaluate the interaction of the Lac-NS with hepatocytes. An increase in fluorescence intensity indicated that the galactose on the Lac-NS was preferentially recognized by ASGPR on the hepatocytes. Figure 2 shows the cell number against log fluorescence intensity as a histogram. This figure shows typical flow cytometry histograms of hepatocytes incubated with LacNS at 4 °C. There was a small amount of background fluorescence with hepatocytes alone (control). The flow cytometry histogram of hepatocytes incubated with LacNS shifted to show more than 10 times higher fluorescence intensity than scan in control hepatocytes, showing the significant interaction of Lac-NS with hepatocytes. The fluorescence intensity of hepatocytes incubated with LacNS at 4 °C was smaller than that at 37 °C (data not shown). ASGPR is known to be inactive at 4 °C. This may explain the observed difference. We attempted to inhibit the interaction using PVLA. Figure 3 shows flow cytometry histograms of hepatocytes incubated with FITC-labeled Lac-NS at 4 °C after 30 min incubation with PVLA at a concentration of 4.3 µM (180 times lager than the molar concentration lactose on the Lac-NS surface). The flow cytometry histogram of hepatocytes incubated with PVLA shifted to lower fluorescence intensity than that with Lac-NS. This phenomenon may indicate specific interaction of Lac-NS with hepatocytes. We analyzed whether Lac-NS was bound to the surface or was incorporated into the hepatocytes by the analysis of
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Figure 4. Confocal laser microscopic views of hepatocytes at 1 h (a, control), hepatocytes incubated with Lac-NS at 1 h (b) and 24 h (c).
Figure 5. Serial optical sections of hepatocytes incubated with fluorescent Lac-NS.
confocal laser micrographs. Confocal laser micrographs of hepatocytes incubated with Lac-NS are shown in Figure 4. In this figure, the upper image represents the phase contrast micrograph image and the lower image represents a superposition of the phase contrast micrograph over the fluorescence micrograph image. FITC incorporated into Lac-NS fluoresces a light green color. A small number of green spots was found on the surface of hepatocytes alone (control). In contrast, a large number of green spots was found on the surface of hepatocytes incubated with Lac-NS. Each green spot on the hepatocytes had approximately the same diameter as that of a single nanosphere (415 nm). Moreover, it should be noted that Lac-NSs were differentially distributed in hepatocytes, depending on the incubation time. We observed the hepatocytes after 1, 4, and 24 h. After 4 h, we found the coagulate. The Lac-NS tended to coagulate with time in hepatocytes. The difference in particle distribution in hepatocytes was distinctly observed in the confocal laser micrographs as a gradual change in focus point from the apical face to the basal faces of the hepatocytes. As shown in Figure 5, Lac-NS was present inside of the cells. These results
showed that Lac-NS was internalized by the hepatocytes after interaction with cell surface receptors at 37 °C. The Lac-NS seemed to be incorporated by the process of endocytosis, although further studies elucidating the details of this interaction are required. Acknowledgment. We acknowledge Dr. A. Maruyama and Dr. Y. Watanabe (Tokyo Institute of Technology, Japan) for their grateful discussions. This work was financially supported in part by Grant-in-Aid for Scientific Researches in the Priority Areas of “Molecular Synchronization for Design of New Materials System” (No. 404/11167270) and Grand-in-Aid for Science Research (11480259) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. We also want to thank M. Nagaoka for their help with our experiments. References and Notes (1) Puisiex, F.; Barrat, G.; Courraze, G.; Couvreur, P.; Devissagut, J. P.; Dubernet, C.; Fattal, E.; Fessi, H.; Vauthier, C.; Benita, S. Polymeric micro- and nanoparticles as drug carriers. In Polymeric
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(2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12)
Biomacromolecules, Vol. 2, No. 4, 2001 Biomaterials; Dumitriu, S., Ed.; Marcel Dekker: New York; 1994; pp 749-794. Buscall, R.; Corner, T.; Stageman, J. F.; Eds. Polymer Colloids; Elsevier Appl. Science Pub.: London and New York, 1985. Akashi, M.; Kirikihira, I.; Miyauchi, N. Angew. Makromol. Chem. 1985, 32, 81. Akashi, M.; Yanagi, T.; Yashima, E.; Miyauchi, N. J. Polym. Sci. Part A: Polym. Chem. Ed. 1989, 27, 377. Akashi, M.; Chao, D.; Yashima, E.; Miyauchi, N. J. Appl. Polym. Sci. 1990, 39, 2027. Riza, M.; Capek, I.; Kishida, A.; Akashi, M. Angew. Makromol. Chem. 1993, 206, 69. Riza, M.; Tokura, S.; Kishida, A.; Akashi, M. New Polym. Mater. 1994, 4, 189. Riza, M.; Tokura, S.; Iwasaki, M.; Yashima, E.; Kishida, A.; Akashi, M. J. Polym. Sci., Part A: Polym. Chem. Ed. 1995, 33, 1219. Chen, M. Q.; Kishida, A.; Akashi, M. J. Polym. Sci., Part A: Polym. Chem. Ed. 1996, 34, 2213. Sakuma, S.; Suzuki, N.; Kikuchi, H.; Hiwatari, K.; Arikawa, K.; Kishida, A.; Akashi, M. Int. J. Pharm. 1997, 158, 69. Akashi, M.; Niikawa, Y.; Serizawa, T.; Hayakawa, T.; Baba, M. Bioimmobilize Chem. 1998, 9, 50. Hayakawa, T.; Kawamura, M.; Okamoto, M.; Baba, M.; Niikawa, T.; Takehara, S.; Serizawa, T.; Akashi, M. J. Med. Virol. 1998, 56, 327.
Notes (13) Serizawa, T.; Chen, M.-Q.; Akashi, M. Langmuir 1998, 14, 1278. (14) Serizawa, T.; Chen, M.-Q.; Akashi, M. J. Polym. Sci., Part A: Polym. Chem. Ed. 1998, 36, 2581. (15) Hiwatari, K.; Serizawa, T.; Kishida, A.; Akashi, M. Manuscript in preparation. (16) Chen, C.-W.; Chen, M.-Q.; Serizawa, T.; Akashi, M. J. Chem. Soc., Chem. Commun. 1998, 831. (17) Chen, C.-W.; Chen, M.-Q.; Serizawa, T.; Akashi, M. AdV. Mater. 1998, 10, 1122. (18) Uchida, T.; Serizawa, T.; Akashi, M. Polym. J. 1999, 31, 970. (19) Serizawa, T.; Uchida, T.; Akashi, M. J. Biomater. Sci. Polym. Edn. 1999, 10, 391. (20) Serizawa, T.; Takehara, S.; Akashi, M. Macromolecules 2000, 33, 1759. (21) Varki, A. Glycobiology 1993, 3, 97. (22) Steer, C. J.; Ashwell, G. J. Biol. Chem. 1980, 255, 3008. (23) Lee, R. T. Biochemistry 1982, 21, 1045. (24) Adach, N.; Maruyama, A.; Ishihara, T.; Akaike, T. J. Biomater. Sci. Polym. Ed. 1994, 6 (5), 463. (25) Dreywood, R. Ind. Eng. Chem., Anal. Ed. 1946, 18, 499. (26) Kobayashi, K.; Sumitomo, H.; Ina, Y. Polym, J. 1985, 17, 567. (27) Seglen, P. O. Methods Cell Biol. 1976, 13, 29.
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