Effect of Biodegradable Polyrotaxanes on Platelet Activation

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Bioconjugate Chem. 1998, 9, 118−125

Effect of Biodegradable Polyrotaxanes on Platelet Activation Nobuhiko Yui,* Tooru Ooya, and Takashi Kumeno School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-12, Japan. Received March 10, 1997; Revised Manuscript Received October 27, 1997X

Cellular response to our designed biodegradable polyrotaxanes was evaluated in terms of physicochemical interaction with plasma membrane and intracellular metabolism of platelets. The polyrotaxanes, in which many hydroxypropylated (HP-) R-cyclodextrins are threaded onto a poly(ethylene glycol) chain capped with a L-phenylalanine moiety via a peptide linkage, were synthesized and characterized. The polyrotaxanes inhibited cytoplasmic calcium increase in platelets, increased plasma membrane fluidity of red blood cell ghosts, and elevated cytoplasmic cyclic-3′,5′-AMP levels in platelets. Such cellular response to the polyrotaxanes was observed, which was more than that to constituent molecules. These results suggest that the supramolecular structure of the polyrotaxanes contributes to acceleration of the physicochemical interaction with plasma membrane and intracellular metabolism of platelets. Thus, biodegradable polyrotaxanes can be useful as new biomaterials for fabricating blood-contacting devices.

INTRODUCTION

Current studies in supramolecular sciences have explored fascinating fields by constructing nanoscale devices as biomimetic systems (1, 2). A polyrotaxane has been recently demonstrated to be a molecular necklace in which many R-cyclodextrins (R-CDs) are threaded onto a poly(ethylene glycol) (PEG) chain capped with bulky end groups (3, 4). From the viewpoint of a polymeric drug carrier aiming for therapeutic application, we previously synthesized a series of biodegradable polyrotaxanes in which hydroxypropylated (HP) R-CDs are threaded onto a PEG chain capped with L-phenylalanine (L-Phe) moieties via peptide linkages (5-7). It was found that the release of HP-R-CDs was achieved only when the terminal peptide moiety was degraded by a hydrolytic enzyme (7). The cellular response to synthetic polymers is of fundamental importance to their possible therapeutic application as pharmacologically active polymers and polymeric carriers for biologically active agents (8). In our previous study, we examined interaction of the polyrotaxanes with hairless rat stratum corneum to demonstrate the feasibility of them being a transdermal penetration enhancer (9, 10). The polyrotaxanes were found to interact with lipid components in the stratum corneum (9) and to enhance the permeation of indomethacin through a full-thickness rat skin (10). Considering these results, such polymers may interact with cellular systems in a certain period until the polymers disappear due to hydrolysis. Thrombogenesis has been shown to be initiated via both activation pathways of coagulation factors and platelets. Platelet response to synthetic polymers forms an important investigative basis for the design of blood compatible polymers (11, 12). It is well-known that platelet activation by extracellular stimuli such as thrombin is mediated by an elevation of cytoplasmic free * Author to whom correspondence should be addressed. Phone: +81-761-51-1621. Fax: +81-761-51-1625. E-mail: yui@ jaist.ac.jp. X Abstract published in Advance ACS Abstracts, December 15, 1997.

calcium concentration ([Ca2+]i) in relation to decreased membrane fluidity (13). Our systemic studies concerning blood-contacting properties of synthetic polymer surfaces have demonstrated that the prevention of contactinduced activation of platelets is a dominant factor for designing nonthrombogenic surfaces (14-16). Through these studies, it has been clarified that nonthrombogenic polymer surfaces showed no increase in [Ca2+]i but enhanced membrane fluidity of platelets (11, 12). Thus, it is suggested that the regulation of intracellular calcium levels in platelets is enhanced by contacting the nonthrombogenic surfaces. In this paper, we report on cellular response to our designed polyrotaxanes by inhibiting thrombin-induced platelet activation. It was found that the polyrotaxanes inhibit thrombin-induced cytoplasmic calcium increase in platelets, increase plasma membrane fluidity of red blood cell ghosts, and, to a certain extent, elevate cytoplasmic cyclic-3′,5′-AMP levels in platelets. This finding has many applications in polymeric supramolecular assemblies for constructing blood-contacting devices. EXPERIMENTAL PROCEDURES

Chemicals. R-Cyclodextrin (R-CD), (benzyloxycarbonyl)(Z)-L-phenylalanine (L-Phe), and propylene oxide were purchased from Wako Pure Chemical Co. Ltd. (Osaka, Japan) and used as received. N-Hydroxysuccinimide (HOSu) was purchased from Peptide Institute, Inc. (Osaka, Japan) and used as received. R-[(2-Amino-2methylethyl)-x-oxypropyl]-ω-(amino-y-oxypropyl)polyoxyethylene (x + y ) 2.5) with a number-average molecular weight (M h n) of 2000 (PEG-BA2000) was kindly supplied from Suntechno Chemical Co. (Tokyo, Japan) as JEFFAMINE ED-2001. R-(3-Aminopropyl)-ω-(3-aminopropyl)polyoxyethylene with an M h n of 4000 (PEG-BA4000) was kindly supplied by Sanyo Chemical Co. Ltd. (Kyoto, Japan) as IONET YB-400. Fura 2-AM [1-[2-(5′-carboxyo x a z o l - 2 ′ - y l ) - 6 - a m i n o b e n z o f u r a n - 5 - o x y ]- 2 - ( 2 ′ amino-5′-methylphenoxy)ethane-N,N,N′,N′-tetraacetic acid pentaacetoxymethyl ester] was purchased from Dojin Chemical Co. (Kumamoto, Japan). Thrombin from bovine plasma (5000 units/bottle) was purchased from

S1043-1802(97)00189-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 01/12/1998

Biodegradable Polyrotaxanes

Bioconjugate Chem., Vol. 9, No. 1, 1998 119

Scheme 1. Synthesis of Biodegradable Polyrotaxanes in Which the M h n of PEG Is 4000 (HP-r/E4-PHE) and 2000 (HP-r/E2-PHE)a

a

A ≈ 6 and B ≈ 2 will be assumed when the degree of HP substitution is ca. 8.

Sankyo Co. Ltd. (Tokyo, Japan). 1,6-Diphenyl-1,3,5hexatriene (DPH) was purchased from Wako Pure Chemical Co. Ltd. Bicinchoninic acid (BCA) protein assay reagents were purchased from Pierce (Rockford, IL). Unless otherwise noted, all chemicals were purchased from Wako Pure Chemical Co. Ltd. Synthesis of the Polyrotaxanes. Two types of biodegradable polyrotaxanes, in which the M h n of PEGs was 2000 and 4000 (Scheme 1), were synthesized according to our previous method (5-7). The synthesis of the polyrotaxanes was as follows. The inclusion complex of R-CD and PEG-BA2000 or PEG-BA4000 was prepared as reported previously (3, 4). Z-L-Phe succinimide (Z-LPhe-OSu), which was prepared as reported previously (17), was dissolved in dry DMSO, followed by the addition

of the inclusion complex. Then, DMSO was added to the suspension until homogeneous conditions were reached and the mixture stirred at room temperature for 48 h. After washing with acetone and water to remove unreacted Z-L-Phe-OSu and the inclusion complex, the obtained polyrotaxanes (R/E2-PHE-Z or R/E4-PHE-Z) were allowed to react with propylene oxide in a 1 N NaOH solution at room temperature for 24 h. After neutralizing with HCl solution, the solution was dialyzed against water and lyophilized. Finally, the Z group in R/E2-PHEZs was deprotected under a H2 atmosphere with paradium-carbon to obtain hydroxypropylated polyrotaxanes (HP-R/E2-PHE and HP-R/E4-PHE) (yield, 40%). As reference samples, HP-R-CD and L-Phe-terminated PEGs (E2-PHE and E4-PHE) were prepared as follows.

120 Bioconjugate Chem., Vol. 9, No. 1, 1998

Figure 1.

1H-NMR

Yui et al.

spectrum of HP-R/E4-PHE (in D2O).

R-CD was hydroxypropylated by the same manner used in the case of the polyrotaxanes. E2-PHE and E4-PHE were synthesized using Z-L-Phe-OSu and PEG-BA2000 or PEG-BA4000 in DMF. The reaction mixture was poured into excess dry ether and then washed with ether to obtain pure E2-PHE or E4-PHE. The purity was checked by 1H-NMR and GPC (yield of HP-R-CD, 64%; yield of E2-PHE, 82%; and yield of E4-PHE, 88%). Characterization of the Polyrotaxanes. The polyrotaxanes were characterized by 1H-NMR and highperformance size exclusion chromatography (SEC) (57). From 1H-NMR spectra of both HP-R/E2-PHE and HPR/E4-PHE, the peaks of R-CDs, PEG, L-Phe, and hydroxypropyl groups were observed. A representative 1H-NMR spectrum of HP-R/E4-PHE is shown in Figure 1. 1H-NMR (D2O): δ 7.50-7.20 (m, 5H, phenyl of L-Phe), 5.22, 5.07 (s, 6H × 23, C-1 H of R-CD), 4.20-3.35 (m, 18H × 23, C-3 H, C-6 H, C-5 H of R-CD, 3H × 184, CH, CH2 of hydroxypropyl groups), 3.72 (s, 4H × 90, CH2 of PEG), 1.18 (d, 3H × 184, CH3 of hydroxypropyl groups). The average number of R-CDs threaded onto a PEG chain was determined to be ca. 11 for HP-R/E2-PHE and ca. 23 for HP-R/E4-PHE from the 1H-NMR, comparing the integration of the signals at δ 5.22 and 5.07 (C-1 H of R-CD) with those at δ 3.72 (CH2 of PEG). With comparison of the integration of the signals at δ 5.22 and 5.07 (C-1 H of R-CD) with those at δ 1.18 (CH3 of hydroxypropyl groups), the average ratio of hydroxypropylation per R-CD molecule was determined to be ca. 8 in HP-R/E2PHE, HP-R/E4-PHE, and HP-R-CD. SEC was performed on a column of G3000SW (30 × 2.5 cm, Tosoh, Tokyo, Japan). A HPLC system (two-line DG-980-51 degasser, Intelligent HPLC PU-980 pump, Column CO-965 oven, and Chiral OR-990 detector, Jasco, Tokyo, Japan) was used at a flow rate of 1.0 mL/min at 40 °C. The eluent was 0.05 mol/L phosphate buffer (PBS, pH 7.0) containing 0.3 mol/L NaCl. The sample solution was prepared by dissolving HP-R/E2-PHE or HP-R/E4PHE (4 mg) in the eluent (1 mL) and passing the solution through a filter (0.45 mm). M h n and the weight-averaged molecular weight (M h w) of HP-R/E2-PHE and HP-R/E4-

PHE were determined form the SEC spectra using the PEG standard. In order to confirm the supramolecular structure (the threading of HP-R-CDs onto a PEG chain) of the polyrotaxanes, the in vitro degradation experiment of the polyrotaxanes by papain was carried out as follows. HPR/E2-PHE or HP-R/E4-PHE (3.3 × 10-3 mmol) was dissolved in 10 mL of a citrate buffer (5 mmol/L citric acid, 58 mmol/L Na2HPO4, 2 mmol/L ethylenediaminetetraacetic acid (EDTA), and 10 mmol/L mercaptoethanol at pH 7.1), followed by the addition of papain (9.5 mg, 200 units per 10 mL), and stirred at 37 °C. The reaction mixture (0.1 mL) was sampled at an appropriate time and diluted with water (0.1 mL), followed by heating to precipitate papain. The hydrolysis of the terminal L-Phe moiety and the release of HP-R-CDs were calculated by the result of HPLC analysis [SEC columns, G2500PWXL (Tosoh) for monitoring the hydrolysis of the terminal L-Phe moiety and G3000SW (Tosoh) for monitoring the release of HP-R-CD measurements] (7). As to the reference experiment, the similar in vitro degradation of the polyrotaxanes without papain was carried out. Measurement of Cytoplasmic Calcium Change in Platelets. A change in the cytoplasmic free calcium concentration ([Ca2+]i) in platelets in contact with the polyrotaxanes was examined using a Fura 2-AM-loaded platelet suspension (11, 12). A platelet suspension in Ca2+- and Mg2+-free Hanks’ balanced salt solution (HBSS) (platelet concentration, 3 × 108/mL) was prepared from citrated blood of male Japanese white rabbits, weighing 2.5-3.0 kg. Fura 2-AM was loaded into platelets by incubating the platelet suspension with a Fura 2-AM solution at 37 °C for 60 min at a Fura 2-AM concentration of 5 mM. The platelets were washed with HBSS and were finally resuspended in HBSS so that the final platelet concentration was 3 × 108/mL. The platelet suspension was recalcified with CaCl2 so that the external calcium concentration was 1 mM just prior to use in the [Ca2+]i measurements. A [Ca2+]i in platelets was measured according to the method of Tsien et al. (18). HP-R/E2-PHE or HP-R/E4-

Biodegradable Polyrotaxanes

PHE in HBSS (10 wt %, 100 µL) was mixed with a Fura 2-AM-loaded platelet suspension (400 µL) in a fluorescence cuvette in a fluorimeter (Japan Spectroscopic Co., Tokyo, Japan, CAF-110) at 37 °C with magnetic stirring. The mixture was excited at both 340 and 380 nm, and emission was measured at 500 nm. Fluorescence intensities at each of the two wavelengths were used to determine the fluorescence dichroic ratio 340/380 (R). [Ca2+]i was calculated on the basis of the R value using the following equation (18):

[Ca2+]i ) Kd[(R - Rmin)/(Rmax - R)]Sf2/Sb2 where Kd is the dissociation constant for Ca2+ binding to the fluorescent dye, Rmin and Rmax are ratios of fluorescence emission intensities at the selected excitation wavelengths (340 and 380 nm, respectively, with very low and very high calcium concentrations, and Sf2 and Sb2 are proportionality coefficients for free dye and Ca2+bound dye measured at the longer excitation wavelength (380 nm), respectively. The Kd for the Ca2+-Fura 2 interaction is assumed to be 224 nM in the cytoplasmic environment. Rmin, Rmax, and Sf2/Sb2 were measured under the same conditions by adding 10 µL of a 1% Triton-X aqueous solution and then 10 µL of a 3 M ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) aqueous solution. As a control, 100-200 nM [Ca2+]i in unused platelets is checked prior to the mixing with the solution. Thrombin was used as an agonist of platelets in order to evaluate the effect of the polyrotaxanes on the platelet activation. Thrombin in HBSS (4 units/mL, 10 µL) was added to the platelet suspension 1 min after mixing with the polyrotaxane solution (final thrombin concentration, 0.1 unit/mL). The magnitude of the increase in [Ca2+]i in platelets, ∆[Ca2+]i, was determined by the following equation:

∆[Ca2+]i ) ([Ca2+]i)thr - ([Ca2+]i)0 where ([Ca2+]i)thr is the [Ca2+]i in platelets after adding thrombin in HBSS and ([Ca2+]i)0 is the [Ca2+]i in platelets before adding thrombin in HBSS. In order to minimize time-dependent effects on platelet functions or leakage of Fura 2-AM, these experiments were completed within 1 h of Fura 2-AM loading. In the same manner, [Ca2+]i was calculated for constituent molecules of the polyrotaxanes and their mixture. Fluorescence Polarization Measurement of DPHLoaded Red Blood Cell (RBC) Ghosts. Hemoglobinfree RBC ghosts were prepared according to Dodge et al. (19). Citrated blood of male Japanese white rabbits weighing 2.5-2.8 kg was collected and centrifuged at 1000 rpm for 15 min to obtain RBCs. RBCs were washed with a 0.155 M NaCl solution at pH 7.4 and centrifuged at 1000 rpm for 10 min two times. The suspension was washed with 15-20 volumes of 0.01 M PBS at pH 7.4 several times. After centrifugation (18 000 rpm for 30 min at 4 °C), the RBC ghost suspension was adjusted at 0.1 mg of protein/mL with a 0.155 M NaCl solution at pH 7.4 by a micro BCA method (20). DPH solution in tetrahydrofuran (2 mM) was diluted into the RBC ghost suspension to 2 µM and incubated with gentle agitation at 37 °C for 60 min in the dark (21). Fluorescence anisotropy of DPH in RBC ghosts was measured in order to assess the membrane fluidity (22). Fluorescence emission spectra (360-500 nm) of DPHloaded RBC ghosts were checked at an excitation wavelength of 360 nm. The polyrotaxane solution in 0.155 M

Bioconjugate Chem., Vol. 9, No. 1, 1998 121

NaCl at pH 7.4 (10 wt %, 100 µL) was mixed with 400 µL of the DPH-loaded RBC ghost suspension in a fluorescence cuvette in a spectrofluorimeter (Japan Spectroscopic Co., FP-777) equipped with a fluorescence polarization accessory (Japan Spectroscopic Co., ADP300) at 37 °C with magnetic stirring. DPH was excited at 360 nm, and the fluorescence was detected at 430 nm. The slit widths for both excitation and emission were 10 nm. Fluorescence intensities were measured with polarizers inserted into the excitation and emission light paths. Fluorescence anisotropy, 〈r〉, was calculated with the following equation (22):

〈r〉 ) (IH - IHb) - G(IV - IVb)/(IH - IHb) + G ) IV′/IH′ 2G(IV - IVb), where IH and IV are emission intensities observed with the analyzing polarizer horizontal and vertical with respect to the polarized excitation beam. IHb and IVb are fluorescence intensities for a blank solution (0.155 M NaCl) at the same position of the polarizer as IH and IV. G is a correction factor, equal to IV′/IH′, the primes indicating excitation polarized in a horizontal direction. In the same manner, 〈r〉 was measured for constituent molecules of the polyrotaxanes and their mixture. Platelet Cyclic-3′,5′-AMP Assay. Cytoplasmic cyclic3′,5′-AMP (cyclic AMP) levels ([cyclic AMP]i) in platelets were measured by means of a 3H-labeled cyclic AMP assay according to the method reported by Beretz et al. (23). HP-R/E2-PHE or HP-R/E4-PHE solution (0.1 g/mL, 200 µL) in HBSS was mixed with the platelet suspension (3 × 108/mL, 800 µL) in HBSS at 37 °C for 1 or 2 min. Cold trichloroacetic acid (50%, 100 µL) was added to the suspension 1 or 2 min after the polyrotaxane addition to stop the cellular metabolism. The suspension was then centrifuged at 12 000 rpm for 1 min. The supernatant was decanted and trichloroacetic acid extracted by five washings with 3 volumes of water-saturated ether. Cyclic AMP was then measured in the supernatant by the protein binding method of Gilman (24) using a commercial kit containing [3H]cyclic AMP (Yamasa Co., Chiba, Japan). RESULTS AND DISCUSSION

Synthesis and Characterization of the Polyrotaxanes. The hydroxypropylation of R-CDs improved the solubility of the polyrotaxanes in PBS at pH 7.4. For example, the solubility of HP-R/E4-PHE was 85 wt %, whereas that of HP-R/E2-PHE was 28 wt % in spite of similar degree of HP substitution (eight per R-CD) (7). On SEC curves of HP-R/E2-PHE and HP-R/E4-PHE, a single peak which was detected by both optical rotation (detection of R-CD) and absorbance at 280 nm (detection of the terminal L-Phe moiety) was obtained (Figure 2). Taking the obtained 1H-NMR spectra of the polyrotaxanes into account, the observed single peak may indicate that R-CDs are threaded onto a PEG chain with the L-Phe moiety at both terminals. M h n, M h w, and M h w/M h n of the polyrotaxanes were summarized in Table 1. M h n values of HP-R/E2-PHE and HP-R/E4-PHE were determined to be 1.29 × 104 and 1.06 × 104, respectively. These values were smaller than the calculated M h n values (1.81 × 104 for HP-R/E2-PHE and 3.75 × 104 for HP-R/E4-PHE). h w values were calculated by both Since these M h n and M h w values of PEG maintaining the random coil M h n and M structure, the M h n value of the polyrotaxanes may not be accurately estimated by SEC. Furthermore, the M hw value of HP-R/E2-PHE was larger than that of HP-R/E4-

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Yui et al.

Figure 4. Relationship between the polyrotaxane concentration and thrombin-induced cytoplasmic calcium increase in platelets (n ) 4, (SEM): HP-R/E2-PHE (O) and HP-R/E4-PHE (4).

Figure 2. SEC curves of polyrotaxanes (a) HP-R/E2-PHE and (b) HP-R/E4-PHE. Column, Tosoh G3000SW 4.4 × 30 cm; eluent, 0.05 M PBS (pH 7.0) containing 0.3 M NaCl; flow rate, 1 mL/ min. Table 1. Molecular Weight of the Polyrotaxanesa polyrotaxane

M h n (×104)

M h w (×104)

M h w/M hn

HP-R/E2-PHEb HP-R/E4-PHEb

1.29 1.06

3.42 1.23

2.65 1.16

a Molecular weights of the polyrotaxanes were determined by SEC as described in Experimental Procedures. A calibration curve was prepared with the PEG standard. b The number of R-CD in HP-R/E2-PHE and HP-R/E4-PHE was determined by 1H-NMR to be ca. 11 and 23, respectively.

Figure 3. HP-R-CD release from HP-R/E4-PHE with (O) and without (b) 20 units/mL papain. HP-R-CD was determined by HPLC analysis (flow rate, 1 mL/min; detect, optical rotation).

PHE (Table 1). Recently, the M h w value of the polyrotaxanes was determined by static light scattering (SLS) measurements, and the M h w value of HP-R/E2-PHE was found to be larger than that of HP-R/E4-PHE (25). From these results, it is suggested that the larger M h w value of HP-R/E2-PHE was caused by the association of HP-R/ E2-PHE molecules under a physiological condition (25). Figure 3 shows HP-R-CD release from HP-R/E4-PHE by the hydrolysis of the terminal L-Phe moiety with or without papain in vitro. It was found that HP-R-CD was released only in the presence of papain. A similar phenomenon was observed in the case of HP-R/E2-PHE. In our previous study, it has been clarified that the HPR-CD release was dominated by the hydrolysis of the

Figure 5. Inhibition of thrombin-induced cytoplasmic calcium increase in platelets by the polyrotaxanes (n ) 5, (SEM). The concentration of constituent molecules (HP-R-CD + E-PHEs, E-PHEs, HP-R-CD, and E2-OH) is equal to the mole percentage in the polyrotaxanes. Entries marked with *, **, and *** were significantly different at P < 0.1, P < 0.05, and P < 0.001, respectively, calculated using the t-test.

terminal L-Phe moiety (7). Therefore, the HP-R-CD release by the enzymatic hydrolysis indicates the threading of HP-R-CDs onto the PEG chain; i.e. the polyrotaxanes form a supramolecular structure. Effect of the Polyrotaxanes on Cytoplasmic Calcium Change in Platelets. With the viewpoint of fabricating blood-contacting devices with these supramolecular-structured surfaces, platelet response to the polyrotaxanes will be the most important basis for preventing thrombus formation. Figure 4 shows a doseresponse relationship between polyrotaxane concentration and thrombin-induced increase in [Ca2+]i. From this result, the most effective concentration of the polyrotaxanes (2 wt %) was selected to compare with the constituent molecules of the polyrotaxanes. With this concentration, an increase in [Ca2+]i was not observed without thrombin, indicating no cytotoxicity and cellular activation (25). The thrombin-induced increase in [Ca2+]i was significantly decreased by the addition of HP-R/E2-PHE or HP-R/E4-PHE solution, as shown in Figure 5. As to reference samples, a mixture of HP-R-CD and L-Pheterminated PEG (HP-R-CD + E2-PHE or HP-R-CD + E4PHE), E2-PHE, and E4-PHE showed a lower inhibitory effect on [Ca2+]i increase, although HP-R-CD and PEG (E2-OH) showed no inhibitory effect (Figure 5). These results indicate that the polyrotaxanes significantly inhibited thrombin-induced platelet activation. Further, in order to confirm the inhibitory effect of the polyrotaxanes on thrombin-induced activation process, a similar experiment was carried out using sodium azide (NaN3)treated platelets. Here, NaN3 was added to the recalcified platelet suspension to 40 mM, followed by incubation at 37 °C for 5 min prior to the addition of the polyrotax-

Biodegradable Polyrotaxanes

Figure 6. Effect of sodium azide on thrombin-induced cytoplasmic calcium increase in platelets by the polyrotaxanes (n ) 3, (SEM). Entries marked with *, **, and *** were significantly different at P < 0.05, P < 0.01, and P < 0.001, respectively, calculated using the t-test.

ane solution or the constituent molecules. The polyrotaxanes showed a significant effect on [Ca2+]i increase in NaN3-treated platelets compared with the constituents (Figure 6). It is recognized that NaN3 inhibits the platelet metabolism via regulation of ATP production in cytoplasmic mitochondria (26). As far as platelet metabolism is inhibited, the interaction of the polyrotaxanes with plasma membranes may induce the increase in [Ca2+]i. Thus, it is suggested that regulation of [Ca2+]i in platelets is enhanced by physicochemical interaction of the polyrotaxanes, the mechanism of which is metabolically directed. Quite recently, static light scattering of the polyrotaxane solutions under a physiological condition was measured (25, 27). Rod-like structure and/or associated natures of these polyrotaxanes were observed, and the association was found to depend upon the hydrophilichydrophobic balance of threaded HP-R-CDs and terminal L-Phe moieties. It was found that HP-R/E2-PHE showed a loosely packed association nature under a physiological condition, although HP-R/E4-PHE showed a little association. E2-PHE and E4-PHE showed a much more closely packed association nature in the physiological condition, and HP-R-CD and E2-OH showed no association (25). From these characteristics, it is considered that supramolecular structure (rod-like structure) of HP-R/ E4-PHE contributes to regulating platelet functions. In addition, supramolecular association of HP-R/E2-PHE may also contribute to physicochemical interaction with cell membrane. Our previous studies have demonstrated that poly(acrylamide-co-methacrylic acid) (PAAmMAc) microparticles exhibit a similar effect on platelet activation (11, 12). Molecular assemblies seen in the polyrotaxanes as well as PAAmMAc particles may induce local interaction with plasma membrane and/or membrane proteins. Changes in Membrane Fluidity of RBC Ghosts Caused by the Polyrotaxanes. It has been well reported that any physicochemical changes in plasma membrane such as membrane fluidity change are related to platelet activation (13). The effect of the polyrotaxanes on the plasma membrane fluidity was examined using a DPH-loaded rabbit RBC ghost suspension. A significant decrease in 〈r〉 was observed when the HP-R/E2-PHE solution was mixed with the RBC ghost suspension (Figure 7). Here, the 〈r〉 decrease indicates an increase in membrane fluidity. It was found that the addition of HP-R-CD + E2-PHE, E2-PHE, HP-R-CD, and E2-OH showed a much smaller decrease in 〈r〉 than HP-R/E2-

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Figure 7. Changes in anisotropy of DPH fluorescent polarization in red blood cell (RBC) ghosts by the polyrotaxanes (n ) 3, (SEM). The concentration of constituent molecules (HP-R-CD + E-PHEs, E-PHEs, HP-R-CD, and E2-OH) is equal to the mole percentange in HP-R/E-PHEs. Entries marked with *, **, and *** were significantly different at P < 0.05, P < 0.01, and P < 0.005, respectively, calculated using the t-test.

Figure 8. Increase in cytoplasmic cyclic AMP levels in platelets by the polyrotaxanes for 1 min (shaded) and 2 min (solid) (n ) 4, (SEM). Entries marked with * and ** were significantly different from HP-R/E4-PHE at P < 0.002 and P < 0.001, respectively, calculated using the t-test.

PHE, as shown in Figure 7. Similar phenomena were observed in the case of samples using PEG with an M hn of 4000. Since the increase in membrane fluidity was enhanced more by HP-R/E2-PHE than by HP-R/E4-PHE, the supramolecular association of HP-R/E2-PHE may lead to a much more enhanced membrane fluidity increase than that of HP-R/E4-PHE (25). This membrane fluidity change was observed within a few seconds after the polyrotaxane addition (data not shown). Taking the rapid increase in membrane fluidity and the results of the [Ca2+]i change in platelets into account, the polyrotaxanes are considered to perturb membrane structure triggering membrane-dependent signaling pathways to regulate intracellular metabolism. Effect of the Polyrotaxanes on Cyclic AMP Levels in Platelets. As mentioned above, it is suggested that the [Ca2+]i increase in platelets by the polyrotaxanes is regulated by the metabolically directed mechanism in relation to the increase in membrane fluidity. One possible way will be used to explain the inhibition as follows. The polyrotaxane can accelerate the biochemical mechanism regulating [Ca2+]i in platelets. A well-known mechanism for regulating [Ca2+]i in platelets is calcium inhibition by cyclic-3′,5′-AMP (cyclic AMP)-dependent protein kinase via the activation of adenylate cyclase or the inhibition of cyclic AMP phosphodiesterase (28). The polyrotaxane induced an increase in [cyclic AMP]i in platelets, although the other references showed almost the same [cyclic AMP]i as the control, as shown in Figure 8. This finding was statistically significant; however, the

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extent of [cyclic AMP]i increase was ca. 20%. This result suggests that the polyrotaxanes inhibit the [Ca2+]i increase in platelets, the mechanism of which may involve the contribution of the [cyclic AMP]i increase. Previously, it was reported that an increase in membrane fluidity of rat reticulocytes allows adenylate cyclase to be activated by enhancing the coupling with β-adrenergic receptor (29). Taking the above report into account, it is likely that the polyrotaxanes could enhance the membrane fluidity of platelets to elevate [cyclic AMP]i. CONCLUSION

Cellular response to our designed biodegradable polyrotaxanes was examined in terms of the intracellular metabolism of platelets and physicochemical interaction with plasma membrane. The polyrotaxanes were characterized by 1H-NMR, SEC, and an in vitro degradation experiment. The inhibition of thrombin-induced cytoplasmic calcium levels in platelets was observed with the addition of our designed polyrotaxanes. Further, the polyrotaxanes were found to enhance membrane fluidity in RBC ghosts. These effects of the polyrotaxanes were statistically more significant than those of their constituent molecules such as a mixture of HP-R-CDs and L-Pheterminated PEGs. The 20% increase in cyclic AMP levels in platlets by the polyrotaxanes suggests that the polyrotaxanes inhibit the [Ca2+]i increase in platelets, the mechanism of which may involve the contribution of the [cyclic AMP]i increase. Although the relation with the supramolecular structure and association is currently under investigation, these findings could contribute to new fields of polyrotaxanes in medical and therapeutic application. These biodegradable polyrotaxanes will degrade into noninteractive components (HP-R-CD, PEG, and L-Phe) within a period of time, and will be naturally removed from the body. It is believed that biodegradable polyrotaxanes can be used to fabricate blood-contacting devices which ultimately can be absorbed in the body. ACKNOWLEDGMENT

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