Biomacromolecules 2000, 1, 515-518
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Control of Cell Death by the Smart Polymeric Vehicle Keiji Fujimoto,*,† Chizu Iwasaki,† Chigusa Arai,† Masayuki Kuwako,† and Etsuko Yasugi‡ Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan, and Research Institute, International Medical Center of Japan,1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan Received August 10, 2000; Revised Manuscript Received October 19, 2000
We report a novel drug delivery system for apoptosis induction by a “smart” polymer vehicle possessing thermosensitivity and bioaffinity. The polymer chain was prepared by copolymerization of N-isopropylacrylamide and N-methacryloyloxysuccinimide. Cell-adhesive RGDS peptide was conjugated with the copolymer as a ligand model for bioaffinity. When the temperature was increased, nanoscale aggregates precipitated from a copolymer aqueous solution. Either dolichyl phosphate (dol-p), which is an apoptotic inducer, or dolichol was added to aggregates at around the precipitation temperature (31 °C), and the temperature was raised to 37 °C for incorporation. Aggregates incorporating dol-p or dolicol were added to a human promonocytic leukemia U937 cell suspension at 37 °C. When the temperature was lowered to 25 °C, cells underwent apoptosis in the presence of Ca2+. Probably, copolymer vehicles were concentrated on a cell surface through the binding of RGDS and integrin and the release of lipid inducers was caused by the disruption of vehicles in response to temperature. Introduction Apoptosis is essential to maintaining the cell population balance in tissue and organ development and is thought to be associated with many diseases such as cancer and disorder of the self-immune system. Recently, it has been reported that apoptosis is induced by some lipid compounds such as ceramide1 and sphingosine.2 It was reported that dihydroprenyl phosphates with more than seven isoprene units are a potent inducer of apoptosis, which stimulates adenylate cyclase and activates caspase-3(CPP32),3 which is activated during apoptotic signaling events by upstream proteases including caspase-6 and caspase-8, in rat glioma C6 cells4 and in human monoblastic leukemia U937 cells.5 Such lipids are amphipathic and behave as a surfactant. In fact, droplets of the submicrometer size are observed for lipid inducers of apoptosis. We found that the transfer of inducers to the cell membrane is facilitated by forming the emulsion, and the transfer efficiency is governed by the emulsion stability.6 Since the emulsion is fluid, it is not so easy to modify this vehicle for the drug delivery. Nanoscale drug vehicles through the interchain aggregation of polymer chains have been reported by Kataoka7 and by Okano.8 Such polymeric micelles forming from amphiphilic copolymers are capable of incorporating a drug through physical entrapment. Therefore, we here propose a novel drug delivery system for apoptosis induction by a “smart” polymeric vehicle engineered to possess a hydrophobic cavity for the drug incorporation and to enable thermal control of the drug release and then to present cell adhesion ligands for the * Corresponding author. Tel & Fax: (+81)45-566-1580. E-mail: fujimoto@ applc.keio.ac.jp. † Keio University. ‡ International Medical Center of Japan.
targeting (Figure 1). This delivery vehicle is composed of the nanoscale polymeric aggregates based on the chemistry of smart polymers. Among them, poly(N-isopropylacrylamide) (polyNIPAM), due to its reversible phase separation corresponding to temperature,9 has been widely studied to produce smart materials in the intramolecular chain,10 the gel,11 and the gel particle forms12 for applications such as immunoassay, bioseparation, and drug delivery.13-16 It is reported that the association of polyNIPAM at low concentrations above its lower critical solution temperature (LCST) leads to stable aggregates formed mainly through the packing of individual collapsed chains.17 The copolymer chains will dissolve by lowering temperature, and thereby the release of lipid inducers of apoptosis from the cavity would be triggered by the disruption of vehicles. Results and Discussion To achieve the specific targeting through the immobilization of various ligands onto the polymer chain, we prepared the reactive and thermosensitive polymer chains (numberaverage molecular weight ) 130 000, Mw/Mn ) 1.75) by copolymerization of N-isopropylacrylamide with N-methacryloyloxysuccinimide. To render the site specificity for the generic cell targeting to the copolymer chain, arginineglycine-aspartate-serine (RGDS) tetrapeptide, which is able to attach to many cells through its binding with integrins and therefore has been utilized for generating a bioaffinity on the synthetic materials,18-20 was chemically linked to the copolymer chain through the amide bonding. We prepared copolymers including 0, 5, and 15 units of RGDS per one chain (RGDS0, RGDS5, and RGDS15 copolymers, respectively). The residual reactive moieties were allowed to react with isopropylamine to convert them to thermosensitive sites.
10.1021/bm000082j CCC: $19.00 © 2000 American Chemical Society Published on Web 11/21/2000
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Figure 3. Changes of the micropolarity for the copolymer solutions as a function of temperature (open circle, 0 unit; filled circle, 5 units; filled triangle, 15 units; open square, water). The sample converted completely to thermosensitive sites using isopropylamine without RGDS was represented as 0 unit.
Figure 1. Schematics of the drug delivery system for apoptosis induction using the smart polymeric vehicle. Targeting sites (e.g., RGDS) can be given to the copolymer chain by immobilizing various ligands. Above their lower critical solution temperature (LCST), the association of the copolymer leads to stable aggregates formed mainly through the packing of individual collapsed chains. The nanoscale delivery vehicle possesses the hydrophobic cavity for the incorporation of lipid inducers of apoptosis. The release of lipid inducers of apoptosis from the cavity is triggered by the disruption of vehicles by lowering temperature. The released inducers are transferred to the cell membrane, leading to apoptosis induction.
Figure 2. Temperature dependence of turbidity for RGDS-carrying vehicles (open circle, 0 unit; filled circle, 5 units; filled triangle, 15 units; open square, polyNIPAM). The sample converted completely to thermosensitive sites using isopropylamine without RGDS was represented as 0 unit. PolyNIPAM was obtained by homopolymerization of NIPAM.
Figure 2 shows the changes in transmittance of the copolymer aqueous solutions with temperature. All curves declined with increasing temperature. This was attributed to aggregation through the packing of individual collapsed polyNIPAM chains. We found that aggregates of RGDS15 copolymers kept monodisperse in size at the temperature region from 33 to 44 °C, and its size was 133 nm at 37 °C. When the temperature was raised and lowered at this temperature
region, monodisperse aggregates shrank and swelled, respectively, with keeping the monodispersity in size. Probably, introduction of the hydrophilic RGDS peptide made the copolymer chain amphiphilic, resulting in the formation of stable aggregates exposing RGDS moieties. Information on the volume transition and the hydrophobicity of polyNIPAM hydrogel particles can be obtained using a fluorescent probe sensitive to environmental micropolarity of its solubilized sites.21 In many cases, spherical aggregates of block copolymers and hydrophobically modified water-soluble polymers are composed of a hydrophobic core and a hydrophilic corona.22-24 From the fluorescence analysis, it was found that the emission maximum was shifted to lower wavelengths when heated and the shifting to 520 nm was observed again when cooled (Figure 3), indicating that aggregates were reversibly solubilized in response to temperature. The blue shift which reflects reduction of environmental micropolarity suggests that aggregates formed by collapsed copolymers would possess the hydrophobic cavity suitable for incorporating hydrophobic drugs. dol-p dissolved in ethanol can neither form the emulsion nor induce the apoptosis.6 Therefore, to incorporate dol-p to the hydrophobic inner core of copolymer aggregates, dol-p dissolved in ethanol was added to Hank’s balanced salt solution (HBSS) of the RGDS15 copolymer at 31 °C and the temperature was gradually heated to 37 °C. dol-p incorporated in the RGDS15 copolymer aggregate was found to be 0.545 units per one polymer chain by 1H NMR, and its average size was 173 nm in diameter determined by dynamic light scattering. The resultant delivery vehicles appeared to dissolve in the medium when cooled to 31 °C. This suggests that copolymer aggregates are capable of releasing passively entrapped dol-p into an aqueous environment. Therefore, we applied them to apoptosis induction as a delivery vehicle for lipid inducers of apoptosis. Cells were treated with the delivery vehicle composed of the RGDS15 copolymer for 30 min at 37 °C. We found no cytotoxicity, which was determined by WST-1 assay, below
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Figure 4. DNA fragmentation induced by RGDS15-copolymer vehicles incorporating dol-p: M, PHY marker; 1, control; 2, dol-p dissolved in ethanol; 3, dol dissolved in ethanol, 4, only vehicle; 5, vehicle incorporating dol-p; 6, vehicle incorporating dol-p (soluble dol-p was removed by centrifugation after incorporation); 7, vehicle incorporating dol; 8, vehicle incorporating dol (soluble dol was removed by centrifugation after incorporation). (a) 25 °C. The cell suspension was cooled from 37 to 25 °C. (b) 37 °C. The temperature was kept at 37 °C. For the upper lines, the operations were performed with Ca2+. For the lower lines, the operations were performed without Ca2+.
4 mg/mL of the copolymer. Cell shrinkage, nuclear collapse, and formation of apoptotic bodies were induced by allowing the cell suspension treated with the RGDS15 copolymer vehicles incorporating dol-p to cool and keep at 25 °C for 1 h (data not shown). Apoptosis induction was also assessed by DNA fragmentation. As is seen in Figure 4, the DNA ladder characteristic of apoptotic cells was clearly seen in the DNA extracted from cells treated with RGDS15 copolymer vehicles incorporating dol-p or dolichol only when the cell suspension was cooled to 25 °C in the presence of Ca2+. On the contrary, when the temperature was maintained at 37 °C, all samples exhibited no characterized feature of apoptosis. As we previously reported,6 the solubilized state of the apoptotic inducer is important to transfer the inducer to the plasma membrane and lead to apoptosis induction. For example, dol-p dissolved in the mixture of ethanol and hexane or n-octane forms the emulsion and nevertheless no apoptosis induction is observed. This is because the emulsion is too stable to transfer lipids to the membrane. Thus, these results indicate that the temperature change caused the drug release and transfer through the disruption of aggregates, resulting in apoptosis induction. Furthermore, it was a
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surprising result that the RGDS15 copolymer vehicle incorporating dolichol could induce apoptosis. Dolichol in solution could never induce apoptosis when added to the cell suspension. This suggests that appropriate incorporation and solubilization of dolichol were achieved using this delivery vehicle and the help of such vehicles might enable some of other lipids so far ignored to induce apoptosis. We carried out an experiment utilizing dop-P incorporated delivery vehicles without RGDS, which was composed of RGDS0 copolymer, to evaluate the influence of biorecognition on inducing apoptosis. Treated cells showed no morphological changes characteristic of apoptosis (data not shown). We can also see from the comparison between the upper and the lower lanes of Figure 4 that Ca2+ was required for apoptosis induction utilizing the RGDS-carrying vehicle. The bioaffinity between RGDS and the integrin requires Ca2+.25 Thus, these results strongly suggest that RGDS-carrying vehicles exposed RGDS moieties from their surface and selectively bound to a cell surface through the binding of RGDS and integrin. Recently, it was reported that soluble RGD peptides induce apoptosis by direct caspase-3 activation.26 However, we observed no apoptosis for cells treated with our synthesized RGDS-carrying copolymer chains in the absence of lipid inducers of apoptosis. These findings indicate that the smartness of the polymer would make it possible to fabricate a delivery vehicle for apoptosis induction. We expect that our developed smart materials will be a promising tool to control apoptosis in response to the stimuli such as the temperature change. Summary We here report a novel drug delivery system for apoptosis induction by a “smart” polymeric vehicle incorporating lipid inducers of apoptosis. This nanometer-sized vehicle possessed the targeting ability, the cavity for incorporation of the drugs. The vehicles incorporating an apoptotic inducer, dolichyl phosphate, were added to human promonocytic leukemia U937 cells at 37 °C. When the temperature was lowered to 25 °C, the release was triggered by the disruption of vehicles and thereby cells underwent apoptosis. This system would be expected to provide a therapeutic application for targeting a drug to the cell and triggering its release in body by cooling. Experimental Section Materials: Dolichol and dolichyl monophosphate (dol-p)were donated by Tsukuba Research Laboratories, Eisai Co. (Ibaraki, Japan). Boc-Arg(Mts)-OH was purchased from Nova biochem Ag (Laufelfingen, Switzerland). Boc-Ser(Bzl)OPac was prepared by coupling Pac-Br with the C-terminal of Boc-Ser(Bzl)-OH. Boc-Gly-OH, Boc-Asp(OBzl)-OH, and Boc-Ser(Bzl)-OH were purchased from Peptide Institute, Inc. (Osaka, Japan). H-RGDS-OBzl was synthesized according to the previous work.25 N-Isopropylacrylamide (NIPAM) was donated by Kohjin Co. (Tokyo, Japan). Preparation of RGDS-Carrying Copolymer Chains. Copolymerization of NIPAM with N-hydroxysuccinimide
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methacrylate was carried out in benzene at 60 °C for 30 min using AIBN as an initiator. Then, H-RGDS-OBzl was linked to the obtained polymer through the amide bonding between its amine group and the succinimide moiety of the polymer. The residual reactive moieties were allowed to react with isopropylamine to convert them to thermosensitive sites. The amount of RGDS conjugated with the polymer chain was estimated by 1H NMR (JNM-LA300 FT-NMR, 300 MHz, JEOL, Tokyo, Japan). Measurements of Turbidity. Optical transmittance of the polymers dissolved in HBSS (1 mg/mL) at various temperatures was measured at 500 nm with a UV-visible spectrometer (Hitachi U-2000, Tokyo, Japan). Dynamic Light Scattering. The hydrodynamic size of each polymer aggregate (1 mg/mL) formed in HBSS was determined by the dynamic light scattering measurement (Otsuka LPA-600, Osaka, Japan). Micropolarity of Polymer Aggregates. 8-Anilino-1naphthalenesulfonic acid, ammonium salt (ANS) was used as a fluorescent probe. A solution of ANS was added to 2.5 mL of the polymer solutions, while the final concentrations of ANS and the polymer were adjusted to 100 µM and 0.5 mg/mL, respectively. Emission spectra were obtained at various temperatures with a spectrophotofluorometer on excitation at 350 nm (Hitachi F-2000, Tokyo, Japan). Incorporation and Release of Lipid Inducers of Apoptosis. The polymer was dissolved at 1 mg/mL in HBSS. dol-p or dol dissolved in ethanol was added to HBSS of the polymer at 31 °C, while the final concentration of dol-p was adjusted to 20 µM. The temperature was gradually raised to 37 °C for 45 min. The amount of lipids incorporated into the polymer aggregate was estimated by 1H NMR (JNM-LA300 FT-NMR, 300 MHz, JEOL, Tokyo, Japan). The resultant polymeric delivery vehicles incorporating dol-p or dol were added to the suspension of U937 cells (5.0 × 105 cells) at 37 °C and the mixture was incubated for 30 min. The final concentration of the polymer was 0.25 mg/ mL. The following operations were performed with Ca2+. (a) The mixture was cooled to 25 °C and incubated for 1 h. Then, the temperature was again raised to 37 °C. After the mixture was incubated at 37 °C for 4 h, the DNA ladder electrophoresis assay was carried out. (b) The temperature was maintained at 37 °C for 5 h. Then, the DNA ladder electrophoresis assay was carried out. On the other hand, the same operations were performed without Ca2+. Cell Morphology. The human promonocytic leukemia cell line U937 was grown in RPMI1640 supplemented with 10% fetal calf serum (FCS) at 37 °C in an atmosphere of 5% CO2. For morphological assessment, cells treated with different agents were pelleted by centrifugation. After the cells were fixed in methanol and stained with Giemza, they were examined under an optical microscope. The occurrence of apoptosis in each group was determined based on the expression of cytoarchitectural characteristics of apoptosis (cell shrinkage, formation of apoptotic bodies, nuclear condensation, and nuclear fragmentation). Detection of DNA Fragmentation. Agarose gel electrophoresis was used to detect internucleosomal DNA cleavage. For U937 cells, 100 µL of lysis buffer containing 10 mM
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Tris-HCl (pH 7.4), 10 mM EDTA, and 0.5% Triton X-100 was added to the cell pellet and the preparation was left at 4 °C for 20 min. After centrifugation at 14 000 rpm for 20 min, the supernatant was incubated for 20 min with 2 µL of 20 mg/mL RNaseA (Sigma) aqueous solution followed by an additional incubation for 30 min with 2 µL of 20 mg/mL proteinase K (Boehringer-Mannheim, Germany) aqueous solution. The fragmented DNA was then precipitated by adding 20 µL of 5 M aqueous NaCl and 120 µL of 2-propanol overnight at -40 °C. After centrifugation at 14 000 rpm for 15 min, DNA was dissolved in TE buffer and analyzed on 1.7% of Nusieve 3:1 agarose gels (FMC BioProducts, Rockland). Acknowledgment. We are grateful to Dr. I. Yamatsu (Tsukuba Research Laboratories, Eisai Co.) for providing derivatives of isoprenoids. We acknowledge financial support from a Grant in Aid for Scientific Research on Priority Areas (A)(2), Ministry of Education, Science, Sports and Culture, Japan. References and Notes (1) Obeid, L. M.; Linardic, C. M.; Karolak, L. A.; Hannun, Y. A. Science 1993, 259, 1769. (2) Ohta, H.; Yatomi, Y.; Sweeney, E.A.; Hakomori, S.; Igarashi, Y. FEBS Lett. 1994, 355, 267. (3) Yokoyama, Y.; Okubo, T.; Ozawa, S.; Nagai, F.; Ushiyama, K.; Kano, I.; Shioda, M.; Kubo, H.; Takemura, M.; Namiki, H.; Yasugi, E.; Oshima, M.; Seyama, Y.; Kano, K. FEBS Lett. 1997, 412, 153. (4) Yasugi, E.; Yokoyama, Y.; Seyama, Y.; Kano, K.; Hayashi, Y.; Oshima, M. Biochem. Biophys. Res. Commun. 1995, 216, 848. (5) Dohi, T.; Yasugi, E.; Oshima, M. Biochem. Biophys. Res. Commun. 1996, 224, 87. (6) Fujimoto, K.; Iwasaki, C.; Kawaguchi, H.; Yasugi, E.; Oshima, M. FEBS Lett. 1999, 446, 113. (7) Yokoyama, M.; Miyauchi, M.; Yamada, N.; Okano, T.; Sakurai, Y.; Kataoka, K.; Inoue, S. J. Contr. Relat. 1990, 11, 269. (8) Chung, J. E.; Yokoyama, M.; Suzuki, K.; Aoyagi, T.; Sakurai, Y.; Okano, T. Colloids Surf. B: Biointerfaces 1997, 9, 37. (9) Heskins, M.; Guillet, J. E. J. Macromol. Sci. Chem. 1968, A2 (8), 1441. (10) Monji, N.; Hoffman, A. S. Appl. Biochem. Biotechnol. 1987, 14, 107. (11) Cussler, E. L.; Stokar, M. R.; Varberg, J. E. Am. Inst. Chem. Eng. J. 1984, 30, 578. (12) Shiroya, T.; Tamura, N.; Yasui, M.; Fujimoto, K.; Kawaguchi, H. Colloids Surf. B: Biointerfaces 1995, 4, 267. (13) Chen, J. P.; Hoffman, A. S. Biomaterials 1990, 11, 631. (14) Yasui, M.; Shiroya, T.; Fujimoto, K.; Kawaguchi, H. Colloids Surf. B: Biointerfaces 1997, 8, 311. (15) Hoffman, A. S. J. Controlled Release 1987, 6, 297. (16) Bae, Y. H.; Okano, T.; Hsu, R.; Kim, S. W. Macromol. Chem., Rapid Commun. 1987, 8, 481. (17) Li, M.; Wu, C. Macromolecules 1999, 32, 4311. (18) Kasuya, Y.; Fujimoto, K.; Miyamoto, M.; Juiji, T.; Otaka, A.; Funakoshi, S.; Fujii, N.; Kawaguchi, H. J. Biomater. Sci., Polym. Ed. 1993, 4, 369. (19) Kasuya, Y.; Fujimoto, K.; Miyamoto, M.; Kawaguchi, H. J. Biomed. Mater. Res. 1994, 28, 397. (20) Kasuya, Y.; Fujimoto, K.; Kawaguchi, H.; Miyamoto, M. Biomaterials 1994, 15, 570. (21) Fujimoto, K.; Nakajima, Y.; Kashiwabara, M.; Kawaguchi, H. Polym. Int. 1993, 30, 237. (22) Yamamoto, H.; Mizusaki, M.; Yoda, K.; Morishima, Y. Macromolecules 1998, 31, 3588. (23) Akiyoshi, K.; Deguchi, S.; Moriguchi, N.; Yamaguchi, S.; Sunamoto, J. Macromolecules 1993, 26, 3062. (24) Harada, A.; Kataoka, K. Science 1999, 283, 65. (25) Pierschbacher, M.D.; Ruoslahti, E. Nature 1984, 309, 30. (26) Buckley, C. D.; Pilling, D.; Henriquez, N. V.; Parsonage, G.; Threlfall, K.; Scheel-Toellner, D.; Simmons, D. L.; Akbar, A. N.; Lord, J. M.; Salmon, M. Nature 1999, 397, 534.
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