Dental Materials Journal 10(2): ,

Transcription

1 Dental Materials Journal 10(2): , Changes in 1H-NMR Chemical Shifts of Bis-GMA and Its Related Methacrylates Induced by Their Interaction with Phosphatidylcholine/Cholesterol Liposomes Seiichiro FUJISAWA*, Yoshinori KADOMA**, and Yasuo KOMODA** *Faculty of Dentistry, Tokyo Medical and Dental University, , Yushima, Bunkyo-ku, Tokyo 113 **Institute for Medical and Dental Engineering, Tokyo Medical and Dental University, , Surugada Kanda, Chiyoda-Ku Tokyo 101, Japan Received on July 1, 1991 Accepted on September 28, 1991 To clarify the hemolytic mechanism of Bis-GMA and its related methacrylates, the interaction of five methacrylates (Bis-GMA, MMA, NPGDMA, TEGDMA, and UDMA) with DPPC/Cholesterol (CS) liposomes as a model for erythrocyte membranes, was studied by 1H-NMR spectroscopy. Exogenous Bis-G partitioning into the DPPC/CS liposomes caused disappearances of its proton signals. With NPGDMA, its signals broadened markedly in DPPC/CS liposomes. UDMA partitioning caused changes in chemical shifts to a higher field, whereas MMA and TEGDMA partitioning did not cause any changes in chemical shifts. It was concluded from these observations that Bis-GMA has a stronger interaction with the DPPC/C liposomes than the other methacrylates used. The high hemolytic activity of Bis-GMA reported previously3,12) seemed to be due to its migration into the lipid bilayer of phospholipids containing CS in erythrocyte membranes. Key wods: Bis-GMA, Phospholipid/Cholesterol Liposomes, NMR. INTRODUCTION Bis-GMA and its related dimethacrylates (TEGDMA, UDMA etc.) are widely used in dentistry as restorative materials and adhesives1). There have been many previous studies of the cytotoxicy2-5), mutagenicity6) and tissue toxicity7,8) of Bis-GMA and its related compounds and these findings have indicated that these compounds have an adverse effect on cells and tissues. It is known that the biological activity of lipidsoluble compounds is intimately related to changes in physical phenomena caused by their interaction with phospholipid liposomes, used as a model for biomembranes9-11). We have previously investigated the interaction of lipid-soluble compounds such as Bis- GMA, TEGDMA, and MMA with dipalmitoylphosphatidylcholine (DPPC) liposomes12) and DPPC/cholesterol (CS) liposomes13) as a model for biomembranes, using differential scanning calorimetry (DSC). The results suggested that Bis-GMA is liable to migrate into the lipid bilayer of DPPC/CS liposomes. There appeared to be a relationship between the hemolytic activity and calorimetric properties of DPPC liposomes (phasetransition temperature Tm, enthalpy, half-height width etc.) caused by Bis-GMA and its related methacrylates12,13). NMR is one of the most powerful methods for studying this interaction at the molecular level14). Also, DPPC/CS liposomes are the best model for erythrocyte membranes in studying this interaction13). Thus, in the present study, we investigated the

2 122 S. FUJISAWA, Y. KADOMA and S. KOMODA changes in NMR-chemical shifts induced by the interaction of Bis-GMA and its related methacrylates (MMA, NPGDMA, TEGDMA, and UDMA) with DPPC/CS liposomes, to clarify the hemolytic mechanism of Bis-GMA and its related monomers. MATERIALS AND METHODS Bis-GMA was separated from a commercial product# by an LC as described previously15). The structure of 2, 2-bis [4-(2-hydroxy-3-methacryloyloxypropoxy) phenyl] propane (Bis-GMA) and its numbering system are shown in Fig. 1. 1H-NMR chemical shifts (ƒâh) and 13C-NMR chemical shifts (ƒâc) of Bis-GMA in CDCl3 were in ppm downfield from tetramethyl silane used as internal reference. [1H-NMR of Bis-GMA, ƒâh: 1.63 (6H, s; H9, H10), 1.96 (6H, br s; H1), 2.57 (2H, d, J=4.9Hz; OH), 4.03 (4H, m; H7), 4.26 (2H, m; H6), 4.35 (4H, double d, J=5.1 and 1.5Hz; H5), 5.60 (2H, m; Ha3), 6.14 (2H, br s; Hb3), 6.81 (4H, d, J=8.9Hz; H2 Œ, H6 Œ) and 7.13 (4H, d, J=8.9Hz; H3 Œ, H5 Œ)]. Bis-GMA NPGDMA Fig. 1 Chemical atructures of Bis-GMA and NPGDMA (neopentylglycol dimethacrylate) and their numbering system. [13C-NMR of Bis-GMA ƒâc: 18.3 (C1), 31.0 (C9, C10), 41.8 (C8), 65.6 (C5), 68.6 (C6), 68.8 (C7), (C2 Œ, C6 Œ), (C3), (C3 Œ, C5 Œ), (C2), (C 4 Œ), (C1 Œ) and (C4)]. Di-2-methacryloyloxyethyl trimethylhexamethylenedicarbamate (UDMA. art resin SH-500 (S) Lot )## was used without further purification. Methyl methacrylate (MMA)###, triethyleneglycol dimethacrylate (TEGDMA)#, and neopentylglycol dimeth acrylate (NPGDMA)# were purifed by HPLC. Preparation of DPPC/CS (4:1) liposomes and NMR studies were carried out by the same methods as those previously reported in this journal14). Bis-GMA, MMA, TEGDMA, UDMA, and NPGDMA were dissolved in CD3OD/D2O (16:84v/v) and each of them was added to DPPC/CS liposomes. Since some dimethacrylates are insoluble in water, the teat compounds in this experiment were #Shin Nakamura Chemical Co. Wakayama Japan, ##Negami Chemical Industrial Co. Ishikawa, Japan, and ###Tokyo Kasei Kogyo Co., Tokyo, Japan.

3 LIPOSOMES/Bis-GMA INTERACTION 123 dissolved in methanol solution, as described above. A16 vol% was determined for the following reasons; NMR signals of methacrylates were observed at this concentration and CD3OD did not extract CS from DPPC/CS liposomes13). RESULTS AND DISCUSSION The NMR chemical-shifts at 37 Ž of Bis-GMA, NPGDMA, DPPC/CS liposomes/bis- GMA, and DPPC/CS liposomes/npgdma are shown in Table 1. Exogenous Bis-GMA partitioning into DPPC/CS liposomes caused disappearance of its proton signals, indicating that saturation occured in this system. It was clear from this that Bis-GMA migrates into liposomes and that its mobility is restricted by the lipid bilayer containing cholesterol. In general, non-purified Bis-GMA is applied to one of the components of composite restorative materials and adhesives. As one of our experiments, the commercial Bis-GMA product was used, in part, without further purification. The 1H-NMR spectrum of its interaction with DPPC/CS liposomes is shown in Fig. 2A. All signals of Bis-GMA disappeared and the signals of Nos. 1-3 were due to ingredients in the commercial product. The commercial product of this compound contains various ingredients such as Iso-Bis-GMA, either the glycidyl ether of bisphenol A (an epoxy molecule)/methacrylic acid or bisphenol A/glycidyl methacrylate, hydroquinones, etc. In particular, iso-bis-gma is the main ingredient in this compound16). Figure 2A shows that some ingredients were easily incorporated into the liposomes. Signals of numbers 1-3 were not due to the moiety of Bis-GMA and iso-bis-gma. This results supported that non-purifed Bis-GMA containing Iso-Bis-GMA had strong hemolytic activity12). For NPGDMA, its signals broadened markedly in DPPC/CS liposomes (see Fig. 2C). The DPPC-NPGDMA interaction appeared to be smaller than that of Bis-GMA. This seemed to be due to differences in hydrophobicity between Bis-GMA (LogPHPLC=3.2) and NPGDMA (LogPHPLC=2.8)17). Table 1 Chemical shifts (ƒâh) of Bis-GMA, NPGDMA, DPPC/CS liposomes (4:1 molar ratio)/bis-gma and DPPC/CS liposomes (4:1 molar ratio)/npgdma.

4 124 S. FUJISAWA, Y. KADOMA and S. KOMODA Fig. 2 1H-NMR spectra of DPPC/CS liposomes/bis-gma(a), DPPC/CS liposomes/udma(b), DPPC /CS lipomes/npgdma(c), and DPPC/CS liposomes/tegdma(d) at 37 Ž in CD3OD/D2O (16:84v/v). A: ingredients 1, 2, and B: 1, proton of the three methyl in the trimethylhexamethylene moiety of isocyanates; 2, CH2=C(CH3); 3, -OCH2CH2OCONH-; 4, CH=C(CH3), and 5, CH=C(CH3)., C: Proton Nos, see Fig. 1.; 1, H7 and H8; 2, H1; 3, H 5; 4, Ha; and 5, Hb., D: 1, CH2=C(CH3); 2, internal -OCH2CH2O-; 3, -CH2OCH2CH2OCH2 -; 4, -COOCH2; 5, CH=C(CH3); and 6, CH=C(CH3)., a, methanol and b, DHO. Changes in the chemical shifts of MMA, TEGDMA, and UDMA in DPPC/CS liposomes are shown in Fig. 2 (B and D) and Table 2. The ƒâh values of free MMA were not different from those of MMA in DPPC/CS liposomes. In our previous report17), MMA did not cause a shift of Tm in DPPC/CS liposomes. It was clear from these findings that the interaction of MMA with DPPC/CS liposomes was small. For TEGDMA, its ƒ ƒâh values were This was almost similar to the case of MMA. On the other hand, the ƒ ƒâh values of UDMA were Its signals were shifted to a higher field, indicating shielding. The mobility of UDMA appeared not to be restricted by the lipid bilayer of DPPC/CS liposomes, and as a result of this, its signals showed a relative decrease in the half-height width of proton signals in DPPC/CS liposomes, compared to those of free UDMA in CD3OD/D2O. This behavior was clearly different from that of Bis-GMA and NPGDMA, which had markedly broadened or abolished signals, as shown in Fig. 2. We have previously reported that the hemolytic activity of Bis-GMA is stronger than that of TEGDMA and MMA, and that the degree of hemolytic activity is dependent

5 LIPOSOMES/Bis-GMA INTERACTION 125 Table 2 Chemical shifts (ƒâh) and chemical-shift differences (ƒ ƒâh) of MMA, TEGDMA, UDMA#, Bis- GMA# induced by their interaction with DPPC/CS (4:1 molar ratio) liposomes at 37 Ž. The ƒâh values are in ppm downfield from TMSPA used as an external reference in D2O/CD3OD (84:16v /v).; The ƒ ƒâh value=ƒâh of methacrylate -ƒâh of DPPC/CS liposomes/methacrylate.; (), ƒ ƒâh; *, TEGDMA; #, the commercial product used without further purification.; +, in trimethylhexamethylene moiety of isocyanates; **, DPPC (50mg): MMA (TEGDMA)=1:1 molar ratio;++, DPPC: UDMA (Bis -GMA)=50mg:10mg.; dd, double doublet; m, multiplet; s, singlet; brs, broad singlet; and NS, no signal. on the hydrophobicity of methacrylates, and, further, that the degree of hemolytic activity of the methacrylates used was as follows: *Bis-GMA, NPGDMA âtegdma> MMA3). We have also investigated the Tm and enthalpy of phospholipid liposomes caused by Bis-GMA and its related methacrylates, using DSC, in order to monitor their biological effects12). This investigation showed that Bis-GMA shifted Tm to a lower temperature and increased the half-height width of the main DSC peak, as its DSC data were compared with those of TEGDMA in the molar ratio of compound/dppc>0.2. Our previous calorimetric studies12,13) revealed that the interaction of Bis-GMA was stronger than that of TEGDMA and MMA. Further, we found, in NMR studies, that Bis -GMA did not diffuse from phospholipid liposomes once it was incorporated into the lipid bilayers of DPPC18). On the other hand, it has been been reported that Bis-GMA and UDMA have a stronger cytotoxicity than that of TEGDMA and MMA5). Our NMR -findins indicate that different toxicological behavior of the Bis-GMA-type and UDMAtype resin systems may be expected. It is known that cholesterol plays a very important role in biological membranes characterized by the so-called dual effect of membrane fluidity19). Since lipid-soluble compounds such as Bis-GMA act intrinsically on the lipid portion of biological membranes, phospholipid liposomes containing cholesterol are the best model for studying the interaction of such compounds with biological membranes. Our results of NMR studies of DPPC/CS liposomes could provide an insight into the role of the chemical structure of methacrylate molecules in clarifying the interaction between methacrylates and erythrocyte membranes. Our NMR data indicated that Bis-GMA was impregnated deeply into the liposomes. This is due to the driving force for incorporation coming from hydrophobic interaction. Whereas TEGDMA (LogPHPLC=1.0)17) may, in part, migrate directly across the lipid bilayer, but may be

6 126 S. FUJISAWA, Y. KADOMA and S. KOMODA located in the surface of liposomes. The interaction of MMA was the smallest, due to its low hydrophobicity (LogPHPLC=0.7)17); it acts on the lipid bilayer at a high concentration. Our present NMR results provided sufficient interpretation of the hemolytic mechanism induced by lipid-soluble compounds such as Bis-GMA and its related monomers. NMR studies have indicated the rationale for chemically determining the degree and nature of interaction of methacrylates with erythrocyte membranes. CONCLUSION The interaction of five methacrylates (Bis-GMA, MMA, NPGDMA, TEGDMA and UDMA) with DPPC/CS liposomes was studied by NMR spectroscopy. Bis-GMA showed strong migration into the lipid bilayer. The NMR-signals of NPGDMA were markedly broadened due to its impregnation into the lipid bilayer. The ĉh values of UDMA shifted to a higher field, indicating shielding, while those of MMA and TEGDMA did not change greatly in DPPC/CS liposomes. REFERENCES 1) von Fraunhofer, J.A.: Scientific aspects of dental materials, Butterworths, London and Boston, 1975, pp , pp ) Spangberg, L., Rodrigues, H., Langeland, L. and Langeland, K.: Biologic effects of dental materials. 2. Toxicity of anterior tooth restorative materials on Hela cells in vitro, Oral Surgery 36: , ) Fujisawa, S., Imai, Y., Kojima, K. and Masuhara, E.: Studies on hemolytic activity of bisphenol A diglycidyl methacrylate (Bis-GMA), J Dent Res 57: , ) Nakamura, M., Imai, K., Oshima, H., Kudo, T. and Kawahara, H.: Biocompatability tests of light-cured composites in vitro, Dent Mater J 4(2): , ) Imai, Y., Watanabe, M., Lee, H-E, Kojima, K. and Kadoma, Y.: Cytotoxicity of monomers used in dental resin, Reports of the Institute for Medical and Dental Engineering 22: 87-90, (in Japanese) 6) Ozawa, K., Kodama, T., and Sato, A.: Mutagenicity of BisGMA and TriEDMA, J J Dent Mater 6: , (in Japanese) 7) Stanley, H.R., Going, R.E. and Chancy, H.H.: Human pulp response to acid pretreatment of dentin and composite restoration, J Am Dent Assoc 91: , ) Schmalz, G.: Biologische Eigenschaften vom Kompositefullungmaterialien, Dtsch Zahnarzt Z40: , ) Sessa, G. and Weismann, G.: Phospholipid spherules (liposomes) as a model for biological membranes, J Lipid Res 9: , ) Jain, M.K., Wu, N.Y.M. and Wray, L.V.: Drug-induced phase change in bilayer as possible mode of action of membrane expanding drugs, Nature 255, , ) Kamaya, H., Matsubayasi, N. and Ueda, I.: Biphasic effect of long-chain n-alkanols on the main phase transition of phospholipid vesicle membrane, J Phys Chem 88: , ) Fujisawa, S. and Kadorna, Y.: Effect of Bis-GMA analogs on hemolytic activity and DSC phase transition of phospholipid liposomes, J J Dent Mater 6: , (in Japanese) 13) Fujisawa, S. and Kadoma, Y.: Effect of Bis-GMA analogs on the DSC phase transition properties of phospholipid/cholesterol liposomes, J J Dent Mater 6: , (in Japanese) 14) Fujisawa, S., Kadoma, Y. and Komoda, Y.: Changes in NMR chemical shifts of methacrylates induced by their interaction with the phospholipid and the phospholipid/cholesterol liposome system, Dent Mater J 9: , 1990.

7 LIPOSOMES/Bis-GMA INTERACTION ) Fujisawa, S., Kadoma, Y., and Masuhara, E.: NMR and DSC studies on the phospholipid interaction with monomers in bonding agents. J J Dent Mater 3: 30-37, (in Japanese) 16) Vankerckhoven, H., Lambrechts, P., van Beylen, M. and Vanherle, G.: Characterization of composite resins by NMR and TEM., J Dent Res 60: , ) Fujisawa, S. and Masuhara, E.: Determination of partition coefficients of acrylates, methacrylates, and vinyl monomers by high performance liquid chromatography (HPLC), J Biomed Mater Res 15: , ) Fujisawa, S., Kadoma, Y. and Komoda, Y.: Nuclear magnetic resonance spectroscopic studies of interaction of Bis-GMA analogues with phosphatidylcholine liposomes as a model for biomembranes, Biomaterials 10: , ) Damel, R.A. and de Kruyff, B.: The function of sterols in membranes, Biochem Biophy Acta 457: , 1976.

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