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Raman and FT-IR studies of photodynamic processes of cholesteryl oleate using IRFELs
Nuclear Instruments and Methods in Physics Research B 144 (1998) 229±235
Raman and FT-IR studies of photodynamic processes of cholesteryl oleate using IRFELs Yuko Fukami a, Yoshihito Maeda a
a,*
, Kunio Awazu
b
Department of Materials Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan b Free Electron Laser Research Institute, Inc. 2-9-1 Tsuda-Yamate, Hirakata, Osaka, 573-01, Japan
Abstract Chemical bond-changes of cholesteryl oleate in infrared free electron laser (IRFEL) photodynamic processes were examined by Raman spectroscopy and Fourier transform infrared microspectroscopy (FT-IR) as a function of the FEL exposure time. We found that the exposure of 5.79 lm-FELs induces not only dissociation of ester (RCOOR0 ) bonds but also the chemical changes from the ester (RCOOR0 ) bonds to aldehyde (RCHO), carboxylic acid (RCOOH), carboxylate (RCOOÿ ) or ketone (R2 C@O) bonds. Ó 1998 Published by Elsevier Science B.V. All rights reserved. PACS: 87.60.-f; 87.70.-k; 33.20.Fb Keywords: Free electron laser; Cholesterol-ester; Photodynamic therapy; Ester
1. Introduction The Free Electron Laser (FEL) has wavelength tunability and novel pulse structures (micro and macropulses) [1,2]. These great advantages are expected to induce some biologic level of interactions between photons and living tissues. The average power of the FEL is sucient for most forms of thermal coagulation therapy and photodynamic therapy (PDT) which is a light-activated chemotherapy for cancer and other indications [3±8]. In advanced atherom atherosclerosis, a large amount of lipids, particularly cholesterol esters, accumu-
* Corresponding author. Tel.: +81 722 54 9709; fax: +81 722 54 9931; e-mail: [email protected].
lates on the arterial wall. In more advanced cases, the lipids also accumulate in the interstitial spaces [9]. Therefore, the PDT is being applied as a less invasive method for eective removal of cholesteryl ester from atheroma using mono-L-aspartyl chlorin e6 (NPe6) as a photoresponsive material and a diode laser of 664 nm in wavelength as an excitation light [10]. Recently, Awazu et al. employed an infrared free electron laser (IRFEL) which is tuned to 5.75 lm of the wavelength corresponding to stretching vibrations of ester bonds to achieve direct dissociation of cholesteryl oleate and removal of cholesterol esters accumulated in rabbit artery without injection of additional chemicals [11]. They obtained very interesting results of wavelength sensitive removal processes in the IRFEL exposure
0168-583X/98/$ ± see front matter Ó 1998 Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 5 8 3 X ( 9 8 ) 0 0 3 6 0 - 7
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Y. Fukami et al. / Nucl. Instr. and Meth. in Phys. Res. B 144 (1998) 229±235
experiments to cholesteryl oleate and albumin. These photodynamic processes are of great importance for biomedical applications such as the PDT to selective removal of cholesterol from living tissues. However, details of physicochemical mechanism have not yet been clari®ed. In this study, cholesteryl oleate was employed as a model material for actual atheroma to understand photodynamic processes induced by the IRFEL exposure. The molecular structure of cholesteryl oleate (C45 H78 O2 ) is shown in Fig. 1 [12]. The cholesterol (C27 H46 O) and oleate (C18 H34 O2 ) are connected to each other through
ester bonds (R±COO±R0 ). It is known that the ester bonds can play a great role in accumulating cholesterol in atheroma [13]. We examined changes of chemical bonds in cholesteryl oleate by Raman spectroscopy and Fourier transform infrared microspectroscopy (FT-IR) as a function of the exposure time of the IRFELs. 2. Experiment Samples of cholesteryl oleate were prepared from dissolution of cholesteryl oleate (Sigma:#C9253) into CCl4 (6.0 ´ 10ÿ2 mol/l). Cholesteryl oleate ®lms for the FEL exposure, FT-IR and Raman spectra measurements were formed on BaF2 crystal substrates (13 mm in diameter and 1 mm in thickness) by coating with 10 ll of the solution on the substrates. The FEL wavelength was maintained to be at 5.79 lm (1727 cmÿ1 ) which is close to stretching vibrations of ester bonds. The FEL was focused with a ZnSe objective lens. The average power (Pex ) was 3.0 mW. The exposed area can be estimated to be 500 lm in radius. The FEL incident angle (h) was 60°. The FEL exposure experiments were carried out in air at room temperature. The FEL exposure times to the sample surface were 10 s, 1 and 5 min. The exposed points were observed through an inverted microscope combined with a CCD-camera (NIKON, Japan). Raman spectra in 2±5 lm area of the sample were measured with a Raman microspectroscope (RAMANOR U-1000, ATAGO BUTSAN-JOBIN YVON) by excitation of a 514.5 nm Ar ion laser (SPECTRA-PHYSICS, Stabilite R 2017) with 100 mW. No damage of the sample surface due to the Ar ion laser was observed during Raman spectra measurements. The Raman measurement dissolution was 2.0 cmÿ1 . The FT-IR transmittance spectra were measured with a FT-IR microscope (HORIBA, FT-520) in dry air at room temperature. The measurement resolution was 4.0 cmÿ1 . 3. Results and discussions
Fig. 1. Molecular structure of cholesteryl oleate (C45 H78 O2 ). Oleate (R) with a long chain of C±C bonds and cholesterol (R0 ) molecules are connected by the ester bond (R±COO±R0 ).
Fig. 2 shows the FT-IR spectra of cholesteryl oleate before (not exposed) and after exposure of
Y. Fukami et al. / Nucl. Instr. and Meth. in Phys. Res. B 144 (1998) 229±235
5.79 lm FELs for 10 s, 1 and 5 min, respectively. The characteristic peak of the ester bonds (RCOOR0 ) was observed at 1737 cmÿ1 . The peak at 1737 cmÿ1 decreased and showed the asymmetric change with broadening to lower frequency as the exposure time increased. The intensity of the broad band as shown by the arrow in 1150±1210 cmÿ1 due to asymmetric stretching of C±O±C of ester (RCOOR0 ) with small contribution of stretching of C±C and wagging of ±CH3 in ±CH(CH3 )2 also decreased. The intensity decrease of the peak at 1737 cmÿ1 showed decrease in the amount of ester bonds, i.e. dissociation of cholesteryl oleate. The dissociation can be con®rmed by marked decrease in the broad band as pointed by the arrow in 1150±1210 cmÿ1 , which comes mainly from dissociation of the C±O±C bonds connecting between cholesterol and oleate mole-
231
cules in cholesteryl oleate. The asymmetric change of the peak near 1734 cmÿ1 can be considered to come from oxidation processes from ester (RCOOR0 ) to aldehyde (RCHO), carboxyl (RCOOH) or ketone (R2 C@O) whose absorption is observed at frequencies lower than 1734 cmÿ1 . Fig. 3 shows the absorption spectra in 1500± 1750 cmÿ1 and their changes depending on the FEL exposure time. The marked chemical bond changes from the ester bonds to carboxylate (RCOOÿ ) can be observed in 1550±1650 cmÿ1 . Especially broad but very clear increase in the absorption in 1620±1700 cmÿ1 corresponds to synthesis of ketone (R2 C@O). The dissociation of the C±O±C bonds produces directly carboxylate (RCOOÿ ) and ketone (R2 C@O) at the edge of oleate and that of cholesterol, respectively. These chemical changes from ester bonds also con®rm
Fig. 2. FT-IR spectra of cholesteryl oleate before and after exposure of 5.79 lm FELs for 10 s, 1 and 5 min, respectively. Some peaks indicated by the arrows (a), (b) and (c) show marked changes after exposure of the IRFEL. The symbols of (st.) denote a stretching, (bend.) a bending, (sciss.) a scissoring, (s.) symmetric, (as.) an asymmetric, (dg.) degenerated vibrations. The dotted line denotes the wavenumber of 5.79 lm (1727 cmÿ1 ) IRFEL.
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Fig. 3. FT-IR spectra of cholesteryl oleate in 1500±1750 cmÿ1 . The broad absorption at 1660 cmÿ1 comes from synthesis of ketone (R2 C@O).
that dissociation of the C±O±C bonds takes place after the FEL exposure. Fig. 4 shows Raman spectra and their changes during exposure of the FELs. In the Raman spectrum in Fig. 4(a), ester bonds show a weak Raman peak at 1735 cmÿ1 . The absorption of ester bonds was observed at 1734±1737 cmÿ1 in the
FT-IR measurement. This discrepancy between them comes from the dierence in their dissolution, and there is no physical meaning in the discrepancy. The Raman peaks at 1200±1400 cmÿ1 can be assigned to the overlap of bending of ±CH in alkenes (>C@C<) and symmetric stretching of ± ÿ1 COÿ 2 in carboxylate, and the peak at 1670 cm double bonds of C@C. The characteristic Raman bands in 2800±3000 cmÿ1 are contributed by stretching vibrations of ±CH, ±CH2 and ±CH3 in cholesteryl oleate. The sharp Raman peak at 3010 cmÿ1 comes from stretching of ±CH in alkenes (>C@C<). The decrease of the Raman bands related to stretching of ±CHn (n 1,2,3), C@C and C@O in ester and bending of ±CH3 were observed after the FEL exposure. After 5 min exposure, some Raman bands at 1300 and in 1320± 1340 cmÿ1 and a broad Raman peak at 2790 cmÿ1 related to further chemical change of ester, i.e. the synthesis of aldehyde (RCHO) were observed in Fig. 4(d). The eects of FEL exposure on the Raman bands near 1300 cmÿ1 were observed as broadening to higher frequency due to increase of aldehyde (RCHO) or carboxylate (RCOOÿ ) synthesized by dissociation of ester bonds. Fig. 5 shows schematic explanation of change of the chemical bonds related to the ester bonds by exposure of 5.79 lm FELs. We found two processes for dissociation of cholesteryl oleate molecules as shown in the processes (a) and (b). The R± C±O±R0 bonds, which are parts of ester bonds (RCOOR0 ) and connect between oleate (R) and cholesterol (R0 ) molecules, respectively dissociate into (a) (carboxylate: RCOOÿ + ±R0 ) and (b) (RCO± + ±OR0 ) as shown in Fig. 5(a) and (b). The dissociated (RCOOÿ + ±R0 ) bonds are oxidized to carboxylic acid (RCOOH) with ketone (R2 C@O) as shown in Fig. 5(c), and the dissociated (RCO± + ±OR0 ) bonds are hydrogenated and/or oxidized to both aldehyde (RCHO) with ketone (R2 C@O) and carboxylate (RCOOÿ ) with ketone (R2 C@O) as shown in Figs. 5(d) and 5(e). The origin of oxidation of the ester bonds has not yet been clari®ed. We speculate that active oxygen or radical oxygen (O*) and radical hydrogen (H*) can be generated through some photochemical reactions induced by strong excitation of the IRFELs
Y. Fukami et al. / Nucl. Instr. and Meth. in Phys. Res. B 144 (1998) 229±235
233
Fig. 4. Raman spectra of cholesteryl oleate before and after exposure of 5.79 lm FELs for 10 s, 1 and 5 min, respectively. The symbols of (st.) denote a stretching, (bend.) a bending, (s.) symmetric, (as.) an asymmetric vibrations. The Raman spectra were excited by a 514.5 nm Ar ion laser at room temperature.
Fig. 5. Schematic explanation of dissociation of ester bonds and syntheses of some functional groups through chemical changes induced by exposure of the IRFEL.
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Y. Fukami et al. / Nucl. Instr. and Meth. in Phys. Res. B 144 (1998) 229±235
(IR-photochemical reactions). One of them can be considered to be IR-photochemical dissociation of H2 O due to excitation of bending vibrations of H±O±H (1640 cmÿ1 ) by the 5.79 lm FELs (1727 cmÿ1 ). Finally let us discuss the thermal eect due to the FEL exposure to cholesteryl oleate. We assume the heat insulation to estimate the maximum temperature Tmax of cholesteryl oleate during exposure time. The Tmax can be calculated by Tmax TRT Pex tex 1 ÿ eÿad cos h= 4:2pr2 dqCv 1 where TRT is room temperature (27°C), Pex is the average power of FEL (3 mW), tex is the exposure time, a is the absorption coecient (20 cmÿ1 ), h is the incident angle of the FEL (60°), d is the thickness of cholesterly oleate ®lms (200 lm), r is the radius of exposed area (500 lm), q is the density of cholesterly oleate (1.052 g/cm3 ), Cv is the speci®c heat (0.35±0.5 cal/g°C). Using Eq. (1), we can obtain the maximum temperature Tmax at each exposure time, Tmax 27±30°C for 10 s, Tmax 58±60°C for 1 min and Tmax 380±400°C for 5 min. For example, the melting point (Tm ) and the boiling point (Tb ) of cholesterolare are respectively 149°C and 360°C. The Tmax ( 380± 400°C) obtained in the 5 min exposure is higher than Tb . However, in the microscopic obsevation of the exposure experiments we did not observe ablation or evaporation of the ®lms but its melting. This shows that the actual maximum temperature may be lower than the calculated Tmax due to the thermal conduction. Therefore, thermal eects such as ablation or evaporation above the boiling point (T>360°C) can be considered not to be related to dissociation of the ester bonds observed in this study. However, the thermal eect such as temperature rise and melting probably enhance their chemical processes such as oxidation and/or hydrogenation of the dissociated ester bonds. Other candidates for the mechanisms can be considered to understand the observed eective dissociation of the ester bonds and their chemical changes. Further advanced studies using some dierent wavelength FELs are needed to solve the
exact mechanism based on the photochemical or photodynamical interactions between molecules and the FELs [14]. 4. Conclusions Chemical bond-changes of cholesteryl oleate in infrared free electron laser (IRFEL) photodynamic processes were investigated by FT-IR and Raman spectroscopic measurements. We found that the exposure of 5.79 lm-FELs induces not only dissociation of the ester bonds (RCOOR0 ) but also the chemical change of the ester bonds to aldehyde (RCHO), carboxylic acid (RCOOH), carboxylate (RCOOÿ ) or ketone (R2 C@O). We think that the chemical changes might be enhanced by active oxygen and hydrogen dissociated from cholesteryl oleate, included water or air at the irradiation portion. We need further study of chemical processes of other cholesteryl esters under the FEL exposure and chemometrical analyses of the IR spectra. Acknowledgements The authors would like to acknowledge Dr. T. Tomimasu and Dr. A. Nagai of the FELI, Inc. and Prof. Y. Hayashi of Osaka Prefecture University for encouragement of this research. They would thank the late Prof. N. Owaki for planning of the FEL research projects. References [1] T. Tomimasu, K. Saeki, Y. Miyauchi, E. Oshita, S. Okuma, K. Wakita, A. Kobayashi, T. Suzuki, A. Zako, S. Nishihara, A. Koga, K. Wakisaka, H. Tongu, A. Nagai, M. Yasumoto, Nucl. Instr. and Meth. A 375 (1996) 626. [2] A. Kobayashi, K. Saeki, E. Oshita, S. Okuma, K. Wakita, A. Zako, A. Koga, Y. Miyauchi, A. Nagai, M. Yasumoto, T. Tomimasu, Nucl. Instr. and Meth. A 375 (1996) 317. [3] K. Awazu, S.L. Jacques, Proceedings of the 12th International Symposium on Analytical Bioscience, 1997, pp. 92± 93. [4] G. Edwards et al., Nature 371 (1994) 416. [5] X.G.D. Dimitrios, K.C. Kennedy, R.H. Pottier, Amer. J. Pathology 136 (1990) 891.
Y. Fukami et al. / Nucl. Instr. and Meth. in Phys. Res. B 144 (1998) 229±235 [6] J. Bedwell, A.J. MacRobert, D. Phillips, S.G. Bown, Br. J. Cancer 65 (1993) 818. [7] C.S. Loh, J. BeA, J. MacRobert, N. Krasner, D. Phillips, S.G. Bown, Br. J. Cancer 66 (1992) 452. [8] X.-Y. He, S.L. Jacques, S.L. Thomsen, Proc. Soc. PhotoOpt. Instr. Eng. (SPIE) 2133 (1994) 18. [9] T. Takano, K. Ananuma, J. Kimura, S. Ohkuma, Acta Histochem Cytochem 1986, 19 (1986) 135. [10] J. Hayashi, T. Saito, K. Aizawa, Lasers Surg. Med. 21 (1997) 287.
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[11] K. Awazu et al., these Proceedings, International Symposium on Free Electron Laser Facilities and Applications, FELFA'98, Kyoto, 1998, Nucl. Instr. and Meth. B 144 (1998) 225. [12] D. Voet, J.G. Voet, Biochemistry, 2nd ed., Wiley, New York, 1995. [13] T. Saito, J. Hayashi, H. Sato, H. Kawabe, K. Aizawa, Med. Electron Microscopy 29 (1997) 137. [14] Y. Maeda, Y. Fukami, K. Awazu, Photochem. Photobiology, to be submitted.
Raman and FT-IR studies of photodynamic processes of cholesteryl oleate using IRFELs Yuko Fukami a, Yoshihito Maeda a
a,*
, Kunio Awazu
b
Department of Materials Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan b Free Electron Laser Research Institute, Inc. 2-9-1 Tsuda-Yamate, Hirakata, Osaka, 573-01, Japan
Abstract Chemical bond-changes of cholesteryl oleate in infrared free electron laser (IRFEL) photodynamic processes were examined by Raman spectroscopy and Fourier transform infrared microspectroscopy (FT-IR) as a function of the FEL exposure time. We found that the exposure of 5.79 lm-FELs induces not only dissociation of ester (RCOOR0 ) bonds but also the chemical changes from the ester (RCOOR0 ) bonds to aldehyde (RCHO), carboxylic acid (RCOOH), carboxylate (RCOOÿ ) or ketone (R2 C@O) bonds. Ó 1998 Published by Elsevier Science B.V. All rights reserved. PACS: 87.60.-f; 87.70.-k; 33.20.Fb Keywords: Free electron laser; Cholesterol-ester; Photodynamic therapy; Ester
1. Introduction The Free Electron Laser (FEL) has wavelength tunability and novel pulse structures (micro and macropulses) [1,2]. These great advantages are expected to induce some biologic level of interactions between photons and living tissues. The average power of the FEL is sucient for most forms of thermal coagulation therapy and photodynamic therapy (PDT) which is a light-activated chemotherapy for cancer and other indications [3±8]. In advanced atherom atherosclerosis, a large amount of lipids, particularly cholesterol esters, accumu-
* Corresponding author. Tel.: +81 722 54 9709; fax: +81 722 54 9931; e-mail: [email protected].
lates on the arterial wall. In more advanced cases, the lipids also accumulate in the interstitial spaces [9]. Therefore, the PDT is being applied as a less invasive method for eective removal of cholesteryl ester from atheroma using mono-L-aspartyl chlorin e6 (NPe6) as a photoresponsive material and a diode laser of 664 nm in wavelength as an excitation light [10]. Recently, Awazu et al. employed an infrared free electron laser (IRFEL) which is tuned to 5.75 lm of the wavelength corresponding to stretching vibrations of ester bonds to achieve direct dissociation of cholesteryl oleate and removal of cholesterol esters accumulated in rabbit artery without injection of additional chemicals [11]. They obtained very interesting results of wavelength sensitive removal processes in the IRFEL exposure
0168-583X/98/$ ± see front matter Ó 1998 Published by Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 5 8 3 X ( 9 8 ) 0 0 3 6 0 - 7
230
Y. Fukami et al. / Nucl. Instr. and Meth. in Phys. Res. B 144 (1998) 229±235
experiments to cholesteryl oleate and albumin. These photodynamic processes are of great importance for biomedical applications such as the PDT to selective removal of cholesterol from living tissues. However, details of physicochemical mechanism have not yet been clari®ed. In this study, cholesteryl oleate was employed as a model material for actual atheroma to understand photodynamic processes induced by the IRFEL exposure. The molecular structure of cholesteryl oleate (C45 H78 O2 ) is shown in Fig. 1 [12]. The cholesterol (C27 H46 O) and oleate (C18 H34 O2 ) are connected to each other through
ester bonds (R±COO±R0 ). It is known that the ester bonds can play a great role in accumulating cholesterol in atheroma [13]. We examined changes of chemical bonds in cholesteryl oleate by Raman spectroscopy and Fourier transform infrared microspectroscopy (FT-IR) as a function of the exposure time of the IRFELs. 2. Experiment Samples of cholesteryl oleate were prepared from dissolution of cholesteryl oleate (Sigma:#C9253) into CCl4 (6.0 ´ 10ÿ2 mol/l). Cholesteryl oleate ®lms for the FEL exposure, FT-IR and Raman spectra measurements were formed on BaF2 crystal substrates (13 mm in diameter and 1 mm in thickness) by coating with 10 ll of the solution on the substrates. The FEL wavelength was maintained to be at 5.79 lm (1727 cmÿ1 ) which is close to stretching vibrations of ester bonds. The FEL was focused with a ZnSe objective lens. The average power (Pex ) was 3.0 mW. The exposed area can be estimated to be 500 lm in radius. The FEL incident angle (h) was 60°. The FEL exposure experiments were carried out in air at room temperature. The FEL exposure times to the sample surface were 10 s, 1 and 5 min. The exposed points were observed through an inverted microscope combined with a CCD-camera (NIKON, Japan). Raman spectra in 2±5 lm area of the sample were measured with a Raman microspectroscope (RAMANOR U-1000, ATAGO BUTSAN-JOBIN YVON) by excitation of a 514.5 nm Ar ion laser (SPECTRA-PHYSICS, Stabilite R 2017) with 100 mW. No damage of the sample surface due to the Ar ion laser was observed during Raman spectra measurements. The Raman measurement dissolution was 2.0 cmÿ1 . The FT-IR transmittance spectra were measured with a FT-IR microscope (HORIBA, FT-520) in dry air at room temperature. The measurement resolution was 4.0 cmÿ1 . 3. Results and discussions
Fig. 1. Molecular structure of cholesteryl oleate (C45 H78 O2 ). Oleate (R) with a long chain of C±C bonds and cholesterol (R0 ) molecules are connected by the ester bond (R±COO±R0 ).
Fig. 2 shows the FT-IR spectra of cholesteryl oleate before (not exposed) and after exposure of
Y. Fukami et al. / Nucl. Instr. and Meth. in Phys. Res. B 144 (1998) 229±235
5.79 lm FELs for 10 s, 1 and 5 min, respectively. The characteristic peak of the ester bonds (RCOOR0 ) was observed at 1737 cmÿ1 . The peak at 1737 cmÿ1 decreased and showed the asymmetric change with broadening to lower frequency as the exposure time increased. The intensity of the broad band as shown by the arrow in 1150±1210 cmÿ1 due to asymmetric stretching of C±O±C of ester (RCOOR0 ) with small contribution of stretching of C±C and wagging of ±CH3 in ±CH(CH3 )2 also decreased. The intensity decrease of the peak at 1737 cmÿ1 showed decrease in the amount of ester bonds, i.e. dissociation of cholesteryl oleate. The dissociation can be con®rmed by marked decrease in the broad band as pointed by the arrow in 1150±1210 cmÿ1 , which comes mainly from dissociation of the C±O±C bonds connecting between cholesterol and oleate mole-
231
cules in cholesteryl oleate. The asymmetric change of the peak near 1734 cmÿ1 can be considered to come from oxidation processes from ester (RCOOR0 ) to aldehyde (RCHO), carboxyl (RCOOH) or ketone (R2 C@O) whose absorption is observed at frequencies lower than 1734 cmÿ1 . Fig. 3 shows the absorption spectra in 1500± 1750 cmÿ1 and their changes depending on the FEL exposure time. The marked chemical bond changes from the ester bonds to carboxylate (RCOOÿ ) can be observed in 1550±1650 cmÿ1 . Especially broad but very clear increase in the absorption in 1620±1700 cmÿ1 corresponds to synthesis of ketone (R2 C@O). The dissociation of the C±O±C bonds produces directly carboxylate (RCOOÿ ) and ketone (R2 C@O) at the edge of oleate and that of cholesterol, respectively. These chemical changes from ester bonds also con®rm
Fig. 2. FT-IR spectra of cholesteryl oleate before and after exposure of 5.79 lm FELs for 10 s, 1 and 5 min, respectively. Some peaks indicated by the arrows (a), (b) and (c) show marked changes after exposure of the IRFEL. The symbols of (st.) denote a stretching, (bend.) a bending, (sciss.) a scissoring, (s.) symmetric, (as.) an asymmetric, (dg.) degenerated vibrations. The dotted line denotes the wavenumber of 5.79 lm (1727 cmÿ1 ) IRFEL.
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Fig. 3. FT-IR spectra of cholesteryl oleate in 1500±1750 cmÿ1 . The broad absorption at 1660 cmÿ1 comes from synthesis of ketone (R2 C@O).
that dissociation of the C±O±C bonds takes place after the FEL exposure. Fig. 4 shows Raman spectra and their changes during exposure of the FELs. In the Raman spectrum in Fig. 4(a), ester bonds show a weak Raman peak at 1735 cmÿ1 . The absorption of ester bonds was observed at 1734±1737 cmÿ1 in the
FT-IR measurement. This discrepancy between them comes from the dierence in their dissolution, and there is no physical meaning in the discrepancy. The Raman peaks at 1200±1400 cmÿ1 can be assigned to the overlap of bending of ±CH in alkenes (>C@C<) and symmetric stretching of ± ÿ1 COÿ 2 in carboxylate, and the peak at 1670 cm double bonds of C@C. The characteristic Raman bands in 2800±3000 cmÿ1 are contributed by stretching vibrations of ±CH, ±CH2 and ±CH3 in cholesteryl oleate. The sharp Raman peak at 3010 cmÿ1 comes from stretching of ±CH in alkenes (>C@C<). The decrease of the Raman bands related to stretching of ±CHn (n 1,2,3), C@C and C@O in ester and bending of ±CH3 were observed after the FEL exposure. After 5 min exposure, some Raman bands at 1300 and in 1320± 1340 cmÿ1 and a broad Raman peak at 2790 cmÿ1 related to further chemical change of ester, i.e. the synthesis of aldehyde (RCHO) were observed in Fig. 4(d). The eects of FEL exposure on the Raman bands near 1300 cmÿ1 were observed as broadening to higher frequency due to increase of aldehyde (RCHO) or carboxylate (RCOOÿ ) synthesized by dissociation of ester bonds. Fig. 5 shows schematic explanation of change of the chemical bonds related to the ester bonds by exposure of 5.79 lm FELs. We found two processes for dissociation of cholesteryl oleate molecules as shown in the processes (a) and (b). The R± C±O±R0 bonds, which are parts of ester bonds (RCOOR0 ) and connect between oleate (R) and cholesterol (R0 ) molecules, respectively dissociate into (a) (carboxylate: RCOOÿ + ±R0 ) and (b) (RCO± + ±OR0 ) as shown in Fig. 5(a) and (b). The dissociated (RCOOÿ + ±R0 ) bonds are oxidized to carboxylic acid (RCOOH) with ketone (R2 C@O) as shown in Fig. 5(c), and the dissociated (RCO± + ±OR0 ) bonds are hydrogenated and/or oxidized to both aldehyde (RCHO) with ketone (R2 C@O) and carboxylate (RCOOÿ ) with ketone (R2 C@O) as shown in Figs. 5(d) and 5(e). The origin of oxidation of the ester bonds has not yet been clari®ed. We speculate that active oxygen or radical oxygen (O*) and radical hydrogen (H*) can be generated through some photochemical reactions induced by strong excitation of the IRFELs
Y. Fukami et al. / Nucl. Instr. and Meth. in Phys. Res. B 144 (1998) 229±235
233
Fig. 4. Raman spectra of cholesteryl oleate before and after exposure of 5.79 lm FELs for 10 s, 1 and 5 min, respectively. The symbols of (st.) denote a stretching, (bend.) a bending, (s.) symmetric, (as.) an asymmetric vibrations. The Raman spectra were excited by a 514.5 nm Ar ion laser at room temperature.
Fig. 5. Schematic explanation of dissociation of ester bonds and syntheses of some functional groups through chemical changes induced by exposure of the IRFEL.
234
Y. Fukami et al. / Nucl. Instr. and Meth. in Phys. Res. B 144 (1998) 229±235
(IR-photochemical reactions). One of them can be considered to be IR-photochemical dissociation of H2 O due to excitation of bending vibrations of H±O±H (1640 cmÿ1 ) by the 5.79 lm FELs (1727 cmÿ1 ). Finally let us discuss the thermal eect due to the FEL exposure to cholesteryl oleate. We assume the heat insulation to estimate the maximum temperature Tmax of cholesteryl oleate during exposure time. The Tmax can be calculated by Tmax TRT Pex tex 1 ÿ eÿad cos h= 4:2pr2 dqCv 1 where TRT is room temperature (27°C), Pex is the average power of FEL (3 mW), tex is the exposure time, a is the absorption coecient (20 cmÿ1 ), h is the incident angle of the FEL (60°), d is the thickness of cholesterly oleate ®lms (200 lm), r is the radius of exposed area (500 lm), q is the density of cholesterly oleate (1.052 g/cm3 ), Cv is the speci®c heat (0.35±0.5 cal/g°C). Using Eq. (1), we can obtain the maximum temperature Tmax at each exposure time, Tmax 27±30°C for 10 s, Tmax 58±60°C for 1 min and Tmax 380±400°C for 5 min. For example, the melting point (Tm ) and the boiling point (Tb ) of cholesterolare are respectively 149°C and 360°C. The Tmax ( 380± 400°C) obtained in the 5 min exposure is higher than Tb . However, in the microscopic obsevation of the exposure experiments we did not observe ablation or evaporation of the ®lms but its melting. This shows that the actual maximum temperature may be lower than the calculated Tmax due to the thermal conduction. Therefore, thermal eects such as ablation or evaporation above the boiling point (T>360°C) can be considered not to be related to dissociation of the ester bonds observed in this study. However, the thermal eect such as temperature rise and melting probably enhance their chemical processes such as oxidation and/or hydrogenation of the dissociated ester bonds. Other candidates for the mechanisms can be considered to understand the observed eective dissociation of the ester bonds and their chemical changes. Further advanced studies using some dierent wavelength FELs are needed to solve the
exact mechanism based on the photochemical or photodynamical interactions between molecules and the FELs [14]. 4. Conclusions Chemical bond-changes of cholesteryl oleate in infrared free electron laser (IRFEL) photodynamic processes were investigated by FT-IR and Raman spectroscopic measurements. We found that the exposure of 5.79 lm-FELs induces not only dissociation of the ester bonds (RCOOR0 ) but also the chemical change of the ester bonds to aldehyde (RCHO), carboxylic acid (RCOOH), carboxylate (RCOOÿ ) or ketone (R2 C@O). We think that the chemical changes might be enhanced by active oxygen and hydrogen dissociated from cholesteryl oleate, included water or air at the irradiation portion. We need further study of chemical processes of other cholesteryl esters under the FEL exposure and chemometrical analyses of the IR spectra. Acknowledgements The authors would like to acknowledge Dr. T. Tomimasu and Dr. A. Nagai of the FELI, Inc. and Prof. Y. Hayashi of Osaka Prefecture University for encouragement of this research. They would thank the late Prof. N. Owaki for planning of the FEL research projects. References [1] T. Tomimasu, K. Saeki, Y. Miyauchi, E. Oshita, S. Okuma, K. Wakita, A. Kobayashi, T. Suzuki, A. Zako, S. Nishihara, A. Koga, K. Wakisaka, H. Tongu, A. Nagai, M. Yasumoto, Nucl. Instr. and Meth. A 375 (1996) 626. [2] A. Kobayashi, K. Saeki, E. Oshita, S. Okuma, K. Wakita, A. Zako, A. Koga, Y. Miyauchi, A. Nagai, M. Yasumoto, T. Tomimasu, Nucl. Instr. and Meth. A 375 (1996) 317. [3] K. Awazu, S.L. Jacques, Proceedings of the 12th International Symposium on Analytical Bioscience, 1997, pp. 92± 93. [4] G. Edwards et al., Nature 371 (1994) 416. [5] X.G.D. Dimitrios, K.C. Kennedy, R.H. Pottier, Amer. J. Pathology 136 (1990) 891.
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