Journal of Photochemistry and Photobiology, B: Biology, 6 (1990) 93-101
93
P H O T O T H E R M A L S E N S I T I Z E R S : P O S S I B L E U S E IN T U M O R THERAPY* GIULIO JORI
Department of Biology, University of Padova, Via Trieste 75, Padova (Italy) JOHN D. SPIKES
Department of Biology, University of Utah, Salt Lake City, UT 84112 (U.S.A.) (Received October 5, 1989; acccpted December 12, 1989)
Keywords.
Phototherapy, photothermal sensitization, tumors, pulsed photo-
excitation.
Summary Photothermal damage of tissues or endotissular compartments may be induced by pulsed irradiation of either endogenous c h r o m o p h o r e s (e.g. hemoglobin, melanin) or externally added dyes; the latter should have short triplet lifetimes and mainly decay from electronically excited states by nonradiative pathways. Potential photothermal sensitizers are some metallo derivatives of porphyrins and porphyrinoid compounds, azo dyes and triphenylmethane derivatives. These dyes have the additional propert y of significant absorbance at wavelengths longer than 600 nm, which can penetrate deep into biological tissues. Spatial confinement of the photothermal process depends on the absorption coefficient of the phot oexci t ed c h r o m o p h o r e and its thermal relaxation time. Present evidence indicates that the selective photothermal damage of macromolecules or subcellular organelles requires pulsed excitation at picosecond or n a n o s e c o n d regimes, while microsecond or millisecond domains are effective in the case of cells or similar structures. The possible use of photothermal sensitization in the treatment of tumors is briefly discussed.
1. I n t r o d u c t i o n A considerable amount of research is presently being carried out on the use of photosensitized reactions in medicine, particularly in the photodynamic *Paper presented at the Congress on Photodynamie Therapy of Tumours, Sofia, Bulgaria, October, 1989.
Elsevier Sequoia/Printed in The Netherlands
94 therapy (PDT) of tumors [1]. In these processes, the photosensitizer is promoted by light to the long-lived triplet state, which may react directly with adjacent substrate molecules and/or produce cytotoxic oxygen derivatives, such as singlet oxygen or superoxide anion [2]. The efficacy of PDT therefore depends on the selectivity of photosensitizer accumulation in the tumor tissue, and on the rate constant and quantum yield of the reactions between the photogenerated transient species and the nearby cell targets. The photosensitizers most frequently used in clinical and pre-clinical investigations are specific porphyrins or porphyrinoid compounds (e.g. phthalocyanines, chlorins), which possess tumor-localizing and photosensitizing properties [3 ]. Several non-photosensitizing dyes, including metalloporphyrins or metallophthalocyanines with paramagnetic metal ions [4], cyanines [5 ] and azo dyes [6] have been shown to be accumulated and retained by tumors in vivo. These dyes generally show very low fluorescence quantum yields and hence probably decay from the electronically excited to the ground state primarily by non-radiative pathways, releasing their energy in several forms, including heat. Thus the possibility exists of producing local photosensitized hyperthermal effects leading to specific damage of the cells and tissues containing the photosensitizer, while all other cells remain unaffected. In this paper, we briefly discuss the principles of selective photothermal sensitization of biological systems and the conditions under which the technique may be developed. We also review the few published studies in this field.
2. General f e a t u r e s o f the p h o t o t h e r m a l s e n s i t i z a t i o n o f biological systems A large number of current therapeutic applications of lasers exploit photothermal processes [7]. Typical examples are removal of unwanted tissues by photoinduced vaporization or the closing of blood vessels through photothermal denaturation of plasma proteins. In general, the nature of the biological damage after irradiation of a tissue depends on the extent and rate of the temperature rise; Table 1 summarizes the most important alterations observed at the molecular and cell or tissue levels with increasing temperature. However, the nature of the damage and its degree of usefulness for medical applications are dictated by spatial confinement of the heating following laser irradiation: if the latter is prolonged for a sufficient period of time, tissues adjacent to the irradiated area are heated and a large number of structures may be injured. The approaches used to obtain high selectivity of photothermal processes at the molecular, cell and tissue levels have been discussed by Anderson and Parrish [8]. The two main parameters to be considered are the tissue absorption coefficient and its thermal relaxation time. In the first place, incident radiation should, if possible, interact with one specific component of the system (either an endogenous pigment or an exogenously added dye) with much stronger optical absorption at the given wavelength than observed
95 TABLE 1 Molecular and cell or tissue alterations caused by increasing the temperature of biological systems T e m p e r a t u r e (°C) M o l e c u l a r targets
Cell o r tissue c h a n g e s
42-45
Conformational changes in several macrobiomolecules
Alteration of membrane permeability Tissue shrinkage
45-50
Reduction or loss of enzyme activity Lipid gelation
Cell mortality
50-60
Protein and DNA denaturation
Tissue coagulation Closing of vessel lumens
80
Collagen denaturation Formation of gaps in cell membranes
Carbonization of tissues
100
Vaporization of water
Decomposition of tissue constituents Vaporization and ablation of tissues
Rupture of several chemical bonds
for the o t h e r c o m p o n e n t s of the s a m e or s u r r o u n d i n g tissues. Most organic m o l e c u l e s and w a t e r have high e x t i n c t i o n coefficients in the UV region. Nucleic acids and p r o t e i n s exhibit m a x i m a l a b s o r p t i o n in the 2 6 0 - 2 9 0 n m range; b e c a u s e of this, and the significant light s c a t t e r i n g in this region, tissues are only v e r y slightly p e n e t r a t e d b y UV light. IR radiation also p e n e t r a t e s poorly, b e c a u s e o f its s t r o n g a b s o r p t i o n b y water. In the visible s p e c t r a l region, light a b s o r p t i o n is mainly due to bilirubin, h e m o p r o t e i n s and melanin. However, as v e r y little e n d o g e n o u s a b s o r p t i o n o c c u r s b e t w e e n 600 and 1000 nm, this s p e c t r a l r a n g e is k n o w n as the " p h o t o t h e r a p e u t i c w i n d o w " and is c h a r a c t e r i z e d b y m a x i m a l light p e n e t r a t i o n into tissues; thus p h o t o s e n s i t i z e r s a b s o r b i n g t h e s e w a v e l e n g t h s m a y b e efficiently e x c i t e d w i t h o u t a p p r e c i a b l e c o m p e t i t i o n f r o m e n d o g e n o u s cell c h r o m o p h o r e s [7]. Thus the c h o i c e of laser w a v e l e n g t h d e t e r m i n e s b o t h the d e p t h of p e n e t r a t i o n and the d a m a g e d sites. The selectivity o f p h o t o t h e r m a l l y sensitized tissue d a m a g e is also controlled b y the d u r a t i o n o f laser e x p o s u r e , w h i c h m u s t b e c h o s e n so as to minimize h e a t t r a n s f e r f r o m the initially e x c i t e d t a r g e t to the m i c r o e n v i r o n m e n t . The scaling p a r a m e t e r for this p r o b l e m is the t h e r m a l r e l a x a t i o n time o f the target, i . e . the time r e q u i r e d for the central t e m p e r a t u r e o f a gaussian t e m p e r a t u r e distribution with a width equal to the t a r g e t d i a m e t e r to d e c r e a s e b y 50% [9]. This quantity is d e p e n d e n t o n w a v e l e n g t h and is m o d u l a t e d b y various factors, including t h e r m a l conductivity, specific h e a t a n d density of the medium. Typical t h e r m a l r e l a x a t i o n t i m e s o f the o r d e r o f p i c o s e c o n d s and milliseconds h a v e b e e n e s t i m a t e d for albumin and melanin r e s p e c t i v e l y [7, 10].
96 It has been estimated [8, 11 ] that the selective photothermal damage of macromolecules or subcellular organelles requires pulsed excitation with picosecond or n a n o s e c o n d regimes, while microsecond or millisecond domains are effective in the case of cells or vessels and similar small structures respectively. Suitable application of this technique may lead to extreme temperature differences between the excited system and its surroundings. Thus 10 ns pulsed laser irradiation at 620 nm in an aqueous solution of malachite green (a triarylmethane dye characterized by very fast internal conversion to the ground state) causes a t em perat ure increase of 130 °C in a 10 nm sphere of water around the dye molecule [12]; on pi cosecond pulse irradiation, the malachite green molecule undergoes transient heating to a temperature greater than 800 °C [13]. However, under these conditions, vaporization and shock-wave effects may cause damage at relatively large distances from the light-absorbing structure. For most mammalian tissues, on irradiation with laser pulse widths selected to make thermal diffusion negligible, a tem pe r at ur e rise of light-absorbing targets of about 40 °C is sufficient to induce significant damage while keeping the overall process confined to within satisfactory spatial limits.
3. Open problems in biomedical applications o f photothermal sensitization In spite of significant advances in our knowledge on the optical and thermal properties of biological tissues, prediction of the detailed effects of laser-induced hyperthermia is still a difficult problem. Not many results are available so far, and their interpretation is often complex due to the inherent inhomogeneity of i n v i v o systems, a situation that hinders refinement of presently available theoretical models [14]. Thus the way in which optical and thermal parameters vary with temperature and tissue state is still only partially defined. For example, during interstitial irradiation of tissues, charring in the vicinity of the fiber tips is often observed: hence, a dramatic rise in scattering and absorption coefficients will be e x p e c t e d [15]. At the same time, thermal conductivity is likely to change as the state of the tissue is altered, e.g. by denaturation of proteins or changes in the proportion of liquid present. In this connection, a major role is played by the degree and rate of perfusion, which normally has a cooling effect since it removes excess heat. Although perfusion initially increases as temp er a t ur e rises, so that the water content of the tissue increases, it falls when the t em pe r at ur e rise b e c o m e s too great [16]; consequently, with higher temperatures, the water content of a tissue decreases and its thermal conductivity drops [ 15 ]. Other factors, whose actual influence on the mechanism and efficiency of photothermal sensitization is still largely unknown, are the extent of oxygenation, the biochemical and conformational features of cells, the degree of compartmentalization of tissues, which creates a num ber of boundaries
97 at a finite distance from the center of the irradiated area, and some physical aspects (e.g. the symmetry with which light penetrates into a tissue from any given point). Therefore there is urgent need for both theoretical and experimental approaches which will allow a more precise evaluation of the separate contribution of individual factors in the overall process and the positive or negative effects of their mutual interactions.
4. E x p e r i m e n t a l s t u d i e s o f p h o t o t h e r m a l s e n s i t i z a t i o n o f biological systems On the basis of the above considerations, it may be argued that an efficient photothermal sensitizer must have a large molar extinction coefficient (preferably in a spectral region where few other tissue c h r o m o p h o r e s absorb), undergo very rapid de-excitation to the ground state mainly via thermal conversion and be photostable. As a first step, it may be assumed that dyes or cell c o m p o n e n t s which show no photochemical activity and which have a very weak fluorescence emission are potential candidates as photothermal sensitizers.
4.1. Endogenous phototherrnal sensitizers Essentially all research on sensitized photothermal effects in biology has b een carried out using e ndoge nous chromophores. It was observed early in the history of laser photomedicine that lasers could be used to destroy small blood vessels [ 17 ]. The immediate effects of irradiation on capillaries include aggregation of erythrocytes, endothelial and transmural necrosis and rupture of the vessel wall with hemorrhaging [ 18]. Typical examples of photothermal sensitization of biological structures containing oxyhemoglobin and/or melanin are give in Table 2. Remarkably, a choice of irradiation wavelength and duration of the light pulse, as discussed earlier, induces specific phot odam age to a restricted n u m b e r of targets. Moreover, the choice of wavelength affects the depth of the observed tissue response: thus the irradiation of cutaneous pigments by a Q-switched Nd:YAG laser at 1064, 532 and 355 nm induces the disruption of melanosomes in all cases, but the effect penetrates d e e p e r into the skin at the longer wavelengths [23]. The importance of the pulsed irradiation mo d e is emphasized by the observation that the exposure of fair Caucasian skin to 488 or 514 nm light from a continuous-wave argon laser causes necrosis of the epidermis and superficial dermis, with no selective effects on blood vessels [19]. Also, when pigmented guinea-pig skin is illuminated with 40 ns pulses from a ruby laser, 750 ns pulses from a dye laser and 400 ns pulses from a dye laser, melanosomal photodisruption occurs only with the first two lasers. The thermal relaxation time of the melanosomes is approximately 0 . 5 - 1 . 0 ps; thus photodisruption occurs only with laser pulses shorter than this value [24]. The mechanism(s) by which melanosomes are disrupted by short laser pulses has not been established,
98
c~
.~
0
~
0
0
N~
~7
~ LO
~ 0
0 LOC~O
0
E
~
99 but shock waves or cavitation produced by the rapid thermal change may be involved.
4.2. E x t e r n a l l y a d d e d p h o t o t h e r m a l s e n s i t i z e r s The exogenous photothermal sensitizers of most interest for in vivo applications should have large molar absorption coefficients in the red spectral region, i.e. corresponding to the phototherapeutic window where the incident light is inefficiently absorbed by most endogenous chromophores of cells and tissues. Unfortunately, very few results of this type of photothermal sensitization have been published. Illumination of enzyme-malachite green covalent complexes with 10 ns pulses of 620 nm light results in enzyme inactivation [ 12 ]. Enzymes irradiated in the presence of free malachite green (which has a very short triplet lifetime, and is hence a very poor photochemical sensitizer) are not affected. It is suggested that the loss of enzyme activity is due to photothermal processes. This may be true; however, it has been shown that the triplet lifetime of malachite green is significantly increased when it is bound to macromolecules [ 25 ]. Other triarylmethane derivatives, such as Victoria Blue BO, are selectively accumulated by the mitochondria of tumor cells and cause cell death when irradiated with an argon-pumped dye laser [26]. Thus these dyes may also be acting as photothermal sensitizers, unless their interaction with submitochondrial structures modifies their photophysical properties. Lastly, it should be mentioned that several metalloporphyrins and metallophthalocyanines [4] and azo dyes [6] (e.g. Evans blue, Trypan blue, etc.) which are non-fluorescent and devoid of appreciable photochemical activity, are excellent tumor localizers. Therefore there is a good possibility that the selective photothermal sensitization of tumors could be obtained i n vivo by using these dyes. Since these photoprocesses probably do not require oxygen, their use may be advantageous, especially in hypo-oxygenated regions of tumors. Photothermal reactions involving exogenous photosensitizers may have application in the treatment of other diseases, in particular, the destruction of arterial plaques. Certain porphyrins as well as other dyes are taken up to a higher concentration by plaques than by the normal arterial wall [27]. Thus the illumination of plaques selectively stained with a photothermal sensitizer using fiber optics and an appropriate laser might result in thermal ablation of the plaque, with relatively little damage to the normal arterial wall.
Acknowledgments This work was supported in part by the Consiglio Nazionale delle Ricerche under the Italy-U.S.A. cooperative program in science (Contract 89.02947.04), the American Cancer Society (Grant PDT-259D), the DOD/SD10 Medical Free Electron Laser Program and the Utah Laser Institute.
100
References 1 T. J. Dougherty, Photosensitizers: therapy and detection of malignant tumors, Photochem. Photobiol., 45 (1987) 8 7 9 - 8 8 9 . 2 C. S. Foote, Photosensitized oxidation and singlet oxygen: consequences in biological systems, in W. A. Pryor (ed.), Free Radicals in Biology, Vol. II, Academic Press, New York, 1976, pp. 8 5 - 1 3 3 . 3 S. Wan, J. A. Parrish, R. R. Anderson and M. Madden, Transmittance of nonionizing radiation in h u m a n tissues, Photochem. Photobiol., 34 (1981) 6 7 9 - 6 8 1 . 4 B. C. V~rflson and J. E. Van Lier, Radiolabelled photosensitizers for t u m o u r imaging and photodynamic therapy, J. Photochem. Photobio£ B, 3 (1989) 459--463. 5 G. Ara, L. Lewandowski and A. R. Oseroff, EDKC selectively photosensitizes respiration of malignant cells in vitro, in T. Hasan (ed.), Pro¢. on Advances in Photochemotherapy, Vol. 997, SPIE, Washington DC, 1989, pp. 6 2 - 6 9 . 6 R.J. Goldacre and B. Sylven, On the access of blood-borne dyes to various t u m o u r regions, Br. J. Cancer, 16 (1962) 306--322. 7 J. L. Boulnois, Photophysical processes in recent medical laser developments: a review, Lasers Med. Sci., 1 (1986) 4 7 - 6 6 . 8 R. R. Anderson and J. A. Parrish, Selective photothermolysis: precise microsurgery by selective absorption of pulsed radiation, Science, 220 (1983) 524--527. 9 L. Svaasand, T. Boerslid and M. Oeveraasen, Thermal and optical properties of living tissue, Lasers Surg. Med., 5 (1985) 5 8 9 - 6 0 2 . 10 A. J. Welch, The thermal response of laser-irradiated tissue, IEEE J. Quantum Electron., 12 (1984) 1 4 7 1 - 1 4 7 5 . 11 J. A. Parrish, R. R. Anderson, T. Harrist, B. Paul and G. F. Murphy, Selective thermal effects with pulsed irradiation from lasers: from organ to organelle, J. Invest. Dermatol., 80 (1983) 75s--80s. 12 D. G. Jay, Selective destruction of protein function by chromophore-assisted laser inactivation, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 5 4 5 4 - 5 4 5 8 . 13 T. Robl and A. Seilmeier, Ground-state recovery of electronically-excited malachite green via transient vibrational heating, Chem. Phys. Lett., 147 (1988) 5 4 4 - 5 5 0 . 14 B. C. Wilson and M. S. Patterson, The physics of photodynamic therapy, Phys. Meal. Biol., 31 (1986) 3 2 7 - 3 6 0 . 15 P. Whiting, J. Dowden and P. Kapadia, A mathematical analysis of the results of experiments on rat livers by local laser hyperthermia, Lasers Med. Sci., 4 (1989) 5 5 - 6 4 . 16 H. C. Barrett, The Regulation of Body Temperatures, Hafner Publishing Co., New York, 1968, pp. 1 0 9 - 1 9 2 . 17 H. Solomon, L. Goldman, B. Henderson, D. Richfield and M. Franzen, Histopathology of the laser t r e a t m e n t of port-wine lesions, J. Invest. Dermatol., 50 (1968) 1 4 1 - 1 4 6 . 18 R. R. Anderson and J. A. Parrish, Microvasculature can be selectively damaged using dye lasers: a basic theory and experimental evidence in h u m a n skin, L a s e r s Surg. Med., 1 (1981) 2 6 3 - 2 7 6 . 19 J. Greenwald, S. Rosen, R. R. Anderson, T. Harrist, F. MacFarland, J. Noe and J. A. Parrish, Comparative histological studies of the tunable dye (at 577 nm) laser and argon laser: the specific vascular effects of the dye laser, J. Invest. Derrnatol., 77 (1981) 3 0 5 - 3 1 0 . 20 J. G. Morelli, O. T. Tan, J. Garden, R. Margolis, Y. Seki, J. Bol, J. M. Carney, i~. R. Anderson, H. Furumoto and J. A. Parrish, Tunable dye laser (577 nm) treatment of port wine stains, Lasers Surg. Med., 6 (1986) 9 4 - 9 9 . 21 G. Ara, R. Anderson, K. Mandel and A. R. Oseroff, Absorption of nsec photoradiation of melanosomes generates acoustic waves and induces pigmented m e l a n o m a cell toxicity, Photochem. Photobiol., 47 (1988) 37S. 22 L. L. Polla, R. J. Margolis, J. S. Dover, D. Whitaker, G. F. Murphy, S. L. Jacques and R. R. Anderson, Melanosomes are a primary target of Q-switched ruby laser irradiation in guinea pig skin, J. Invest. Derrr~tol., 89 (1987) 2 8 1 - 2 8 6 .
101 23 R. R. Anderson, R. J. Margolis, S. Watenabe, T. Flotta, G. J. Hruza and J. S. Dover, Selective photothermolysis of cutaneous pigmentation by Q-switched Nd:YAG laser pulses at 106.4, 532 and 355 nm, J. Invest. Dermatol., 93 (1989) 28--32. 24 J. S. Dover, L. L. Polla, R. J. Margolis, D. Whitaker, S. Watenabe, G. F. Murphy, J. A. Parrish and R. R. Anderson, Pulse width dependence of pigment cell damage at 694 nm in guinea pig skin, in S. N. Joffe, J. A. Parrish and R. S. Scott (eds.), Proc. o n L a s e r s i n M e d i c i n e , Vol. 712, SPIE, Washington DC, 1987, pp. 200-205. 25 G. Oster, Dye binding to high polymers, J.. P o l y m . Sci., 36 (1955) 235-244. 26 K. Wadwa, S. Smith and A. R. Oseroff, Cationic triarylmethane photosensitizers for selective photochemotherapy, in T. Hasan (ed.), Proc. on A d v a n c e s i n P h o t o c h e m o t h e r a p y , Vol. 997, SPIE, Washington DC, 1989, pp. 154-161. 27 A. Vison~, V. Cuomo, L. Lusiani, A. Pagnan and G. Jori, Accumulation of zinc-phthalocyanine in the aorta of atherosclerotic rabbits, Lasers Med. Sci., 4 (1989) 167-170.
93
P H O T O T H E R M A L S E N S I T I Z E R S : P O S S I B L E U S E IN T U M O R THERAPY* GIULIO JORI
Department of Biology, University of Padova, Via Trieste 75, Padova (Italy) JOHN D. SPIKES
Department of Biology, University of Utah, Salt Lake City, UT 84112 (U.S.A.) (Received October 5, 1989; acccpted December 12, 1989)
Keywords.
Phototherapy, photothermal sensitization, tumors, pulsed photo-
excitation.
Summary Photothermal damage of tissues or endotissular compartments may be induced by pulsed irradiation of either endogenous c h r o m o p h o r e s (e.g. hemoglobin, melanin) or externally added dyes; the latter should have short triplet lifetimes and mainly decay from electronically excited states by nonradiative pathways. Potential photothermal sensitizers are some metallo derivatives of porphyrins and porphyrinoid compounds, azo dyes and triphenylmethane derivatives. These dyes have the additional propert y of significant absorbance at wavelengths longer than 600 nm, which can penetrate deep into biological tissues. Spatial confinement of the photothermal process depends on the absorption coefficient of the phot oexci t ed c h r o m o p h o r e and its thermal relaxation time. Present evidence indicates that the selective photothermal damage of macromolecules or subcellular organelles requires pulsed excitation at picosecond or n a n o s e c o n d regimes, while microsecond or millisecond domains are effective in the case of cells or similar structures. The possible use of photothermal sensitization in the treatment of tumors is briefly discussed.
1. I n t r o d u c t i o n A considerable amount of research is presently being carried out on the use of photosensitized reactions in medicine, particularly in the photodynamic *Paper presented at the Congress on Photodynamie Therapy of Tumours, Sofia, Bulgaria, October, 1989.
Elsevier Sequoia/Printed in The Netherlands
94 therapy (PDT) of tumors [1]. In these processes, the photosensitizer is promoted by light to the long-lived triplet state, which may react directly with adjacent substrate molecules and/or produce cytotoxic oxygen derivatives, such as singlet oxygen or superoxide anion [2]. The efficacy of PDT therefore depends on the selectivity of photosensitizer accumulation in the tumor tissue, and on the rate constant and quantum yield of the reactions between the photogenerated transient species and the nearby cell targets. The photosensitizers most frequently used in clinical and pre-clinical investigations are specific porphyrins or porphyrinoid compounds (e.g. phthalocyanines, chlorins), which possess tumor-localizing and photosensitizing properties [3 ]. Several non-photosensitizing dyes, including metalloporphyrins or metallophthalocyanines with paramagnetic metal ions [4], cyanines [5 ] and azo dyes [6] have been shown to be accumulated and retained by tumors in vivo. These dyes generally show very low fluorescence quantum yields and hence probably decay from the electronically excited to the ground state primarily by non-radiative pathways, releasing their energy in several forms, including heat. Thus the possibility exists of producing local photosensitized hyperthermal effects leading to specific damage of the cells and tissues containing the photosensitizer, while all other cells remain unaffected. In this paper, we briefly discuss the principles of selective photothermal sensitization of biological systems and the conditions under which the technique may be developed. We also review the few published studies in this field.
2. General f e a t u r e s o f the p h o t o t h e r m a l s e n s i t i z a t i o n o f biological systems A large number of current therapeutic applications of lasers exploit photothermal processes [7]. Typical examples are removal of unwanted tissues by photoinduced vaporization or the closing of blood vessels through photothermal denaturation of plasma proteins. In general, the nature of the biological damage after irradiation of a tissue depends on the extent and rate of the temperature rise; Table 1 summarizes the most important alterations observed at the molecular and cell or tissue levels with increasing temperature. However, the nature of the damage and its degree of usefulness for medical applications are dictated by spatial confinement of the heating following laser irradiation: if the latter is prolonged for a sufficient period of time, tissues adjacent to the irradiated area are heated and a large number of structures may be injured. The approaches used to obtain high selectivity of photothermal processes at the molecular, cell and tissue levels have been discussed by Anderson and Parrish [8]. The two main parameters to be considered are the tissue absorption coefficient and its thermal relaxation time. In the first place, incident radiation should, if possible, interact with one specific component of the system (either an endogenous pigment or an exogenously added dye) with much stronger optical absorption at the given wavelength than observed
95 TABLE 1 Molecular and cell or tissue alterations caused by increasing the temperature of biological systems T e m p e r a t u r e (°C) M o l e c u l a r targets
Cell o r tissue c h a n g e s
42-45
Conformational changes in several macrobiomolecules
Alteration of membrane permeability Tissue shrinkage
45-50
Reduction or loss of enzyme activity Lipid gelation
Cell mortality
50-60
Protein and DNA denaturation
Tissue coagulation Closing of vessel lumens
80
Collagen denaturation Formation of gaps in cell membranes
Carbonization of tissues
100
Vaporization of water
Decomposition of tissue constituents Vaporization and ablation of tissues
Rupture of several chemical bonds
for the o t h e r c o m p o n e n t s of the s a m e or s u r r o u n d i n g tissues. Most organic m o l e c u l e s and w a t e r have high e x t i n c t i o n coefficients in the UV region. Nucleic acids and p r o t e i n s exhibit m a x i m a l a b s o r p t i o n in the 2 6 0 - 2 9 0 n m range; b e c a u s e of this, and the significant light s c a t t e r i n g in this region, tissues are only v e r y slightly p e n e t r a t e d b y UV light. IR radiation also p e n e t r a t e s poorly, b e c a u s e o f its s t r o n g a b s o r p t i o n b y water. In the visible s p e c t r a l region, light a b s o r p t i o n is mainly due to bilirubin, h e m o p r o t e i n s and melanin. However, as v e r y little e n d o g e n o u s a b s o r p t i o n o c c u r s b e t w e e n 600 and 1000 nm, this s p e c t r a l r a n g e is k n o w n as the " p h o t o t h e r a p e u t i c w i n d o w " and is c h a r a c t e r i z e d b y m a x i m a l light p e n e t r a t i o n into tissues; thus p h o t o s e n s i t i z e r s a b s o r b i n g t h e s e w a v e l e n g t h s m a y b e efficiently e x c i t e d w i t h o u t a p p r e c i a b l e c o m p e t i t i o n f r o m e n d o g e n o u s cell c h r o m o p h o r e s [7]. Thus the c h o i c e of laser w a v e l e n g t h d e t e r m i n e s b o t h the d e p t h of p e n e t r a t i o n and the d a m a g e d sites. The selectivity o f p h o t o t h e r m a l l y sensitized tissue d a m a g e is also controlled b y the d u r a t i o n o f laser e x p o s u r e , w h i c h m u s t b e c h o s e n so as to minimize h e a t t r a n s f e r f r o m the initially e x c i t e d t a r g e t to the m i c r o e n v i r o n m e n t . The scaling p a r a m e t e r for this p r o b l e m is the t h e r m a l r e l a x a t i o n time o f the target, i . e . the time r e q u i r e d for the central t e m p e r a t u r e o f a gaussian t e m p e r a t u r e distribution with a width equal to the t a r g e t d i a m e t e r to d e c r e a s e b y 50% [9]. This quantity is d e p e n d e n t o n w a v e l e n g t h and is m o d u l a t e d b y various factors, including t h e r m a l conductivity, specific h e a t a n d density of the medium. Typical t h e r m a l r e l a x a t i o n t i m e s o f the o r d e r o f p i c o s e c o n d s and milliseconds h a v e b e e n e s t i m a t e d for albumin and melanin r e s p e c t i v e l y [7, 10].
96 It has been estimated [8, 11 ] that the selective photothermal damage of macromolecules or subcellular organelles requires pulsed excitation with picosecond or n a n o s e c o n d regimes, while microsecond or millisecond domains are effective in the case of cells or vessels and similar small structures respectively. Suitable application of this technique may lead to extreme temperature differences between the excited system and its surroundings. Thus 10 ns pulsed laser irradiation at 620 nm in an aqueous solution of malachite green (a triarylmethane dye characterized by very fast internal conversion to the ground state) causes a t em perat ure increase of 130 °C in a 10 nm sphere of water around the dye molecule [12]; on pi cosecond pulse irradiation, the malachite green molecule undergoes transient heating to a temperature greater than 800 °C [13]. However, under these conditions, vaporization and shock-wave effects may cause damage at relatively large distances from the light-absorbing structure. For most mammalian tissues, on irradiation with laser pulse widths selected to make thermal diffusion negligible, a tem pe r at ur e rise of light-absorbing targets of about 40 °C is sufficient to induce significant damage while keeping the overall process confined to within satisfactory spatial limits.
3. Open problems in biomedical applications o f photothermal sensitization In spite of significant advances in our knowledge on the optical and thermal properties of biological tissues, prediction of the detailed effects of laser-induced hyperthermia is still a difficult problem. Not many results are available so far, and their interpretation is often complex due to the inherent inhomogeneity of i n v i v o systems, a situation that hinders refinement of presently available theoretical models [14]. Thus the way in which optical and thermal parameters vary with temperature and tissue state is still only partially defined. For example, during interstitial irradiation of tissues, charring in the vicinity of the fiber tips is often observed: hence, a dramatic rise in scattering and absorption coefficients will be e x p e c t e d [15]. At the same time, thermal conductivity is likely to change as the state of the tissue is altered, e.g. by denaturation of proteins or changes in the proportion of liquid present. In this connection, a major role is played by the degree and rate of perfusion, which normally has a cooling effect since it removes excess heat. Although perfusion initially increases as temp er a t ur e rises, so that the water content of the tissue increases, it falls when the t em pe r at ur e rise b e c o m e s too great [16]; consequently, with higher temperatures, the water content of a tissue decreases and its thermal conductivity drops [ 15 ]. Other factors, whose actual influence on the mechanism and efficiency of photothermal sensitization is still largely unknown, are the extent of oxygenation, the biochemical and conformational features of cells, the degree of compartmentalization of tissues, which creates a num ber of boundaries
97 at a finite distance from the center of the irradiated area, and some physical aspects (e.g. the symmetry with which light penetrates into a tissue from any given point). Therefore there is urgent need for both theoretical and experimental approaches which will allow a more precise evaluation of the separate contribution of individual factors in the overall process and the positive or negative effects of their mutual interactions.
4. E x p e r i m e n t a l s t u d i e s o f p h o t o t h e r m a l s e n s i t i z a t i o n o f biological systems On the basis of the above considerations, it may be argued that an efficient photothermal sensitizer must have a large molar extinction coefficient (preferably in a spectral region where few other tissue c h r o m o p h o r e s absorb), undergo very rapid de-excitation to the ground state mainly via thermal conversion and be photostable. As a first step, it may be assumed that dyes or cell c o m p o n e n t s which show no photochemical activity and which have a very weak fluorescence emission are potential candidates as photothermal sensitizers.
4.1. Endogenous phototherrnal sensitizers Essentially all research on sensitized photothermal effects in biology has b een carried out using e ndoge nous chromophores. It was observed early in the history of laser photomedicine that lasers could be used to destroy small blood vessels [ 17 ]. The immediate effects of irradiation on capillaries include aggregation of erythrocytes, endothelial and transmural necrosis and rupture of the vessel wall with hemorrhaging [ 18]. Typical examples of photothermal sensitization of biological structures containing oxyhemoglobin and/or melanin are give in Table 2. Remarkably, a choice of irradiation wavelength and duration of the light pulse, as discussed earlier, induces specific phot odam age to a restricted n u m b e r of targets. Moreover, the choice of wavelength affects the depth of the observed tissue response: thus the irradiation of cutaneous pigments by a Q-switched Nd:YAG laser at 1064, 532 and 355 nm induces the disruption of melanosomes in all cases, but the effect penetrates d e e p e r into the skin at the longer wavelengths [23]. The importance of the pulsed irradiation mo d e is emphasized by the observation that the exposure of fair Caucasian skin to 488 or 514 nm light from a continuous-wave argon laser causes necrosis of the epidermis and superficial dermis, with no selective effects on blood vessels [19]. Also, when pigmented guinea-pig skin is illuminated with 40 ns pulses from a ruby laser, 750 ns pulses from a dye laser and 400 ns pulses from a dye laser, melanosomal photodisruption occurs only with the first two lasers. The thermal relaxation time of the melanosomes is approximately 0 . 5 - 1 . 0 ps; thus photodisruption occurs only with laser pulses shorter than this value [24]. The mechanism(s) by which melanosomes are disrupted by short laser pulses has not been established,
98
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~
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~ LO
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99 but shock waves or cavitation produced by the rapid thermal change may be involved.
4.2. E x t e r n a l l y a d d e d p h o t o t h e r m a l s e n s i t i z e r s The exogenous photothermal sensitizers of most interest for in vivo applications should have large molar absorption coefficients in the red spectral region, i.e. corresponding to the phototherapeutic window where the incident light is inefficiently absorbed by most endogenous chromophores of cells and tissues. Unfortunately, very few results of this type of photothermal sensitization have been published. Illumination of enzyme-malachite green covalent complexes with 10 ns pulses of 620 nm light results in enzyme inactivation [ 12 ]. Enzymes irradiated in the presence of free malachite green (which has a very short triplet lifetime, and is hence a very poor photochemical sensitizer) are not affected. It is suggested that the loss of enzyme activity is due to photothermal processes. This may be true; however, it has been shown that the triplet lifetime of malachite green is significantly increased when it is bound to macromolecules [ 25 ]. Other triarylmethane derivatives, such as Victoria Blue BO, are selectively accumulated by the mitochondria of tumor cells and cause cell death when irradiated with an argon-pumped dye laser [26]. Thus these dyes may also be acting as photothermal sensitizers, unless their interaction with submitochondrial structures modifies their photophysical properties. Lastly, it should be mentioned that several metalloporphyrins and metallophthalocyanines [4] and azo dyes [6] (e.g. Evans blue, Trypan blue, etc.) which are non-fluorescent and devoid of appreciable photochemical activity, are excellent tumor localizers. Therefore there is a good possibility that the selective photothermal sensitization of tumors could be obtained i n vivo by using these dyes. Since these photoprocesses probably do not require oxygen, their use may be advantageous, especially in hypo-oxygenated regions of tumors. Photothermal reactions involving exogenous photosensitizers may have application in the treatment of other diseases, in particular, the destruction of arterial plaques. Certain porphyrins as well as other dyes are taken up to a higher concentration by plaques than by the normal arterial wall [27]. Thus the illumination of plaques selectively stained with a photothermal sensitizer using fiber optics and an appropriate laser might result in thermal ablation of the plaque, with relatively little damage to the normal arterial wall.
Acknowledgments This work was supported in part by the Consiglio Nazionale delle Ricerche under the Italy-U.S.A. cooperative program in science (Contract 89.02947.04), the American Cancer Society (Grant PDT-259D), the DOD/SD10 Medical Free Electron Laser Program and the Utah Laser Institute.
100
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