Technology and Health Care, 1 (1994) 219-222 0928-7329/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved

219

Laser diode coupled with optical fiber for applications in photodynamic therapy WaIter Cecchetti a

a, Roberta Biolo b, Giulio Jori b

Dipartimento di Chimica Fisica, Universita di Venezia, s. Marta 2137, 30123 Venice, Italy; b Dipartimento di Biologia, Universita di Padova, via Trieste 75, 35121 Padua, Italy

(Received in final form 12 September 1993)

Abstract A diode laser with roughly 480 mW / cm 2 emission at 776 nm was developed and engineered to obtain a uniform illumination

of tumour cells deposited on Petri dishes having an external diameter of 5 cm. The efficacy of the apparatus in inducing photocytotoxic effects was successfully checked by irradiation of pigmented melanoma cells in the presence of a photosensitizer, Si(IV)-naphthalocyanine, having intense absorption bands at 776 nm. Our results open interesting prospects for extending photodynamic therapy to highly pigmented tumours. Key words: Laser diode; Optical fiber; Photodynamic therapy; Melanoma, pigmented

1. Introduction The spectral region spanning the 670-830 nm wavelength range is especially suitable for the photodynamic therapy of tumours (PDT) owing to the relatively high transparency of most mammalian tissues to far-red light, which ensures the uniform illumination of large tissular volumes [1]. In this field, interesting new prospects are now being opened by the availability of diode lasers having an emission power of the order of 1 Watt. These laser sources are compact, and are characterized by high yield and affordability, as compared with the complex technology and high costs

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typical of the argon-pumped dye lasers which are most frequently used for PDT [2]. However, diode lasers are characterized by an inhomogeneous emitted beam and high divergence, generally with one or more closely-spaced lines, as a consequence of their intrinsic WAFER-type structure. This situation generates several problems when the diode lasers are to be used as PDT sources for in vitro or in vivo experiments. To address these problems, the emitted radiation can be piloted to the target site by multimode optical fibers: the mixing of the propagation mode within the fiber core eliminates the spatial inhomogeneity of the diode emission so that the light distribution at the fiber end has a nearly gaussian shape with a dispersion angle which is proportional to the numerical aperture of the fiber. The

W. Cecchetti et al. / Technology and Health Care 1 (1994) 219-222

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H

--+

SH

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Fig. 1. Coupling scheme of laser diode-pipe housing by optical fiber.

coupling of the SMA 90S connectors to the fiber tips allows the focusing of the diode light onto small sites (Fig. 1). A major difficulty is represented by the low coupling efficiency of the diode emission with an optical fiber when traditional lenses are used (coupling losses may be larger than 80%). 2. Experimental procedures The diode laser (Sony, Heidelberg, Germany) emitted 776 nm light with a power of 480 mW at 1SoC; the emission is characterized by the presence of two closely spaced lines. The radiation was piloted into the optical fiber by a Gradient Index Rod Lens (Melles Griot) having a Numerical Aperture (N.A.) of 0.46, Pitch 0.29, diameter 1.8 mm, length S.4 mm. Such GRIN microlenses are of optical material with a gradient refraction index, hence the radiation follows predetermined pathways. The relative position of the diode and the fiber tips in both the axial and longitudinal directions is critical: in Fig. 2 the curve showing the longitudinal distances for the best coupling between the laser diode-GRIN and GRIN-optical fiber is given. The GRIN is glued inside a small threaded cilinder which is screwed behind a fe-

male SMA connector. The precision of the GRIN positioning to the laser diode was obtained by an XYZ micrometric system. Behind the small cylinder there is a conical insert for the truing of the GRIN with the male SMA connector containing the fiber tip (Fig. 1, sect. A-A'). The optical fiber used was a PCS 600 multimode plastic clad-silice, with N.A. 0.4, core diameter 600 jLm, and length 2.S m. The fiber tips were connected with two male SMA 90S connectors. Cells to be irradiated were deposited on Petri dishes. To obtain a uniform illumination we used the experimental arrangement outlined in Fig. 1. The cell-loaded disc was placed on the top of a cylinder and a plug with a diffusive internal sur-

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d 1 (mm) Fig. 2. GRIN Lens pitch 0.29. coupling length laser diodeoptical fiber. O.F., optical fiber; L.D., laser diode; R.D., rod diameter; d! LD-GRIN, distance; d z GRIN-OF, distance; L, rod length.

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W. Cecchetti et al./Technology and Health Care 1 (1994) 219-222

After incubation cells were washed three times with phosphate-buffered saline and irradiated with the diode laser at 776 nm for 20 min. at a dose-rate of 7.5 mW / cm 2 • The survival of irradiated cells was estimated by colony forming assay [5].

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Fig. 3. Spatial distribution of radiation out of optical fiber and on disk surface. A-A!, amplified section; B, irradiation box; C, cells; DA, dispersion angle; DS, diffusive surface; Dl GRIN-diode length; D2 GRIN-fiber length; G, GRIN lens; LD, laser diode; LDH, laser diode head; GC, gas cooling; PS, power supply; OF; optical fiber; S, 905 SMA connector; FS, female SMA connector; H, pipe housing; P, plug; SH, shutter; RS, reflective surface; XYZ, multi axis translators.

face was screwed onto the disc. A female SMA connector over a manual shutter was fixed below the cylinder. To increase the homogeneity of the illumination field, the inner surface of the cylinder was layered with a reflective material in order to utilize the slopes of the gaussian spot (Fig. 3a). The homogeneity of the laser diode radiation on the surface of the cell-loaded disc was measured and reported in Fig. 3b. The efficacy of our apparatus for irradiating biological systems was checked by using cultured B16 pigmented melanoma cells and a well-known tumour-photosensitizing agent, Si(IV)-naphthalocyanine (SiNc) [3]; this napthalocyanine (kindly supplied by prof. M.A.J. Rodgers, University of Ohio at Bowling Green) has an intense absorption band peaking at 776 nm (Emax = 5.6 . 10 5 M-I cm-I) [4], i.e. matching the emission from the diode laser. Cells were obtained by explantation of a B16 melanoma tumour from C57 mice and were cultivated in DMEM enriched with 10% bovine fetal serum. For irradiation 10 3 cells were deposited on Petri dishes having a diameter of 5 cm and incubated for 18 h at 37°C with 2.56 JLM SiNc incorporated into unilamellar liposomes of DL-a-dipalmitoyl-phosphatidylcholine (DPPC).

3. Results and discussion Our experimental arrangement led to a very efficient coupling of the diode laser with the optical fiber: the energy losses measured at the fiber end were lower than 30%; the proportion of the losses due to reflectance of light by the optical surfaces was around 16%. The spatial distribution of the irradiation at the fiber exit (a) and at the surface of the cell-loaded disc (b) are reported in Fig. 3. Under these conditions SiNc efficiently photosensitizes the B16 melanoma cells. In a typical experiment, cell survival decreased by ca. 70% after 2 min. irradiation and beyond 95% after 20 min. irradiation. The photosensitization of cells having a high content of melanin is an important result: usually, highly pigmented cells and tissues are poorly sensitive to the effects of UV and visible light even in the presence of photosensitizing agents [6]; melanin is known to limit the penetration of light into biological systems and to act as a quencher of electronically excited species. The efficacy of 776 nm light and SiNc in inducing photo toxicity of B16 melanoma cells appears to be the consequence of two factors: (i) the enhanced transparency of pigmented melanoma at wavelengths longer than ca. 700 nm [7]; and (ii) the high molar extinction coefficient of SiNc at 776 nm so that this dye can favourably compete with melanin for light absorption. Thus, the diode laser described in this paper allows one to extend the scope of phototherapeutic methods involving red light-activation of dyes to include pigmented tumours. Actually, preliminary experiments performed in our laboratory show that the concerted action of systemically administered SiNc and 776 nm-diode laser induces the regression of B16 pigmented melanoma subcutaneously transplanted to C57 mice; this tumour is not responsive to photodynamic therapy

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performed in the presence of photosensitizers, such as hematoporphyrin derivatives, wich absorb at lower light wavelengths. Acknowledgment

This work was financially supported by Consiglio N azionale delle Ricerche, Special Project "Tecnologie Elettroottiche", contract. No. 92.02564.PF65. References 1 Moan, J. and Berg, K. (1992) Photochemotherapy of cancer: experimental research. Photochem. Photobio!' 55:931948.

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2 Marcus, S.L. (1992) Photodynamic therapy of human cancer. Proc IEEE 80:869-889. 3 Cuomo, V., Jori, G., Rihter, B., Kenney, M.E. and Rodgers, M.A.J. (1990) Liposome-delivered Si-naphthalocyanine as a photodynamic sensitizer for experimental tumours. Br. J. Canc. 62:966-970. 4 Firey, P.A. and Rodgers, M.A.J. (1987) Photoproperties of Si-naphthalocyanine, a potential photosensitizer for photodynamic therapy. Photochem. Photobio!' 45:535-538. 5 Bertoloni, G., Rossi, F., Valduga, G., Jori, G., All, H. and Van Lier, J.E. (1992) Photosensitizing activity of waterand lipid-soluble phthalocyanines on prokaryotic and eukaryotic microbial cells. Microbios 71:33-46. 6 Zhou, C. (1989) Mechanisms of tumour necrosis induced by photodynamic therapy. J. Photochem. Photobio!., B:Bio!., 3:299-318. 7 Wilson, B.C. and Jeeves, W.P. (1987) Photodynamic therapy of cancer. In: E. Ben-Hur and I. Rosenthal (Eds.), Photomedicine, Vo!. 2, p. 217, CRC Press, Boca Raton.

Laser diode coupled with optical fiber for applications in photodynamic therapy.

A diode laser with roughly 480 mW/cm2 emission at 776 nm was developed and engineered to obtain a uniform illumination of tumour cells deposited on Pe...
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