Determination of the activation spectrum of aluminium phthalocyanine chloride against cultured meningioma cells using a tunable laser

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1 Determnaton of the actvaton spectrum of alumnum phthalocyanne chlorde aganst cultured menngoma cells usng a tunable laser Andrew C. Wlson MSc* Gregory M. Malham MB ChB t Raewyn J. Thomsen NZCS* John D. Harvey PhO* Bruce C. Baguley PhD *Department of Physcs, Unversty of Auckland, tdepartment of Neurosurgery, Auckland Hosptal, ;Department of Pathology and ~Cancer Research Laboratory, Unversty of Auckland School of Medcne, Auckland, New Zealand. The actvaton spectrum of the photosenstsng agent alumnum phthalocyanne chlorde (CLMPc) was determned usng cultured menngoma cells as the bologcal target. Cells were rradated wth tunable lasers over the wavelength range um and photocytotoxcty was measured 24 h later by a tetrazolum assay. The photoactvaton spectrum was found to be relatvely narrow wth a sharply defned maxmum at nm, a readly accessble wavelength for commercally avalable dode lasers. The absorpton spectrum of CAPc n aqueous soluton was broad and featureless, contrastng markedly wth the actvaton spectrum. However, as the content of an organc solvent (dmethylsulphoxde) was ncreased, the absorpton spectrum progressvely resembled the actvaton spectrum. t s concluded that the absorpton spectrum of C1APc n aqueous soluton s determned by drug aggregaton, whch can be prevented by an organc envronment. Ths mples that the actve form of CAPc n cells s the monomerc speces probably located n cellular membranes. Journal of Clncal Neuroscence 1996, 3 (3) : Pearson Professonal 1996 Keywords" Menngoma, Culture, Absorpton, Actvaton spectrum, Laser, Photodynamc therapy ntroducton Photodynamc therapy (PDT) s a promsng new tumour treatment modalty where cell death s ntated by the photoactvaton of a tumour localsng photosenstsng agent. The drug s nert untl llumnated by lght of an approprate wavelength. Absorpton of a photon by the agent results n the promoton of an electron from the ground state to an excted state, whch can ether decay back to the ground state or promote photonduced chemcal reactons. n the phthalocyanne system the excted snglet state can undergo ntersystem crossng to the excted trplet state whch can undergo ether a type electron reacton to produce free radcal speces, or a type energy transfer process to produce snglet oxygen speces. 1 The man objectve of PDT s to nduce selectve tumour cytotoxcty wth mnmal systemc sde effects. 2 Phthalocyanne dyes offer consderable potental as senstsers for PDT both n vtro and n vvo. 3 Several dervatves, whch dffer n ther degree of sulphonaton from the unsulphonated to the tetrasulphonated forms, have been nvestgated. Whle n vvo the photodynamc effcacy correlates postvely wth the degree of sulphonaton, the stuaton n vtro s the reverse wth the unsulphonated dervatve beng the most potent photocytotoxn. 4 An alumnum-chelated phthalocyanne dervatve, alumnum phthalocyanne chlorde (C1A1Pc) s a convenent agent for n vtro studes on PDT and has the advantage of beng easly synthessed, nexpensve, commercally avalable, composed of a sngle molecular speces, and hghly cytotoxc aganst cultured cells. 4 C1A1Pc, whch we have found to be soluble n aqueous soluton to at least 10 gm, s cytotoxc to cultured cells at submcromolar concentratons. 5 n a prevous study we have demonstrated detectable kllng of menngoma cells by CA1Pc at a concentraton of 10 nm 6. Lasers provde the optmal source of photorradaton, beng hghly ntense, monochromatc and effcently transmtted by an optcal fbre.v, 8 Laser dodes, whch are compact and economcal, are partcularly sutable for the treatment of sold tumors, and the long wavelengths avalable ( nm) offer excellent tssue penetraton. 9 However, snce only certan wavelength bands are avalable, t s mportant to choose a photodynamc dye whch s optmally actvated wthn ths range. Menngomas are the most frequently occurrng bengn CNS tumour2 They are potentally sutable targets for PDT snce surgcal removal s not always 252 J. Cln. Neuroscence Volume 3 Number 3 July 1996

2 CAPc actvaton spectrum possble 11,12 and the avalable adjuvant therapes are suboptmal.13,14 n a prevous study we have used broad band red lght to demonstrate the photocytotoxcty of CA1Pc aganst cultured menngoma cells. 6 n ths study we have the same bologcal target, n conjuncton wth tunable lasers, to determne the actvaton spectrum of C1A1Pc. We have also determned the lght ntensty requred for C1MPc cytotoxcty. Materals and methods Photosenstsng agents CA1Pc was obtaned from Eastman Kodak Company (Rochester, NY), and 5 mm stock solutons, prepared by dssoluton n dmethylsulphoxde (DMSO), were stored at-20 C n the absence of lght. Cell culture Menngomas removed neurosurgcally from two patents were studed. Tumour specmens were collected n culture medum and dsaggregated the same day by mncng wth crossed scalpel blades followed by enzymatc dgeston wth pronase (1 mg/ml Calbochem), collagenase (1 mg/ml; Sgma Chemcal Co. Clostrdum hstolytcum, crude), and DNAase (1 mg/ml; Sgma, type 1, pancreatc, crude). Cells were centrfuged to remove debrs and proteases, washed twce and seeded nto separate culture flasks. The cells were grown to confluence and mantaned n an alpha modfed mnmal essental medum (t>mem; Gbco, New Zealand) contanng 20% heat nactvated fetal calf serum (FCS; Gbco), penclln (100 U/ml; Sgma) and streptomycn (100 mg/ml; Sgma) at 37 C n a humdfed 5% CO 2 atmosphere. Growth medum was changed every 7 days. Cells were passaged by detachng them from the plastc usng trypsn (0.07% w/v; Dfco) n ctrate salne soluton (trsodum ctrate dhydrate 4.4 g/l, KC1 10 g/l, ph 7.3). dentfcaton of cultured cells as menngoma n prevous studes has been establshed by lght mcroscopc, electron mcroscopc and chromosomal studes. 15 For experments, the cells were trypsnsed, resuspended n fresh medum, washed and plated nto 96 well trays at 104 cells per well (150 ml/well) wthn one to two passages from explantng. Cultures were ncubated for 24 h, after whch the medum was asprated and 150 ml of phenol red-free medum (a-mem; Gbco) contanng FCS (20%) and CA1Pc (0.02 mm) were added to each well. Each plate contaned wells to whch the photosenstsng agent was not added to serve as controls. Cells were ncubated wth CA1Pc for 16 h pror to photorradaton. Determnaton of absorpton spectrum CA1Pc was dssolved ether n water, n DMSO or n 10%, 20% or 50% DMSO n water (v/v). A spectrophotometer (Shmadzu model UV3101PC) was used to obtan absorpton spectra at wavelengths rangng from the ultravolet to the near nfrared. The spectral resoluton of the spectrophotometer was 0.1 am. Determnaton of actvaton spectrum Laboratory studes The optmal actvaton wavelength of C1A1Pc was determned usng tunable lasers. The laser output beam was coupled nto a 1 m long fbre optc bundle wth the emergng cone of lght drected at the sample mcroculture plate. The survvng cell populaton of each plate was assessed by a cytotoxcty assay (see below). The wavelength range nm (red part of spectrum) utlsed a CW dye laser (Coherent model 599) runnng on DCM laser dye (Lambda Physk LC6500), whch was optcally pumped by an argon on laser (Coherent model nnova ). The wavelength range nm was obtaned usng a ttanum doped sapphre laser (Spectra- Physcs model 3900) optcally pumped by the same argon on laser. The output power of the CW dye laser and of the ttanum doped sapphre laser were both approxmately 1 W over the respectve tunng ranges. Wavelength tunng of both lasers was acheved usng a three plate brefrngent flter and wavelength measurements were made wth an optcal multchannel analyser (EG&G Parc model 1453A) wth a spectral resoluton of 0.5 nm. The cells were exposed to the central regon of the beam extng the fbre bundle, where an ntensty of 140 mw/cm2was measured (usng an Ophr model 30A laser power meter) at the mcroculture plate contanng the cells to be rradated. Only the central regon of the lght cone extng the bundle was used to ensure that the ntensty vared by no more than 10% over the wells n use. Cytotoxcty assay After photorradaton the medum was asprated and fresh la-mem/fcs was added. The survvng cell populaton of each plate well was assessed by the MTT (3-[4, 5- dmethylthazol-2-yl]-2, 5-dphenyltetrazolum bromde; Sgma) assay. 16,17 MTT was dssolved n PBS at 5 mg/ml, flter-sterlsed and stored at-20 C n the absence of lght. MTT soluton was added to each well (15 ml/well) 24 h followng lght exposure. The plates were ncubated at 37 C for a further 2 h to allow MTT metabolsm, after whch the medum was asprated and the formazan crystals produced by vable cells were dssolved by the addton of DMSO (100 ml/well). Control groups receved dentcal concentratons of the photosenstsng agent and MTT wthout photoactvaton. Absorbance was measured usng a mcroplate reader (Dynatech MR 600) and results expressed as a percentage of control values. Expermets were performed usng quadruplcate cub tures and standard errors were typcally <15% of mean values. Dosage dependence The ntensty of photollumnaton to whch the menngoma cells treated wth C1MPc.were exposed, was vared by attenuatng the lght beam between the laser and the fbre optc bundle usng an alumnum coated glass slde of varyng optcal densty. J. Cln. Neuroscence Volume 3 Number 3 July

3 Laboratory studes CAPc actvaton spectrum t 0.3- "~ < ? Wavelength(nm) Fg. 1 Actvaton spectrum of CAPc at a concentraton of 0.02 mm, as measured by photocytotoxdty of cultured menngtoma cells (dashed lne; rght axs). The absorpton spectrum of CAPc n aqueous soluton (10 mm) s shown by the sold lne (left axs). Results The actvaton spectrum of CA1Pc was determned usng tunable laser sources. Prevous experments usng broad band red lght had determned the optmal concentraton of C1A1Pc to be 0.02 mm, whch was the C50 value (concentraton requred for 50% kllng of cells). t should be noted that ths concentraton s more than two orders of magntude less than that commonly used wth sulphonated C1A1Pc dervatves. 6 Other experments had demonstrated that a concentraton of C1A1Pc of 0.02 mm had no effect on the vablty of cells n the absence of lght, and that the cells were not senstve to lght n the absence of C1A1Pc. 6 Menngoma cells were exposed to laser lght n the presence of 0.02 mm CA1Pc for 1 mn at defned wavelengths, at 5 nm ntervals, over the range nm. Cells were then grown for a further 24 h and the survvng cells measured usng the MTT assay. The actvaton spectrum for CA1Pc exhbted a sharply defned maxmum between 675 nm and 680 nm, together wth mnor peaks of actvaton (30% cytotoxcty) at 610 nm, 640 nm and 655 nm, and no sgnfcant cell kll at wavelengths greater than 700 nm (Fg. 1). The absorpton spectrum for C1A1Pc n aqueous soluton over wavelengths nm showed a broad, relatvely featureless pattern (Fgs. 1 & 2). As the content of DMSO was ncreased, the absorpton spectrum progressvely resembled the actvaton spectrum wth peaks appearng at 612 and 680 nm (Fg. 2, Table 1). The effect of varyng exposure tme to photollumnaton of the cell sample was also nvestgated. Menngoma cells were exposed to laser lght at a wavelength of 670 nm for 10, 20, 30, 40, 50, 60, 90, 120, 180 and 300 seconds. Cells were then cultured and assayed as before. Exposure for less than 35 seconds produced : *-... r" Un~'Et~M~1H " ' " ' ' ' -... " ' 1 1,2 t ' l, 1._1., : n''''""ll '... B " 'm- " W" n... l..., "'-''''~ll... S Jll m~t~,,, l..t..~'~... Fg. 2 The absorpton spectrum of CAPc (10 mm) n aqueous soluton (A), 10% aqueous DMSO (B), 20% aqueous DMSO (C), and 50% aqueous DMSO (D). The spectrum n 100% DMSO was smlar to (D) above. t~l D 1 t l 254 J. Cln. Neuroscence Volume 3 Number 3 July 1996

4 CAPc actvaton spectrum Laboratory studes Table 1 Absorpton of CAPc (10 mm) n the DMSO-water mxtures Solvent Absorbance at 612 nm Absorbance at 680 nm Absorbance at 800 nm Water DMSO 10% n water DMSO 20% n water DMSO 50% n water DMSO 100% neglgble cell kll, whle exposure for longer than 120 seconds caused no further cell kllng (Fg. 3). Although 670 nm s not at the peak of the actvaton spectrum, ths wavelength was used as t corresponds to that of readly avalable laser dodes. The effect of varaton of ntensty of exposure was also measured at a constant photollumnaton tme of 1 mn. Exposure at an ntensty of less than 70 mw/cm 2 produced neglgble cell kll, whle exposures above 85 mw/cm 2 caused only a small further ncrease n cell kllng (Fg. 4). Dscusson A tunable laser system has been utlsed to provde an accurate actvaton spectrum of C1MPc for cultured menngoma cells. ts optmal actvatng wavelength was approxmately 675 nm, smlar to that reported for cultured V79 Chnese hamster cells. TM Ths wavelength provdes a dstnct advantage n that t s readly accessble by dode lasers. The latter are easly obtanable, avalable at hgh power wth good relablty, and provde excellent tssue penetraton. 9 C1MPc s a hghly potent photosenstser of menngoma cells, 6 s easly synthessed, nexpensve, commercally avalable and composed of a sngle molecular speces. ts low aqueous solublty has made t unattractve as a potental clncal photosenstser, but the present results suggest on the bass of ts hgh potency that t mght usefully be re-evaluated, and should provde an mproved and more defned methodology for PDT research. The results demonstrate that the actvaton spectrum of C1A1Pc n soluton does not correspond to the absorpton spectrum n aqueous soluton, but does correspond to that n DMSO-water (50% v/v) or n pure DMSO (Fg. 2, Table 1). t s lkely that the absorpton spectrum of C1MPc n aqueous soluton s broadened by drug aggregaton, gvng rse to the spectral changes reported n Table 1. Ths drug aggregaton can be prevented by an organc envronment. 19 The results are consstent wth other studes whch ndcate that aggregated forms of the phthalocyanne dyes are photodynamcally nactve. 2 Our prevous study, employng confocal laser scannng mcroscopy, has shown that C1MPc s taken up rather slowly by menngoma cells, and that cell-assocated C1MPc s dffusely dstrbuted throughout the cytoplasm. Ths behavour contrasts wth that of sulphonated C1A1Pc dervatves, whch are concentrated n lysosomes. 6 The much greater n vtro potency of C1MPc n comparson to the less lpophlc sulphonated C1A1Pc dervatves suggests that the actve form of C1MPc n cells s the monomerc speces dssolved n cellular membranes. Ths s the form thought to provde the hghest yeld of snglet oxygen, whch s proposed to be the domnant cytotoxc speces medatng photodynamc therapy. 19 Acknowledgements Ths work was supported by the New Zealand Neurologcal Foundaton, nc. and by the Auckland Dvson of the Cancer Socety of New Zealand, nc. Receved 24 March 1995 Accepted for publcaton 17 July { { {. l 50 [ [ F ' 60- & ~40-0 {{ o { {{ Exposure(s).=_ ~a 4O [ntensty(mw/cm 2) Fg. 3 Dependence of photocytotoxcty of CAPc (0.02 mm) on exposure tme, usng an llumnaton wavelength of 670 nm and an exposure ntensty of 140 mw/cm 2. Vertcal bars represent estmated standard errors of determnatons. Fg. 4 Dependence of photocytotoxcty of CAPc (0.02 mm) on exposure ntensty usng an llumnaton wavelength of 670 nm and an exposure tme of 1 mnute. Vertcal bars represent estmated standard errors of determnatons. J. Cln, Neuroscence Volume 3 Number 3 July

5 Laboratory studes CAPc actvaton spectrum Correspondence and requests for offprnts: Dr Gregory M Malham Department of Neurosurgery Auckland Hosptal Prvate Bag Auckland New Zealand Te1:(64-9) exm Fax: (64-9) References 1. Ln C-W. Photodynamc therapy of malgnant tumors-recent developments. Cancer Cells 1991; 3: Kessel D. Photodynamc therapy and neoplastc dsease. Oncol Res 1992; 4: Chan WS, MarshallJF, Svensen R, et al. Effect of sulfonaton on the cell and tssue dstrbuton of the photosenstzer alumnum phthalocyanne. Cancer Res 1990; 50: Rosenthal. Phthalocyannes as photodynamc senstzers. Photochem Photobol 1991; 53: Ben-Hur E, Rosenthal. The phthalocyannes: a new class of mammalan cells photosenstzers wth a potental for cancer phototherapy. ntj Radat Bol 1985; 47: Malham GM, Thomsen RJ, Fnlay GJ, et al. Subcellular dstrbuton and photocytotoxcty of alumnum phthalocyannes and haematoporphyrn dervatve n cultured human menngoma cells. BrJ Neurosurg 1996; 10: Demn-6der W. Laser Spectroscopy. Berln: Sprnger Verlag, Dougherty T. Yearly Revew. Photodynamc Therapy. Photochem Photobol 1993; 58: Abernathey CD, Anderson RE, Koostra KL, et al. Actvty of phthalocyanne photosenstzers aganst human globlastoma n vtro. Neurosurgery 1987; 21: ReyJ, Pestana A, Bello M. Cytogenetcs and molecular genetcs of nervous system turnouts. Oncol Res 1992; 4: Mayberg MR, Symon L. Menngomas of the clvus and apcal petrous bone. Report of 35 cases. J Neurosurg 1986; 65: Black PM. Bran tumors (second of two parts). New EnglandJ Med 1991; 324: 22: A1-Mefty O, Routh A, Smth RR. The long term sde effects of radaton therapy for bengn bran turnouts n adults.j Neurosurg 1990; 73: Schrell UMH, Fahlbusch R. Hormonal manpulaton of cerebral menngomas. n: A1-Mefty O, ed. Menngomas. New York: Raven Press, 1991: Malham GM, Thomsen RJ, Synek BJ, et al. Dagnostc markers demonstratng successful cuhure of human menngoma cells. Journal of Clncal Neuroscence 1995; 2: Mosmann T. Rapd colormetrc assay for cellular growth and survval: applcaton to prolferaton and cytotoxcty assays.j mmunol Meth 1983; 65: Fnlay GJ, Wlson WR, Baguley BC. Comparson of n vtro actvty of cytotoxc drugs toward human carcnoma and leukaema cell lnes. EurJ Cancer Cln Oncol 1986; 22: SpkesJD. Phthalocyannes as photosenstsers n bologcal systems and for the photodynamc therapy of tumors. Photochem Photobol 1986; 43: Berg K, BommerJC, MoanJ. Evaluaton of sulfonated alumnum phthalocyannes for use n photochemotherapy. A study on the relatve effcences of photonactvaton. Photochem Photobol 1989; 49: Moan J, Peng O~ Evensen F, et al. Photosenstzng effcences, tumor- and cellular uptake of dfferent photosenstzng drugs relevant for photodynamc therapy of cancer. Photochem Photobol 1987; 46: J. Cln. Neuroscence Volume 3 Number 3 July 1996