J. Photochem. Photobiol. B: Bioi., 22 (1994) 105-117 105

Time-resolved fluorescence spectroscopy and intracellular imaging ofdisulphonated aluminium phthalocyanine

M.AmbrozDepartment of Chemical Physics, Charles University, Prague (Czech Republic)

A.J. MacRobertt and J. MorganThe Rayne Institute, University College London Medical School, 5 University St., London WCIE 6JJ (UK)

G. Rumbles, M.S.C. Foley and D. PhillipsDepartment of Chemistry, Imperial College of Science, Technology and Medicine, Exhibition Rd., London SW7 2AY (UK)

(Received June 15, 1993; accepted October 11, 1993)

Abstract

Spectroscopic studies were carried out on the photosensitizer disulphonated aluminium phthalocyanine (A1SzPc)which has prospective applications in photodynamic therapy. The fluorescence lifetimes of AISzPc were measuredin a range of model systems and cultured leukaemic cells using laser excitation and time-correlated, single­photon-counting detection. In an investigation of non-covalent protein binding, we studied AISzPc in the presenceof human serum albumin (HSA) in 0.1 M phosphate-buffered saline at pH 7.4. On addition of excess concentrationsof HSA, small red shifts in the fluorescence and absorbance spectra were observed, together with an increasein fluorescence polarization anisotropy, consistent with binding of the phthalocyanine. Fluorescence decays couldbe resolved into two lifetimes for bound AISzPc with a dominant component of 5.5 ns and a minor componentof 1 ns. Fluorescence imaging and time-resolved microfluorometry were carried out on intracellular AISzPc usingleukaemic 1<562 cells. Microscopic imaging with a charge-coupled device (CCD) camera revealed that AISzPcfluorescence predominated in a discrete perinuclear region which was then probed selectively by a focused laserspot for fluorescence lifetime measurements. Bi-exponential decays with lifetime components of 6.1 and 2.2 nswere observed. On irradiation at 633 nm, the fluorescence intensity increased initially and subsequently declineddue to photodegradation.

Key words: Phthalocyanine; Fluorescence lifetime; Fluorescence imaging; Photodynamic therapy

1. Introduction

Photodynamic therapy (PDT) is a promlsmgtreatment for certain cancers and is based on thesystematic administration of a photosensitizer fol­lowed by laser irradiation of the sensitized tumour.Preparations based on haematoporphyrin deriv­ative (HpD) are being used at present as clinicalphotosensitizers, but it is now widely recognizedthat these compounds suffer from several disad­vantages. Consequently, there has been a vigoroussearch for new photosensitizers, and among themost actively investigated are phthalocyanine dyes[1], in particular the water-soluble aluminium sul­phonated phthalocyanine (AISPc). The principal

tAuthor to whom correspondence should be addressed.

1011-1344/94/$07.00 © 1994 Elsevier Sequoia. All rights reservedSSDl 1011-1344(93 )06955-3

advantage over porphyrin sensitizers is the muchstronger absorption of AISPc (€::::: 105 M - 1 cm -1 )

at longer wavelengths near 700 nm. Furthermore,AISPc is a good fluorophore with a peak emissionnear 680 nm which has enabled in vitro and invivo detection [2, 3]. Promising results have beenobtained with experimental animal tumours [4, 5]but, until recently, the majority of these studiesemployed AlSPc in the form of a mixture com­prising mono-, di-, tri- and tetrasulphonated com­ponents. This compound can now be preparedwith a defined degree of sulphonation and a con­siderable amount of work [6, 7] has been devotedto comparative studies of these components,AISnPc (n = 1, 2, 3, and 4). The degree of sul­phonation affects the lipid solubility and aggre­gation properties, both of which are known to

106 M. Ambroz et al. / Fluorescence spectroscopy and intracellular imaging of AlS2Pc

influence cellular uptake and phototoxicity, withthe lesser sulphonated, more lipophilic componentsexhibiting higher in vitro photoactivities [6]. Thedisulphonated aluminium phthalocyanine com­ponent (designated hereafter as S2) exhibits theoptimum combination of in vivo pharmacokineticsand phototoxicity and appears to be the mostsuitable for clinical PDT [6-8], whereas the mon­osulphonated component (the most active com­ponent in vitro) undergoes relatively high uptakein vivo by the reticuloendothelial system, partic­ularly the liver, resulting in poor tumour sensi­tization.

The fluorescence from intrinsic fluorophores ofbiomolecules (e.g. the aromatic amino acid residuesin proteins) or from fluorophores added to bio­logical samples has been widely used to probe thestructure, environment and dynamics of a varietyof systems of biological importance. In particular,the fluorescence spectrum, intensity, polarizationand lifetime of fluorophores may all be influencedby environmental factors. Thus such studies canprovide information on the binding of photosen­sitizing fluorophores to biological substrates andlocalization within membranes, which are believedto be among the primary targets of PDT owingto lipid peroxidation. The application of fluor­escence microscopy and, more recently, scanningconfocal fluorescence microscopy has enabled thedirect observation of fluorescence from isolatedcells and intracellular components. With a fewexceptions, microfluorometric studies have beenconcerned with monitoring only the fluorescenceintensity and spectra of fluorophores in cellularsystems. However, the steady state fluorescenceintensity is a function of both the concentrationand fluorescence quantum yield, which is depen­dent on the fluorescence lifetime and may varyfrom site to site owing to quenching and aggregationprocesses. Therefore, in order to quantify micro­scopic distributions of a given fluorophore, it isnecessary to measure the fluorescence lifetimes atthe microscopic level. A sensitive technique forfluorescence microscopic lifetime measurementshas now been developed using time-correlatedsingle-photon counting with laser excitation [9]and several groups have employed this method instudies of intracellular porphyrins. Streak camerastudies have also been reported although thistechnique is generally less sensitive. Research inthis field has been reviewed recently [10].

This work is concerned with fluorescence spec­troscopic studies of disulphonated aluminiumphthalocyanine, the aim being to determine howthe fluorescence properties of the sensitizer are

influenced by microenvironmental factors. Suchstudies are of practical relevance since fluorescencedetection of sensitizers in vitro and in vivo is ahighly sensitive means of quantifying cellular up­take and localization as demonstrated recently forS2 [7, 11, 12]. Two complementary experimentalapproaches have been adopted here as part of acomprehensive study of the photoproperties ofsulphonated phthalocyanines: (i) measurements inmodel systems; and (ii) direct microscopic studieson individual cells containing the sensitizer. In aninvestigation of binding to serum proteins, we havemade steady state and time-resolved studies of S2fluorescence in the presence of an excess of humanserum albumin (HSA), and similarly with micelles,which act as rudimentary models of membranelipid environments. For the work on cells, we havecombined fluorescence imaging of intracellular S2with spatially resolved measurements of fluores­cence decays.

2. Experimental details

2.1. MaterialsAluminium sulphonated phthalocyanine of

mixed sulphonation was prepared by oleum sul­phonation of chloroaluminium phthalocyanine.The mixture was then separated into the AISnPccomponents using reverse-phase medium-pressureliquid chromatography, with purity assessed usingreverse-phase high-performance liquid chroma­tography (HPLC) [13]. Similar procedures havebeen used elsewhere and the preparation of theS2 component as a well-defined peak in HPLCchromatograms (the most hydrophobic) appearsto be reproducible between different laboratories[14]. This S2 component is commonly supposedto contain sulphonates with an adjacent config­uration [13,14]. AIS3Pc and AIS4Pc were preparedas described previously [13]. HSA, essentially fattyacid free, was obtained from Sigma; Triton Xl00,Nonidet P40 detergent and cetyl ammonium bro­mide (CTAB) were obtained from BDH; air-equil­ibrated aqueous solutions were prepared usingdistilled water at pH 7.4 in 0.1 M phosphate­buffered saline (PBS) (Sigma).

For the microscopy experiments, cells from theK562line suspended in RPMl-1640 with 2% foetalcalf serum (FCS) at a concentration of 2 X 106

ml- I were incubated with AISPc at 10 J.LM for 60min at 37 °C in the dark. The cells were thenwashed in RPMl-1640 with 2% PCS, centrifuged,resuspended in PBS and then placed onto a glassslide for microscopic examination at ambient tem-

M. Ambroz et al. / Fluorescence spectroscopy and intracellular imaging of AIS2Pc 107

perature (25°C). Further details may be foundelsewhere [15].

Data were recorded to a minimum of 20 000 countsin the channel of maximum intensity for the modelsystem studies and analysed by a non-linear, least­squares iterative reconvolution program using thereduced X2 coefficients, plots of weighted residualsand autocorrelation function for assessment of thedata. Global analysis was also employed in certaincases [16].

For the fluorescence decay microscopic studieson individual K562 cells, the dye laser beam (wave­length, 610 nm) was reflected off a dichroic mirror

2.2. MethodsAbsorbance spectra were measured using a Per­

kin-Elmer Lambda 15 spectrometer and fluor­escence spectra with a Perkin-Elmer LS-5B spec­trofluorometer controlled by a PC which couldgenerate emission spectra corrected for variationsin wavelength response and polarization anisot­ropy. From the parallel (III) and perpendicular(I L) anisotropy components, the steady state an­isotropy of each sample was evaluated using thestandard equation, r= (III - GI L)/(III + 2GIL)'where G is the polarization correction factor de­rived from the ratio 111 /1 L using horizontally po­larized excitation.

2.2.1. Fluorescence decay measurementsFor fluorescence decay lifetime studies in model

systems, phthalocyanine concentrations of 0.5 JLMin 0.1 M PBS (pH 7.4) were employed using laserexcitation at 610 nm. Lifetimes were measuredusing time-correlated single-photon counting, andemission was detected via a long-pass Schott RG645filter with a Hamamatsu red-sensitive R928 pho­tomultiplier tube, with a convoluted instrumentresponse time of approximately 350 ps. The laserapparatus consisted of a frequency-doubled, mode­locked Nd:YAG laser (Coherent, Antares 76-S)which synchronously pumped a cavity-dumped dyelaser (Coherent 701-3CD, rhodamine 60 dye)supplying output pulses of less than 10 ps at arepetition rate of 3.8 MHz. Time-resolved fluor­escence decays I(t) and polarization anisotropydecays r(t) were analysed as a sum of exponentialsusing eqns. (1) and (2), where for the ith com­ponent, 'TeoT is the rotational correlation time andr a is the fraction of the total anisotropy

I(t) = LA; exp( -t/7";) (1);

(DF650, Omega Optical Inc.) mounted on anOlympus IMT-2 microscope and directed througha 40 X objective (NA 0.95) to produce a laserspot of 1 JLm radius on a pre-selected cell. Fluor­escence was collected by the same objective andtransmitted through the dichroic mirror to an R928photomultiplier for time-correlated single-photon­counting analysis via barrier filters (Schott RG665)and an iris to eliminate scattered light; both sandp polarizations were equally transmitted by thedichroic mirror over the detection wavelength band(660-700 nm). In order to minimize AISPc pho­todegradation, the power of the excitation beamwas attenuated to less than 1 JLW, which never­theless yielded fluorescence counting rates up to104

S -1 due to the high detection efficiency of themicroscope objective.

2.2.2. Fluorescence imagingAn inverted microscope (Olympus IMT-2), with

epifluorescence and phase contrast attachments,was used with excitation light provided by an 8mW helium-neon laser emitting at 632.8 nm. Fluor­esC,(ence was imaged using a cooled, slow-scan,charge-coupled device (CCD) camera (Wright In­struments, model 1, resolution 600 X 400 pixels, 14bit). A liquid light guide and a condenser wereused to direct the laser output (via a 10 nm bandpassfilter centred at 633 nm to remove extraneouslight) into the dichroic mirror housing for standardepifluorescence excitation [6, 7]. The phthalocy­anine fluorescence was detected in the range660-700 nm covering the main fluorescence bandof these sensitizers. The advantages of using acooled, slow-scan, CCD camera over video imagingsystems include much higher sensitivity, directdigital image integration and a high dynamic rangeof over 104

• The high sensitivity allows low-powerexcitation and short integration times minimizingsensitizer bleaching that may distort the fluores­cence image. An IBM PC with a high-resolutioncolour monitor controlled the camera operationand was used for digital image processing, displayand storage. Autofluorescence from control cellsamounts to only 1-2 counts on an image scale of103 employed in this work. Fluorescence was dig­itally quantified by either line or box superim­position on areas of interest.

3.1. Model systems3.1.1. Human serum albumin (HSA)Binding of porphyrins to this protein has been

widely studied owing to its importance in the

3. Results

(2)r(t) = Lro, exp( - t/'Teor;);

108 M. Ambroz et aL I Fluorescence spectroscopy and intracellular imaging of AlS2Pc

pharmacokinetics of the circulating sensitizer anddelivery to tissues. The interaction of 52 with H5Awas investigated using a combination of steadystate and time-resolved techniques, namely mea­surements of alterations in absorbance and fluor­escence spectra, and changes in fluorescence decayand fluorescence emission anisotropy. Since themain objective of this work was to investigate thefluorescence properties of 52, the 52 concentrationswere in general restricted to 0.5 ILM in order tominimize re-absorption over the 1 cm path lengthof the cuvettes (optical density (OD)<O.l at 675nm). Therefore, for these binding studies, it wasfound necessary to use excess concentrations ofHSA which were added to phthalocyanine solutionsin successive aliquots, freshly prepared in 0.1 MPBS, to give H5A concentrations ranging from 0.5to 100 J.LM. For the absorbance experiments, acuvette with H5A at the same concentration wasplaced in the reference beam to compensate forlight scattering. Experiments at higher HSA con­centrations up to physiological values were notattempted owing to the marked light scatteringwhich could have affected polarization anisotropy,amongst other effects. Figure 1 shows that the S2absorption spectra in the presence of H5A areconsistent with monomeric phthalocyanine, withthe Q-band absorbance slightly broadened and redshifted from 672 to 676 nm at the highest HSAto S2 concentration ratio; an isosbestic point isapparent at 676 nm, although closer examinationreveals that the traces are not exactly coincident.Fluorescence emission spectra (Fig. 2) also exhibita small red shift on addition of excess H5A,reaching a maximum at 685 nm compared with680 nm in 0.1 M PB5. Steady state fluorescence

0.2

0.18

0.16

0.14

""u 0.12z...'"'" 0.10en

'"... 0.08

0.06

0.04

0.02

0

640 680 680 700

WAVELENGTH Inm)

Fig. 1. Absorption spectra in 0.1 M PBS of S2 (1.0 JLM) in thepresence of excess concentrations of HSA (S2:HSA shown inparentheses): spectrum 1 (no HSA); 2 (1:10); 3 (1:25); 4 (1:50);5 (1:100). Spectra 4 and 5 are virtually indistinguishable.

~'c

3".....~

I 2.5

.. 2uz..u

~ 1.5

~

840 880 880 700 720 740 780 780 800

WAYElZNGTB [DUll

Fig. 2. Fluorescence emission spectra in 0.1 M PBS of S2 (0.5JLM) in the presence of excess concentrations of HSA withexcitation at 610 nm (vertical polarization excitation and magicangle detection). For ease of inspection each spectrum is separatedon the intensity scale by 0.5 units (at 640 nm the intensity wasnegligible in all cases). A small red shift in the spectra (un­corrected) is apparent with increasing HSA.

emission anisotropy was observed with increasingHSA concentration and converged to an anisotropyvalue of r = 0.06. For the higher H5A concentra­tions, a small reduction (typically 10%) in theintegrated fluorescence intensity (corrected forchanges in emission wavelength response and ab­sorbance at 610 nm) was found in these mea­surements; however, some variation was evidentbetween experimental runs probably because ofthe residual aggregated S2 before addition of H5A.From the bathochromic shifts in the 52, absorbanceand fluorescence emission spectra, we estimatethat greater than 90% association was present in50-100 JLM of HSA. Although we did not attempta definitive study with a range of different 52 andHSA concentrations, the binding constant for 52may be estimated as K::::: 1as M - 1 which is an orderof magnitude lower than for a haematoporphyrin[17]. A limited number of experiments were carriedout with the tri- and tetrasulphonated components(53 and S4) which both exhibited lower bindingaffinities to HSA judging from the spectral shifts.It is interesting to note that the 54 component,which is present in both monomeric and dimeric

M. Ambroz et al. / Fluorescence spectroscopy and intracellular imaging of AISzPc 109

forms of 0.1 M PBS [13], did not monomerize onaddition of excess HSA, in contrast with solutionscontaining aggregated S2. Systematic studies willbe reported elsewhere [18].

From time-resolved measurements, S2 in PBSalone exhibited a mono-exponential fluorescencedecay with a lifetime of 5.0±0.1 ns, but at highHSA to S2 concentration ratios (50 and 100) bi­exponential decays were observed with lifetimesof 7"1 = 5.5 ± 0.2 ns and T2 = 1.0 ± 0.2 ns, with nor­malized pre-exponential A factors converging toAl =0.92±0.1 andA2 =0.08±0.01 (X2 coefficientsof 1.05-0.95). At intermediate HSA to S2 ratios(1,2.5,5, 10 and 25), we observed multi-exponentialdecays which could be fitted satisfactorily usingthe 7"1 and T2 values together with the free dyelifetime of 5 ns. It should be noted that thesedecays were measured without a monochromatorunder conditions which were independent of po­larization anisotropy (G factor of unity); a con­tribution from scattered light can also be elimi­nated. Of course, even at the higher HSAconcentrations the dye is never completely bound,but it is difficult to resolve the relatively smallcontribution from the free dye especially whenthe lifetime does not differ significantly. At highHSA to S2 ratios the time dependence of thepolarization anisotropy [19] was studied, and wederived a rotational correlation time of Tcor = 45 ± 5ns; the value for free dye in PBS was Tcor ::::: 0.3ns corresponding to out-of-plane rotation (in-planewould exceed the resolution of our system).

3.1.2. MicellesThe incorporation of S2 was examined in two

micellar solutions: CTAB (10- 2 M) and TritonX100 (1%) in 0.1 M PBS. A 20 J,LI aliquot of S2stock solution was added to the micellar solutionsto give an S2 concentration of 0.5 JLM. In bothcases a small spectral red shift was observed: thepeak absorbance in Triton X100 is at 675 nmcompared with 672 nm in aqueous solution. Theaddition of Triton X100 to a more concentratedS2 aqueous solution (10 J,LM), in which the S2was partially aggregated, resulted in monomeri­zation. The fluorescence lifetimes were found tobe: T=5.9±0.1 ns and Tcor =8.0±0.7 ns for TritonX100; T=6.0±0.1 ns for CTAB. For comparison,in glycerol T=5.4 ns and 7"cor= 5.7 ±0.5 ns.

3.2. Microspectrojluorometry of K562 cells3.2.1. Steady state spectroscopy and imagingstudiesSpectroscopic studies were carried out on cul­

tured leukaemic cells, the 1<562 human erythro-

leukaemic progenitor line. This particular cell linewas selected for investigation because it has beenshown to be highly susceptible to Al8Pc photo­sensitized destruction [15, 20] which is hoped mayprovide a means of selective elimination of leu­kaemic cells from infected bone marrow (pho­topurging). In this work, cells were incubated underthe same conditions as used in the photosensi­tization studies, i.e. 10 JLM of S2for 1h. Usingthe CCD imaging system to examine the intra­cellular 82 fluorescence distribution, it is clearlyevident from Fig. 3(a) that the dye is taken upinside the cell (diameter, approximately 20 JLm)at extranuclear sites. The conspicuous feature isa highly fluorescent region adjacent to the nucleuscontributing over 50% of the integrated intensity,which proved ideal for spatially selective probingby a focused laser spot in subsequent time-resolvedstudies. Owing to the high sensitivity of the CCD,which afforded a short exposure time of 1 s, noperturbation in the 82 distribution due to pho­todegradation was apparent. A few cells wereexamined using a 100 X objective which suggesteda granular structure in the fluorescence distri­bution. Further studies using cytospins demon­strated good correlation between the fluorescencedistribution and histochemical staining for acidphosphatase, implying that the 82 was associatedwith lysosomes. In comparative studies with 84,a much weaker fluorescence intensity was observed,whereas S3 exhibited an intensity distribution whichappeared to be intermediate between the S2 and84 components.

On prolonged irradiation at 633 nm (40 X ob­jective), it was apparent that the 82 fluorescencedistribution became perturbed, and a series ofimages were taken during irradiation of the samecell as shown in Fig. 3(b) (recorded (with a 1 sexposure) after 30 s irradiation) and 3(c) (aftera further 60 s). The power incident on the cellsat 633 nm was estimated to be 2 mW over 0.2mm2

, giving an irradiance of about 1 W cm- 2•

Comparing Figs. 3(a) and 3(b), it is clearly seenthat the perinuclear feature intensified and ex­panded: quantitative analysis showed an increasein overall intensity of this feature by about 50%between the two images. This intensity increasewas reproducible in all cells examined after 20-40s irradiation and, in exceptional cases, a factor oftwo was found (but a 50% increase was the typicalvalue). In Fig. 3(c) the fluorescence has spreadthroughout the cell and appears to have penetratedinto the nucleus. Figure 3(d) shows a three-di­mensional intensity plot of Fig. 3(b). These ob­servations, which were found in the many cells

(c) (d)

......(5

~~;:l<:J-i3N

~..:-

:::Jl::C~~~R

~<")

~~

~'"§l:l..

S'~~~:>....3'~.~

~~

§;~

Fig. 3. Using the CCD fluorescence imaging system a series of false colour images (white, highest intensity) with an exposure time of 1 s were recorded during irradiationat 633 nm (1 W cm-2) of the same 1<562 cell (diameter, approximately 20 /Lm) incubated for 1 h with 10 /LM S2: (a) 1 s irradiation; (b) 30 s irradiation; (c) 90 s irradiation.Comparing (a) and (b), it is clearly seen that the perinuclear feature intensifies and expands; (d) shows a three-dimensional plot of (b), in which the low-intensity intranuclearregion is distinctly delineated. In (c) the fluorescence distribution has become more diffuse and weaker as the S2 undergoes photodegradation. Scale in micrographs, 50 X 35pm.

M. Ambroz et al. / Fluorescence spectroscopy and intracellular imaging of AIS2Pc 111

examined from different batches and which wereapparently irreversible, would be consistent withthe photosensitized destruction of organelles as­sociated with S2 such as mitochondria and lyso­somes. The integrated cellular fluorescence in­tensity in Fig. 3(c), and for yet longer irradiationtimes, declined progressively signifying that pho­todegradation of S2 was taking place in parallelwith its redistribution.

The fluorescence emission spectrum of cell sus­pensions excited at 610 nm closely resembled thatobserved with HSA with a peak intensity at 683nm. The addition of Nonidet P40 detergent (1%),which lysed with cells releasing the dye in mon­omeric form, resulted in up to a twofold increasein fluorescence emission, and this effect was ob­served over a range of S2 incubation concentrationsfrom 2 to 20 ILM using 2 ml suspensions containingapproximately 106 cells. With a calibrated plot ofS2 fluorescence intensity vs. concentration in thedetergent solution, we were able to estimate themean cellular S2 uptake to be approximately 107

molecules per cell.

3.2.2. Time-resolved studiesFor time-resolved microfluorometry, the focused

laser beam was aligned initially by adjusting theposition of the cell under phase contrast relativeto the laser spot. The CCD camera, which wasattached to another port on the inverted micro­scope orthogonal to the photomultiplier tube, wasthen used to assess the fluorescence distributionand thus guide the positioning of the laser spotov~r the selected intracellular region taking careto minimize irradiation of the cell. In addition toits inherent spatial resolution, this microscopictechnique is more reliable than bulk measurementson a cell suspension, which are susceptible tointerference from sensitizer leakage from the re­suspended cells and polarization anisotropy effects.The irradiation power of the laser spot was ap­proximately 10 W em -z at 610 nm and experimentswere confined to cells incubated with 10 ,uM ofS2 for 1 h in a series of studies conducted overseveral days. In most of this work, S2 fluorescencedecays were recorded with the laser spot (2 ,urnin diameter) incident on the highly fluorescentcellular region (typically 4 ,urn in diameter) asshown in Fig. 3(a). Counting rates up to 5000 s - 1

were detected and decays were accumulated to amaximum of 10 000 counts in the peak channelin the first instance. It was found that, under theseconditions, the S2 fluorescence at this site exhibitedbi-exponential decays with lifetime componentsnear 2 and 6 ns. Modelling of the data from 15

cells including global analysis confirmed values of2.2 ± 0.4 and 6.1 ±0.2 ns, with similar amplitudesfor each component; good fits were obtained withthe normalized A factor of the shorter componentin the range 0.3-0.5 (X Z coefficients of 1.2-1.4),although in some cases [4] tri-exponential fits gavea slightly better agreement with the inclusion ofa minor 1 ns component. The data quality waslimited by the relatively short accumulation times;these were required because, on prolonged irra­diation, the bi-exponential dependence evolvedessentially to a mono-exponential decay with justthe longer component of 6.1 ns remaining, i.e.counting to 20 000 in the peak channel reducedthe A factor of the shorter component to less than0.1. Thus the time-resolved data may be char­acterized by two regimes of fluorescence decaybehaviour with case 1 (initial excitation) giving abi-exponential dependence and case 2 (excitationafter irradiation) giving a mono-exponential de­pendence. The relative fluorescence yields for case1 compared with case 2 may be estimated fromthe respective A factors (taking equal values forcase 1) and lifetimes by the equation: Y= LA;r;;for case 1, Y1 = (0.5 X2) + (0.5 x6) =4 andYz=(IX6)=6, giving a 50% increase from case1 to 2. However, in general, the counting ratefrom the laser spot showed little change, presum­ably due to diffusion and photodegradation, al­though in a few cases it did increase by 10% whenthe laser intensity was sufficiently attenuated. Thisis an important point, since it has often beenassumed in previous studies that a near-constantcounting rate implies no perturbation to the flue­rophore lifetime dynamics. Cells examined by theCCD system after laser spot probing exhibitedfluorescence distributions similar to those shownin Figs. 3(b) and 3(c) demonstrating that, duringthe course of the lifetime measurements, S2 under­went intracellular redistribution. Lifetime mea­surements were attempted at other subcellularsites but with varying success due to signal lim­itations: probing at the cell periphery outside thehighly fluorescent region yielded a lifetime of about6 ns.

4. Discussion

In previous papers, we have investigated theexcited singlet and triplet state properties of theAISnPc components using steady state and time­resolved techniques [13, 21-23]. Comparative stud­ies were carried out in methanol and phosphate­buffered saline which demonstrated that pertur-

112 M. Ambroz et al. / Fluorescence spectroscopy and intracellular imaging of AlS2Pc

bations to the phthalocyanine photoproperties,induced by the addition of varying numbers ofsulphonate groups, are negligible [13]. However,differences in the fluorescence quantum yields andlifetimes of S2 in these solvents were found: inaqueous solution 7=5.1±0.1 ns, <P=0.4±0.04; inmethanol 7=6.1 ±0.1 ns, <P=0.56±0.05. It wasconcluded that this effect was due to an increasein the quantum yield of internal conversion to 0.4in aqueous solution, consequently limiting the trip­let state yield to 0.2. In this work, the interactionof S2 with HSA has been studied at pH 7.4 in0.1 M PBS. Both the steady state and time-resolvedspectroscopic studies provide convincing evidenceof binding, although the affinity of S2 for HSAis weaker than found for haematoporphyrin. Bath­ochromic spectral modifications have also beenobserved for several porphyrins on binding to HSA[24] and have been attributed to the lower overalldielectric constant of the protein microenviron­ment. HSA is predominantly an a-helical globinprotein comprising three major domains, each ofwhich contains two subdomains. This morphologyoffers a variety of binding sites, with two particularsubdomains separated by approximately 8 nm iden­tified as being suitable conformations for smallmolecules [25]. From absorbance and fluorescencespectroscopic studies [24], the binding of hae­matoporphyrin to HSA is characterized by onehigh-affinity site, which is adjacent to the soletryptophan residue (204-Trp) on the basis of fluor­escence energy transfer from this residue to hae­matoporphyrin, together with several other un­specified lower affinity sites. We attempted thesame fluorescence energy transfer experiment usingS2 but the results were unconvincing. Nevertheless,the presence of one dominant decay componentclearly favours the presence of a specific bindingsite for S2. Our results may be compared withanother study [26] of HSA binding with AISPccontaining a mixture of components giving anaverage sulphonation of about three. Althoughthese results are not strictly comparable with thosepresented here, because the mixture componentshave different binding affinities, similar spectralchanges were reported in the presence of HSA;time-resolved studies were confined to the tripletstate and they concluded that the bound phthal­ocyanines were accessible to quenching speciesresiding in the solvent phase. Binding of S2 toHSA to form a non-covalent complex is an im­portant factor influencing the biodistribution ofthe sensitizer, since it is thought that HSA deliveryfavours interstitial localization, and S2 is certainlyknown to partition to these regions, especially

submucosal sites [7, 12). Binding of S2 with lipo­proteins also occurs at similar concentrations [21]but, given the large excess of HSA in serum,binding to albumin should predominate in vivo.

In the time-resolved fluorescence studies at thehighest HSA concentrations, S2 fluorescence de­cays could be fitted reproducibly within the timeresolution of the system using two lifetimes: onemajor component of 5.5 ns and one minor com­ponent of 1 ns. These results imply two bindingsites for S2. Time-resolved fluorescence studies ofhaematoporphyrin complexed with HSA at pH 7.4have also shown the presence of two lifetimecomponents with decay times near 9 and 17 ns[24]. It has been established that protein structuralfluctuations occur in the nanosecond time scale;thus single lifetimes resolved from simple expo­nential fitting may represent mean values for thevarious conformations in the dye/protein microen­vironment [19]. As shown in Table 1 and theaccompanying references, the 5.5 ns value of themajor S2 lifetime component is intermediate be­tween the lifetimes measured in H20 and othersolvents including micelles for which higher valuesaround 6 ns are consistently found. As mentionedabove, it has been shown recently that the fluor­escence quantum yield and lifetime in H20 arereduced owing to an enhancement in the rate ofinternal conversion. We therefore propose thatthis effect is inhibited partially when the phthal­ocyanine is protein bound, yielding the interme­diate lifetime value observed here in the presenceof HSA, with the assumption that the intrinsicsinglet state lifetime is not perturbed significantlyon binding. A similar shielding effect has beenobserved in the triplet state kinetics of AlSPcstudied using a tunable nanosecond flash photolysissystem [21]. In argon-saturated PBS, S2 exhibitsa triplet-triplet absorption band centred at 480nm which decays as a single exponential with alifetime of 600 ±40 I-LS. However, in the presenceof excess HSA, the lifetime of the triplet stateundergoes a substantial increase: in 100 I-LM HSA,

TABLE 1. Fluorescence lifetime measurements of 0.5 JLM S2in a range of solvent/model systems

Solvent TF (ns) Reference(A factor)

MeOH 6.1 ±0.1 [13, 22]

0.1 M PBS (pH 7.4) 5.0±0.1 [13]

Triton XIOO/PBS 5.9±0.1 This work

50 ILM HSA/PBS 5.5 ± 0.2 (0.92), This work1.0 ± 0.2 (0.08)

M. Ambroz et al. / Fluorescence spectroscopy and intracellular imaging of A1SzPc 113

T = 875 ±50 j.LS. This stabilization of the tripletstate was ascribed to a reduction in the rate ofintersystem crossing to the ground state of theprotein-bound dye compared with the free dye inH 20. In addition, the rate constant for oxygenquenching of the triplet state is decreased by afactor of ten when AISPc is protein bound (vs.unbound) as a result of the lower rate of oxygendiffusion through the protein matrix. It should benoted that the S2 fluorescence lifetime is insensitiveto quenching by oxygen and this could not explainour results [19].

However, the minor 1 ns lifetime component(A ::::: 0.1) appears to be anomalous and has noconvincing explanation; it is also intriguing to notethat the A factors for both components exhibiteda similar correlation with HSA concentration, in­dicating comparable affinities assuming the pres­ence of two binding sites. The short lifetime couldarise from binding at a site which induces efficientquenching by distortion of the phthalocyanine ma­crocycle, resulting in an enhanced rate of internalconversion. An alternative explanation would in­volve interaction with amino acid residues, but aseries of experiments in aqueous solution withseveral amino acids (trytophan, tyrosine, cysteine)did not provide any evidence for singlet statequenching, although admittedly we are comparingdifferent microenvironments. It is conceivable thatS2 may contain two isomers with distinctly differentbinding properties, but reproducible results wereobtained between different S2 preparations withdifferent isomeric compositions. In an attempt toresolve the components from differences in thepolarization anisotropy, we studied the time-re­solved polarization anisotropy of the bound dye.However, optimal fitting yielded a rotational cor­relation time of 45 ns which is the expected valuefor an immobilized fluorophore bound to a globularprotein of 65 kDa [19]. Of course, fitting with twolifetimes does not necessarily preclude anothertype of decay function, and the weak shortercomponent found here may merely allude to moresubtle decay dynamics of a single population. Forexample, deviations from simple exponential be­haviour can result from excited state dipole re­laxation processes in the protein matrix, and dif­fusion-controlled quenching which introduces anextra t 1/2 dependence [18]. The latter explanationis a possibility if the surrounding H2 0 solventmolecules may be considered to be a quenchingspecies, since solvation (and complexation of H20with the central AI) enhances the rate of internalconversion. MCP detection, with its superior timeresolution, may offer further refinement to these

provisional conclusions. The slight degree of steadystate fluorescence quenching (typically 10%), ob­served on addition of HSA, was near the limit ofthe experimental resolution and difficult to re­produce satisfactorily. In principle, from the smallcounterbalancing changes in lifetime behaviour,observed on binding, the overall fluorescence yieldof bound S2 should be indistinguishable from thefree dye. However, in practice, the increased scat­tering of the spectrometer excitation beam at higherHSA concentrations could have resulted in a smallapparent attenuation of the detected fluorescence.Adsorption of HSA onto the sides of the silicacuvette and subsequent binding of S2 is anotherpossible factor: with orthogonal excitation/detec­tion geometry, the fluorescence of S2 adsorbedon the cuvette would be undetectable. Adsorptionon silica is known to occur for bovine serum albuminand was investigated recently using the techniqueof evanescent-wave-induced fluorescence spec­troscopy [27].

As expected for an amphiphilic dye, S2 is in­corporated into Triton XlOO and crAB micelles;the partition coefficient (K) of S2 between TritonX114 and water has been studied previously [14]as a function of pH: at pH 7 and 37°C, K = 200,with the value increasing at lower pH as thesulphonate groups with pKa ::::: 3 become progres­sively acidified. The longer fluorescence lifetimeobserved in micellar solutions (approximately 6ns) compared with aqueous solutions is consistentwith exclusion from the aqueous phase, which wasalso demonstrated by a correlation time (approx­imately 8 ns) indicative of hindered rotation andsimilar to that observed in glycerol. For comparison,time-resolved studies of haematoporphyrin in Tri­ton XlOO revealed a single fluorescence lifetimeof 16 ns typical of the monomeric species [24].

These studies on model systems were followedby an in vitro investigation of intra-cellular S2using the leukaemic K562 cell line. Imaging witha CCD camera revealed that S2 fluorescence pre­dominated in a discrete perinuclear region whichwas then selectively probed in isolated cells by afocused laser spot for fluorescence decay mea­surements. Two-component fits were observed giv­ing lifetimes of 2.2 and 6.1 ns with comparablepre-exponential factors. However, on extendedirradiation, the fluorescence decays were char­acterized by a single lifetime of 6.1 us, with theshorter 2.2 ns component being excluded. Thisevolution from bi-exponential to mono-exponentialdecay behaviour suggests that a major change inthe S2 microenvironment occurs during irradiation,which must be due in part to photosensitized

114 M. Ambroz et al. / Fluorescence spectroscopy and intracellular imaging of AlS2Pc

oxidation of the subcellular sites (e.g. organelles)associated with S2, and probably combined withdiffusion of S2 away from these sites. The fluor­escence lifetimes of A1SPc (mean sulphonation oftwo) have been measured in murine ascitic tumourcells [28} incubated in vivo but studied ex vivoin a suspension. The bulk fluorescence decays weremulti-exponential with lifetimes close to the mi­croscopic values found here. The presence of theshorter lifetime components in that study and oursshows that Al5Pc may undergo a significant degreeof fluorescence quenching in vitro at certain in­tracellular sites with lysosomes being prime can­didates. Assuming that lysosome localization issignificant, the observed fluorescence redistributioncan be explained by photo-oxidation of lysosomalmembranes leading to the release of S2 togetherwith hydrolytic enzymes into the cytosol whichwould ultimately result in cell autolysis. The mech­anism of AlSPc sensitization of cell membraneshas recently been studied [29] using erythrocyteghosts, which were shown to sustain damage fromextensive lipid peroxidation in addition to cho­lesterol degradation specific to a type 2 processmediated by singlet oxygen. The subcellular lo­calization of S2 and 54 has been studied previously[30] in carcinoma cells (NHIK 3025) which ex­hibited uptake mainly by lysosomes; furthermore,it was proposed that S2 partitioned mainly tolysosomal membranes, whereas the hydrophilictetrasulphonated component resided in the centralaqueous compartment. In another recent study[31] on S2 localization in a human melanoma cellline using confocal scanning, uptake in mitochon­dria as well as lysosomes was implicated. We alsonote that localization in the Golgi apparatus wassuggested in a recent study of AISPc (mixed sul­phonation) photosensitization of K562 cells [32].In view of these potential complications, the par­titioning of S2 between subcellular sites remainsunresolved and further studies using confocal scan­ning of S2 in K562 cells would be desirable.

Regarding the origin of the fluorescence quench­ing mechanism, we can only speculate which sub­strates might be involved. Assuming that locali­zation in lysosomes is important, the influence ofpH should be considered since the intralysosomalcompartment is relatively acidic (about pH 5).Such an effect, resulting in a marked lifetimeshortening of lysomotropic sulphonated tetra­phenyl porphyrin in RR1022 epithelial cells [33],has been observed using time-resolved microfluo­rometry and was ascribed to the presence of aprotonated monocationic species at the lysosomalpH. However, from previous studies [13] we can

eliminate any perturbation in the S2 lifetime atthis pH; a shorter lifetime is observed under moreacidic conditions (below pH 3), but this is ac­companied by pronounced spectral changes whichwere not observed here. A more plausible quench­ing mechanism would involve energy transfer orelectron transfer between S2 and substrates at ornear the S2 sites. For example, we already knowthat highly efficient quenching of the Al5Pc singletstate occurs with the electron acceptors methylviologen and anthraquinone in aqueous solution[34]. On photosensitization, the binding sites wouldpresumably be destroyed, thus eliminating thequenching, and resulting in the longer fluorescencedecay observed on extended irradiation. The dif­ficulty with this explanation is that, for a highproportion (nearly half) of the heterogeneous S2sites, a suitable substrate must reside in closeproximity to S2; moreover, without a precise knowl­edge of S2 localization it is obviously impossibleto identify a specific class of substrates in cellswhich could account for either of these quenchingprocesses.

A further possibility is the interaction betweenadjacent phthalocyanine molecules, resulting inconcentration quenching of the excited state byground state S2. In the preceding analysis, wehave assumed that fluorescence from sulphonatedphthalocyanine aggregates is undetectable, as otherworkers have concluded [30, 31], and would there­fore exhibit an unresolvable lifetime on the pi­cosecond time scale. Certainly the 52 fluorescenceand excitation spectra observed from suspensionsof K562 cells appeared to be consistent with mon­omeric 52, although it should be noted that theemission spectrum of dimeric unsulphonated zincphthalocyanine in liposome membranes closelyresembles that of the ZnPc monomer [35]. Froman analysis of the fluorescence decays as a functionof ZnPc concentration, it was concluded that thedimeric species in liposomes exhibits a lifetimeapproximately half that of the monomer. Somedegree of aggregation of intracellular S2 wouldbe expected: the estimated cellular uptake wasapproximately 107 molecules per cell which wouldgive a local concentration approaching 10-3 M inthe region probed. At such a concentration inaqueous solution, 52 would be completely aggre­gated, but intracellular binding to substrates and/or association with membrane structures would beexpected to promote partial monomerization [30].It is therefore conceivable that self-quenching be­tween S2 molecules either associated or in closeproximity (e.g. in membranes) may be possible atsuch high concentrations; alternatively, a distinct

M. Ambroz et al. / Fluorescence spectroscopy and intracellular imaging of AIS2PC 115

type of intracellular fluorescent aggregate couldbe invoked. It would be desirable to replicate suchbehaviour in model systems, such as liposomalmembranes, with high dye to lipid ratios in orderto induce S2 aggregation within the membrane.Studies of the photophysics of S2 in reversedmicelles [36] may also aid the interpretation ofthe non-exponential fluorescence decay dynamicsof S2 in heterogeneous environments.

The attractive feature of a concentration quench­ing mechanism is that it could explain the transitionto the longer mono-exponential lifetime behaviourwhen S2 undergoes dilution following photosen­sitized redistribution. In order to verify conclusivelythe contribution of such a mechanism, experimentsat lower concentrations would have been desirable;however, the limited fluorescence intensities avail­able with the present detection system precludeda more complete investigation. The increase in S2fluorescence intensity after the addition of de­tergent to unirradiated cellular suspensions is alsoconsistent with the presence of fluorescencequenching in whole cells. The lack of any distinctconcentration dependence using S2 concentrationsin the range 2-20 IJ-M (although only a factor offour difference in cellular uptake was observed)would appear to argue against a self-quenchingmechanism, but it is possible that localization inthe relevant sites becomes saturated even at thelowest concentration, thus limiting correlation withconcentration. The small Stokes shift of the fluor­escence emission from the S2 Q-band may favouran energy transfer mechanism, and fluorescencedepolarization studies (not attempted here) could,in principle, provide evidence of such a mechanism.It is interesting to note that, from transient ab­sorption studies [21, 22], we have established thatground state quenching limits the S2 triplet statelifetime with a rate constant of approximately2x lOS M- 1 S-l in water.

Following extended irradiation, the evolution ofthe lifetime to just one longer component near 6ns may be interpreted in terms of the photosen­sitized elimination of the nascent fluorescencequenching process; equivalently, we can infer that(before photodegradation becomes significant) theoverall S2 fluorescence efficiency is increased dur­ing irradiation. The transition between these twolifetime regimes appears to be consistent withfluorescence imaging studies on whole cells inwhich photosensitized alterations in fluorescenceintensity and distribution were observed using ex­tended irradiation at 633 nm, although thesechanges occurred over a longer time scale thanwith the focused laser spot at 610 nm which had

a much higher irradiance (factor of ten); moreover,the S2 absorbance at 610 nm is twice that at 633nm. It is also pertinent to note that irradiationat 633 nm for over 90 s (see Fig. 3(c)) at 1 Wcm - 2, giving an energy dose of 90 J cm - 2, isequivalent to approximately 10 J cm -2 at 675 nm,which is comparable with that employed in previousin vitro studies [15] of AISPc photosensitizationof K562 cells: irradiation with approximately 30J cm -2 (using 675 nm) was lethal to more than95% of the cells under the same incubation con­ditions as used here. Although a precise comparisonbetween the different sets of microscopic data issomewhat conjectural, the increases in microscopicsteady state fluorescence, as shown in Figs. 3(a)and 3(b), are similar to the approximate 50%increase in the fluorescence yield from case 1 to2 in the lifetime studies. We therefore proposethat the transition between the two lifetime regimesoccurs concomitantly with the intensification andredistribution of intracellular S2 as a result of thephotosensitized rupture of organelles. This viewis reinforced by the observation that, after theacquisition of case 2 data, the intracellular dis­tribution closely parallelled that of Fig. 3(b). Theseresults may have phototherapeutic implicationssince, if significant fluorescence quenching to theground state is present, the triplet and singletoxygen yields will be correspondingly reduced anda lower photosensitization efficiency will be ob­tained. Moreover, differential fluorescence quench­ing at various sites would affect the correlationof sensitizer localization with fluorescence imagingof cells and frozen sections. Further studies shouldinvestigate the degree of quenching in other celllines and tissue explants.

Similar photosensitization effects have been re­ported with AISPc in other cell lines using quan­titative microscopic imaging. The behaviour of amixture of S3 and S4 was studied [37] in cells atcomparable concentrations as in this study, butafter a much longer incubation time of 24 h fortwo cell lines: RR1022 rat epithelial cells and 3T3murine fibroblasts. Following irradiation at 675nm with 3.8 W em -2, a fluorescence increase (factorof two) was observed for RR1022 cells, but notfor the fibroblast line (this disparity may be relatedto different subcellular localization), and photo­bleaching was subsequently observed in each linewith prolonged irradiation. In another study, com­parative experiments were perfonned on a humanmelanoma cell line with S2 and S4 [31]; a largeinitial increase in cellular fluorescence was ob­served for both S2 and S4 components, but thiseffect was attributed to disaggregation to form

116 M. Ambroz et al. / Fluorescence spectroscopy and intracellular imaging of AIS2Pc

fluorescent monomers following photosensitizationof organelles containing non-fluorescent aggre­gates. This explanation is reasonable since an 18h incubation time was used, which should resultin much higher intracellular concentrations andtherefore favour aggregation. Our results indicatea slightly different conclusion, since our studiesinvestigated a much lower concentration regime,but in the absence of time-resolved data from theother cell lines we believe that the two interpre­tations are compatible. The common feature ofall these in vitro studies is the ultimate processof photodegradation or photobleaching. Photo­degradation of AISPc in aqueous solution is min­imal with a quantum yield of only approximately10- 6 [38], since phthalocyanines are relatively re­sistant to singlet oxygen attack. In the presentcase, we irradiated the cells with about 1 W cm- 2

giving an incident fluence of approximately 1020

hv cm - 2 delivered over an estimated photodeg­radation half-life of approximately 102 s. In com­bination with the absorption cross-section of S2(10- 16 molecule -I cm2 at 633 nm), we can estimatethat the photodegradation quantum yield in K562cells is in excess of 10- 5

, most probably occurringvia type 1 processes [24]. Photodegradation ofAISPc has also been shown to occur in vivo attherapeutic light doses, and this process is nowrecognized to be a key factor in improving theselectivity of PDT, since it allows normal tissueadjacent to tumour to be spared if the AISPc doseis below a certain threshold level. However, incontrast with the in vitro results reported hereand elsewhere, an initial increase in fluorescenceintensity has not been observed upon irradiationin vivo. Several factors may be involved in thisdisparity: firstly, much lower sensitizer concentra­tions are generally present in vivo and the presenceof non-fluorescent aggregates is consequentlylower, and secondly, a substantial amount of thesensitizer may be bound to extracellular compo­nents. In the case of AISPc we know from CCDfluorescence imaging of frozen tissue sections thata major fraction is retained in interstitial regionscontaining macromolecular substrates such as col­lagen [12].

5. Conclusions

This work has demonstrated the value of thecombination of time-resolved microfluorometryand quantitative fluorescence imaging to unravelcomplex processes in cellular systems. This tech­nique has many other potential applications in

studies of heterogeneous samples (not only bio­logical) on the microscopic scale with the nextstage being to assemble a time-resolved fluores­cence imaging system with subnanosecond timeresolution and submicrometre spatial resolutionusing confocal optics. Fluorescence decays andtime-gated spectra would be acquired using single­photon-counting techniques with detectors (e.g.microchannel plates and photon-counting ava­lanche photodiodes) offering enhanced time res­olution and sensitivity.

Acknowledgments

We are grateful to the Imperial Cancer ResearchFund, and also acknowledge generous support fromthe Waldburg Trust, the Science and EngineeringResearch Council and The British Council for theaward of a scholarship to M. Ambroz. Assistancefrom C.l.R. Singer and A. Beeby is acknowledged.

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