Intracellular Uptake and Phototoxicity of 31,32-Didehydrophytochlorin-fullerene Hexaadducts

Intracellular Uptake and Phototoxicity of31,32-Didehydrophytochlorin-fullerene Hexaadducts

Fiorenza Rancan*1,2, Matthias Helmreich3, Andreas Molich4, Norbert Jux3, Andreas Hirsch3,Beate Roder2 and Fritz Bohm1

1Hautklinik, Photobiologisches Labor, Universitatsklinikum Charite, Berlin, Germany2Institut fur Physik, Humboldt Universitat, Berlin, Germany3Institut fur Organische Chemie, Universitat Erlangen-Nurnberg, Erlangen, Germany4Institut fur Biologie, Tierphysiologie, Humboldt Universitat, Berlin, Germany

Received 5 March 2007; accepted 8 May 2007; DOI: 10.1111/j.1751-1097.2007.00163.x

ABSTRACT

C60-fullerene derivatives are potential building blocks in modular

carrier systems for selective tumor targeting. In [5:1] fullerene

hexakis adducts, one position can be occupied by an addressing

unit (e.g. monoclonal antibody) while the other five positions are

suitable for dendrimers or spacers loaded with several drug

moieties. This article reports intracellular uptake and photo-

toxicity of three fullerene hexakis adducts coupled with a

different number of photosensitizers: a bis(31,32-didehydrophyto-

chlorin)-fullerene [5:1]-hexaadduct (FHP1), a fullerene [5:1]-

hexaadduct with six 31,32-didehydrophytochlorin groups (FHP6)

and a fullerene [6:0]-hexaadduct that carries 12 31,32-dide-

hydrophytochlorin units (FHP12).The most promising complex,

the hexa-31,32-didehydrophytochlorin fullerene hexaadduct

FHP6, was also compared with its fullerene-free analogous

derivative P6. It was found that the extent of intracellular uptake

is influenced by both nanomolecular size and asymmetry

(amphiphilicity) of the fullerene complexes. The degree and

mechanism of phototoxicity was found to depend on intracellular

concentrations and singlet oxygen quantum yields.

INTRODUCTION

Several drug delivery systems have been developed to increasedrug selectivity toward a specific target. The accumulation of a

drug exclusively at its site of action can improve its efficacy andreduce undesired side effects (1,2). Carrier systems might bevery useful also in photodynamic therapy (PDT) (3–5). In fact,an insufficient phototoxic activity and side effects such as skin

photosensitization and chest pain (6) are the reasons for alimited use of this treatment in cancer therapy. Photosensitizers(PSs) with higher selectivity for their target tissue might be

therefore more effective and at once less harmful. A newgeneration of ‘‘intelligent’’ carriers for PS has been proposed(4,7,8). These should localize preferentially in the target tissue,

thanks to their ability of recognizing specific proteins orreceptors expressed by tumor cells. Monoclonal antibodies(MAbs) or their fragments were found to be the most selective

carrier system (9–11). However, a disadvantage of MAbs isthat, once loaded with several PSs, they may loose affinity fortheir antigen (12). To solve this problem the concept ofmodular drug delivery system (MDDS) was proposed (13). An

MDDS is a combination of units or modules with diversespecific functions. Besides the therapeutic unit, an addressingunit serves to direct the drug to the target and a multiplying unit

has the role of increasing the number of drug moieties thesystem can carry without affecting the recognizing ability of theaddressing unit. Following this model, by means of one

multiplying unit, a number of drug moieties can be loaded toone monoclonal antibody (mAb) with a minimal loss of mAb’santigen-binding ability. C60-fullerene derivatives are potentialmultiplying units for such delivery systems. Fullerene hexakis

adducts have an octahedral addition pattern that allows the sixsubstituents to occupy spread positions with minimal stericinteractions (14). In [5:1]C60 hexakis adducts, one position can

be occupied by the addressing unit while the other five positionsare suitable for dendrimers or spacers loaded with several drugmoieties. The suitability of fullerene adducts as multiplying

units for MDDSs was already the matter of our investigations.First, we synthesized two fullerene derivatives, a mono- and ahexaadduct (FP1, FHP1, Fig. 1) coupled through spacers to

two 31,32-didehydrophytochlorin (short Pyro, from the trivialname pyropheophorbide-a) moieties (15). Studies of the photo-physical properties of these derivatives revealed a strongreduction in singlet oxygen quantum yield for the fullerene

monoadduct. This occurs as a result of the high electron-accepting capabilities of C60. On the contrary, in the fullerenehexaadduct the p-system is broken up and C60 does not act as

electron acceptor anymore (15,16). As a direct consequence, thein vitro phototoxicity of the fullerene monoadduct FP1 wasdrastically inferior to that of the fullerene hexaadduct FHP1

carrying the same number of PSs (17). Next, we synthesizedfullerene hexaadducts carrying six (FHP6) (18,19) and 12(FHP12) (20) 31,32-didehydrophytochlorin moieties (Fig. 1).

The investigation of the photophysical properties of the twohexaadducts revealed that neighboring dye molecules cova-lently linked to one fullerene moiety could interact with eachother forming energy traps. A very fast and efficient delivery of

the excitation energy to a trap can therefore occur via dipole–dipole Forster energy transfer (18–20). This explains the

*Corresponding author email: [email protected] (Fiorenza Rancan)� 2007TheAuthors. JournalCompilation.TheAmericanSociety ofPhotobiology 0031-8655/07

Photochemistry and Photobiology, 2007, 83: 1330–1338

1330

Figure

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icalstructuresofphotosensitizer31,3

2-didehydrophytochlorin,hexa-3

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2-didehydrophytochlorinsystem

P6andfullerenehexakisadductsFHP1,FHP6andFHP12.

Photochemistry and Photobiology, 2007, 83 1331

reduction in fluorescence as well as singlet oxygen quantumyields with increasing number of dye molecules per complex(21).

This article reports the photobiological properties of the

three aforementioned fullerene hexaadducts FP1, FHP1 andFHP6 in comparison with those of the uncoupled PS (31,32-didehydrophytochlorin, short Pyro). In addition, the most

promising fullerene hexaadduct FHP6 was compared with itsanalogous fullerene-free hexa-31,32-didehydrophytochlorindendron derivative (P6).

MATERIALS AND METHODS

Studied compounds. Figure 1 reports the chemical structures of thestudied compounds. 31,32-didehydrophytochlorin (Pyro) was obtainedby in situ hydrolysis and decarboxylation of pheophorbide-a accordingto a literature protocol (22,23). The syntheses and photophysicalproperties of FHP1 were recently reported (15,16), while the photo-biological properties are reported in (17). Synthesis and photophysicalproperties are described in Refs. (18,19) for FHP6 and in Ref. (20) forFHP12. A comparative study of the photophysical behavior of thecompounds is reported in Roder (21). All compounds were dissolved indimethylformamide (DMF; Sigma, Germany).

Cell culture and incubation conditions. A special line of humanT-lymphocytes (Jurkat cells: clone E 6-1, human acute T-cell leukemia,ECACC-Catalogue) were cultivated in 50 mL flasks in 8 mL RPMI1640 medium containing Glutamax-I, supplemented with 10%fetal calf serum, 100 lg mL)1 streptomycin and 100 IE ⁄mLpenicillin. Cells were cultivated at 37�C in 100% humidity and 5%CO2 and were seeded in new medium every 2–3 days. Jurkat cells(2 · 105 cells mL)1) were incubated in RPMI 1640 medium with a0.5% DMF solution of the studied derivative. Different incubationconcentrations were used. Cells incubated in growing medium with0.5% DMF were used as reference.

Intracellular uptake. The intracellular uptake of sensitizers wasinvestigated using a confocal laser scanning microscope (CLSM 510;Zeiss) equipped with a Helium–Neon laser (kexc = 633 nm). Pictureswere taken capturing the fluorescence at an emission wavelengthkem > 655 nm. After 24 h incubation with the studied compounds(1 lMM), cells were washed twice with Dulbecco’s phosphate buffersolution (PBS; Sigma) and analyzed. At least four different fields wereobserved for each sample. The amount of PS taken up by Jurkat cellswas determined measuring the fluorescence intensity of cell extracts.Cells were harvested after different incubation times, washed twice withPBS, counted using a hemocytometer (Neubauer improved) andcentrifuged. The cell pellet was stored at )20�C for 20 min. Cells werethen thawed and DMF was added. The fluorescence intensity of cellextracts was measured using a fluorescence microplate reader GeminiEM (Molecular Devices, Germany), using kexc = 400 nm andkem > 672 nm. The PS concentration in the cell extracts was quantifiedby comparison with standard curves. The intracellular concentrationwas calculated considering the cell number after each incubation time.

Subcellular localization. Jurkat cells were incubated with thestudied compounds and successively with the dyes with affinity for aspecific cell organelle (Molecular Probes, Inc.). Mitochondria werestained with MitoTraker-Green FM (200 nMM, 45 min, 37�C), lyso-somes with LysoSensor-Green DND-189 (75 nMM, 60 min, 37�C),the Golgi apparatus and the endoplasmic reticulum with NBDC6-sphingomyelin (200 nMM, 60 min, 37�C). The fluorescence of theorganelle probes was detected using kexc = 458 nm and kem = 475–525 nm (BP 475–525), while the fluorescence of the PS was detectedusing kexc = 633 nm and kem > 650 nm (LP 650). For each fieldimages were recovered of transmitted light, 31,32-didehydrophyto-chlorin fluorescence (red) and organelle probe fluorescence (green).Images of superimposed fluorescence (merged) were created using thePaint Shop Pro 7 software to illustrate the extent of co-localization.

Irradiation conditions. A special setup was used for cell irradiationexperiments. After incubation with or without PS, cells were washedtwice, resuspended in a red phenol-free medium and placed in a 96-wellculture plate (Falcon), 100 lL per well. The 96-well culture plate waspositioned on a plane having a circular hole with a surface of 0.32 cm2,

which corresponds to the surface of a single well. A laser diode withemission at 668 nm is embedded in a cylinder whose walls are coveredwith barium sulfate. The cylinder is fixed on the bottom of the plane incorrespondence with the above mentioned hole. The output of the laserthrough the hole and a culture plate cover was 0.60 mW, so that theresulting irradiance was 2.12 mW cm)2. Irradiation times of 3 min, 60and 30 s were used, corresponding to light doses of 400, 192 and64 mJ cm)2, respectively. Fresh medium was added and cells wereincubated at 37�C, 5%CO2and100%humidityprior to further analysis.

Cell vitality and proliferation. Cell vitality and proliferation wereassessed using the XTT test (Roche, Germany). The assay is based onthe conversion of the yellow tetrazolium salt XTT into an orangeformazan dye by the activity of mitochondria dehydrogenase in viablecells (24). The amount of formazan production was analyzed spectro-photometrically at the absorbance of 492 nm. The reference wavelengthwas 650 nm. The blankODwas subtracted from the sample OD and thepercentage of cell vitality and proliferation was determined as follows:(ODsample ⁄ODreference) · 100. The results are the mean of three inde-pendent experiments. Statistics were performed with the Mann–Whit-ney U-test. Differences were considered significant when P < 0.05.

Caspase-3 ⁄ 7 assay. Caspase 3 ⁄ 7 activity was assessed using afluorimetric assay kit (Apo-ONETM Homogeneous Caspase-3 ⁄ 7Assay; Promega). The assay provides a caspase-3 ⁄ 7 substrate(Z-DEVD-R110) as a pro-fluorescent substrate. The cleavage andremoval of the DEVD peptide by caspase 3 and 7 leads to theproduction of free rhodamine 110. Its fluorescence intensity isproportional to the enzyme’s activity. The fluorescence intensity wasmeasured using a fluorescence microplate reader Gemini EM (Molecu-lar Devices), using kexc = 485 nm and kem > 530 nm. Cells incubatedat 37�C with 1.5 lMM staurosporine (Sigma) were used as positivecontrol. The maximum caspase 3 ⁄ 7 activity was detected after 3–4 hincubation. The caspase 3 ⁄ 7 activity of the samples is expressed aspercentage of the positive control values after 4 h stimulation. Barsrepresent the standard deviation of three experiments.

RESULTS

Intracellular uptake

The intracellular uptake of 31,32-didehydrophytochlorin andthe fullerene hexaadducts was first investigated with a CLSM.For all compounds, fluorescence was detected in sections

belonging to intracellular compartments. Successively, thefluorescence intensity of cell extracts was measured to followthe compounds’ intracellular accumulation in dependence on

the incubation concentration (Fig. 2).As the studied fullerene adducts carry a different number of

PSs, a distinction was made between the concentration of the

entire molecule and that of the carried PS. The intracellularuptake is reported both as concentration of entire molecules(Fig. 2a) and as concentration of Pyro moieties (Fig. 2b).A dependency of intracellular uptake on the incubation

concentration was detected for all compounds except forFHP12, which was taken up by cells in very low amounts.Considering the intracellular uptake of the whole molecules,

the free PS (Pyro) was found to reach higher intracellularconcentrations than the fullerene hexaadducts (Fig. 2a). Sig-nificantly different intracellular uptake was also found when

comparing the three fullerene derivatives. At an incubationconcentration of 1 lMM, FHP6 reached intracellular concentra-tions about five times higher than FHP1 and about 40 timeshigher than FHP12. The quantity of Pyro moieties that FHP6

carries into Jurkat cells is comparable to that reached bythe free PS at almost all incubation concentrations (Fig. 2b).On the contrary, the amount of PS moieties internalized by

FHP1 and FHP12 is extremely low in comparison with thatmeasured with FHP6 and Pyro. For instance, at the incubation

1332 Fiorenza Rancan et al.

concentration of 1.0 lMM FHP6, Pyro intracellular concentra-tions are about 15 and 20 times higher than those obtained

with FHP1 and FHP12, respectively.

Subcellular localization

The subcellular localization of fullerene hexaadducts was

studied with a CLSM by comparison of the fluorescencepattern of the fullerene adducts with those of dyes withspecific affinity for cell organelles like mitochondria, lyso-

somes and the Golgi apparatus. The three complexesshowed an accumulation pattern similar to that of Lyso-Sensor-Green, the dye which localizes specifically in lyso-somes. Figure 3 reports a summary of images captured for

cells incubated with the fullerene adducts and LysoSensor-Green.

The first column (Fig. 3a) shows transmitted light pictures.

The cell nuclei are well visible, as well as some globularvesicles in a region near the nuclei. Column B shows theimages of the red fluorescence of the PS taken using a long-

pass (LP) filter transmitting light with wavelengths >650 mm.For all three fullerene hexaadducts fluorescence was detectedin sections belonging to cell internal compartments. The red

fluorescence of 31,32-didehydrophytochlorin molecules has ahigh intensity in a defined region near cell nuclei, whichcorresponds to the vesicles visible in the transmitted lightimages. Fluorescence is also visible in the perinuclear region

and sometimes near the plasma membrane. Column C reportsthe images of fluorescence registered in the green spectralregion using a bandpass filter that transmits light of wave-

length between 475 and 525 nm. Fluorescence was detected indefined regions near cell nuclei that correspond to those wherePS fluorescence has the highest intensity. In the merged

images (Fig. 3d) the fluorescence of the two compounds (redand green) are merged. The orange-yellow color illustrates theextent of co-localization. In most of the cases, a yellow color

was evident in the region near the nuclei but not in theperinuclear region. In cells incubated with fullerene hexaad-ducts and organelle probes specific to mitochondria and theGolgi apparatus, the green fluorescence pattern did not match

those of PS (data not shown). No fluorescence was detected incell nuclei.

Phototoxicity

Figure 4 reports the phototoxicity toward Jurkat cells offullerene hexaadducts and free PS (Pyro) at different incuba-tion concentrations. Phototoxicity was determined measuring

cell vitality and proliferation (Fig. 4a) as well as apoptosisinduction (Fig. 4b). Cell vitality and proliferation decreasedwith increasing incubation concentrations for compoundsFHP6, FHP1 and Pyro. No significant cell vitality decrease

was found for FHP12 (Fig. 4a). The free PS exerts a highphototoxic effect. At a Pyro concentration of 0.33 lMM, only40% of living cells were detected after photosensitization. At

this concentration, most of the dead cells had undergoneapoptosis (Fig.4b). At higher incubation concentrations theratio of cell vitality decreases and at a concentration of

1.67 lMM almost all cells were lethally damaged. Further, thepercentage of apoptotic cells decreases at higher incubationconcentrations. As determined with the trypan blue test, at

incubation concentrations higher than 0.33 lMM almost all deadcells were found to be necrotic. At a Pyro concentration of1.67 lMM approximately 99% of necrotic cells were detected(data not shown).

Considering the three fullerene hexaadducts, FHP6 wasthe most phototoxic nanosized complex. At the lowestincubation concentration, 67% of the living cells were

detected for samples treated with FHP6, while at the sameconcentration FHP1 and FHP12 exerted no significantphototoxicity. FHP1 was phototoxic only at incubation

concentrations higher than 0.33 lMM. Cells treated withFHP12 had percentages of living and proliferating cellssimilar to those of reference cells. Nevertheless, caspase 3 ⁄ 7activity was found for all three fullerene adducts. At

increasing incubation concentrations, the number of apop-totic cells increases, and at the highest incubation concentra-tion 47%, 27% and 23% of apoptotic cells were measured

for FHP6, FHP1 and FHP12, respectively.

Comparison of FHP6 with fullerene-free analogous P6

The intracellular uptake and phototoxicity of FHP6 were

compared to those of P6, the analogous derivative without

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Figure 2. Intracellular uptake of fullerene hexaadducts (FHPn, a) andof 31,32-didehydrophytochlorin (Pyro) moieties (b) in dependence oncompound incubation concentration of the studied compounds. Theamount of compounds taken up by Jurkat cells was determinedmeasuring cell extract fluorescence intensity at 668 nm. The compoundconcentrations in cell extracts were extrapolated from fluorescenceintensity standard curves.


C60. Figure 5 shows the subcellular localization of P6 as well

as its intracellular uptake in comparison with those of FHP6

and Pyro. The accumulation pattern of P6 in Jurkat cells issimilar to that of FHP6 (Figs. 3 and 5). The compounds aredistributed in globular vesicles in a region near the cell

nucleus. The fluorescence distribution pattern of P6 is similarto that of LysoSensor-Green. This is indicative of thelysosomal localization of the dendritic compound. The

intracellular uptake of FHP6 is evidently higher than thatof its analogous P6, approximately seven times higher. Theuptake kinetic of Pyro showed increasing concentration after

incubation times of 2 and 4 h, and a small decrease after alonger incubation time (24 h). On the contrary, the intracel-lular concentrations of P6 and FHP6 did not demonstrate

significant changes with incubation times.Figure 6 illustrates the phototoxic activity of P6 and

FHP6.Under the used conditions, no dependency on light dose

was found for P6, which induces approximately 10% ofcell death. FHP6 phototoxicity was light dose dependentand significantly higher than that of P6. After a light dose of

64 mJ cm)2, 78% of living cells were detected for FHP6, withno significant decrease after a three times stronger light dose(192 mJ cm)2) but with a remarked phototoxic effect (36.3%

viable cells) after a light dose of 400 mJ cm)2.

DISCUSSION

Intracellular uptake and localization

The images recovered with the CLSM are a clear proof that

these nanosized fullerene derivatives can be taken up by Jurkatcells. All fullerene–PS complexes were found to localize mainlyin lysosomes. Nevertheless, fluorescence was also detected in the

perinuclear region. This indicates that some of these moleculesare able to exit lysosomes or to enter cells via a nonendocyticmechanism, localizingmostly on the nuclear membrane without

entering the cell nuclei. The higher intracellular accumulation ofPyro with respect to C60 derivatives is mainly due to thedifferent sizes of the studied derivatives. The molecular weight(MW) of Pyro is 536 g · mol)1 whereas that of the C60

derivatives ranges from 4300 g · mol)1 (P6) to 8700 g · mol)1

(FHP12). In general, small molecules (MW < 1000 g mol)1 or1 kDa) can diffuse through cell membrane, whereas molecules

with MW higher than 1 kDa may enter cells by means ofendocytosis (25). The lower intracellular concentrationsreached by the fullerene hexaadducts with respect to Pyro

would suggest that their uptake occurs via endocytosis. Afurther proof that these nanosized compounds are endocytosedby Jurkat cells is their accumulation in lysosomes (Fig. 3). In

fact, endocytosed molecules are accumulated in endosomes,which then fuse with lysosomes (25,26).

FHP6

FHP12

(d)(c)(b)(a)

FHP1

Figure 3. Subcellular localization of fullerene hexaadducts FHP1, FHP6 and FHP12. Jurkat cells were incubated 24 h with the studied compoundsat a molar concentration of 1 lMM. The organelle probe (LysoSensor-Green) was then added to the cells. After 2 h incubation cells were washedtwice and observed with a confocal laser scanning microscope. For each field, images were recovered of transmitted light (a), 31,32-didehydrophytochlorin fluorescence (b) and organelle probe fluorescence (c). Images of superimposed fluorescence (d) were created to illustrate theextent of co-localization.


The comparison of the three fullerene hexaadducts in regardto their intracellular uptake reveals consistent differences(Fig. 2a). Such different uptake ratios cannot be adduced to

the different molecular sizes of the complexes; otherwise theintracellular uptake of P6 would be higher than those of FHP1

and FHP6. The chemical structure of FHP6 and FHP1 is

probably the reason for their higher uptake with respect to P6

and FHP12. The last ones are symmetric molecules. On thecontrary, FHP1 and FHP6 are asymmetric structures in which

a lipophilic part (fullerene) is bound to a more hydrophilic part(spacers and PS moieties). The amphiphilicity of these com-pounds might favor their intracellular uptake by means of a

special endocytic mechanism. It has been reported that thespherical shape of fullerene and its lipophilicity confer it theability to adapt to enzymes or receptor active sites (27).Shraiman (28) found a structural analogy between the fullerene

cage and that of the clathrin-coated vesicles, which are involvedin one of the several endocytosis mechanisms. These can beroughly classified as: non–receptor-mediated pinocytosis,

adsorptive pinocytosis and receptor-mediated endocytosis.

The first is merely an internalization of fluids without anyinteraction of the uptaken molecules with membrane compo-

nents. This process results in low intracellular concentrations(29) and it could be involved in the intracellular uptake ofFHP12 and P6. Adsorptive and receptor-mediated endocytosisrequire an interaction with the cell membrane. As binding of

the molecule to the membrane is a major concentrating step, itresults in a higher intracellular uptake than non–receptor-mediated endocytosis (29–31). Thus, it could be possible that

fullerene derivatives FHP6 and FHP1 accumulate in Jurkatcells by adsorptive endocytosis, thanks to their structures thatfacilitate interactions with special plasma membrane domains.

Furthermore, asymmetrical, amphiphilic compounds may formmicelles. Fullerene derivatives that can form micelles in waterhave already been reported (32). It is possible that FHP1 and

FHP6, once added to cell culture medium, form micelles whichfind a special way for cellular internalization.

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Figure 4. Cell proliferation (a) and apoptosis induction (b) of fullerenehexaadducts and 31,32-didehydrophytochlorin (Pyro) at differentincubation concentrations. Cell vitality and proliferation percentageswere determined with the XTT assay 24 h after cell irradiation(668 nm, 2.12 mW cm)2, 64 mJ cm)2). Apoptosis was determinedmeasuring caspase 3 ⁄ 7 activity 4 h after irradiation with the same lightdoses. The percentage of caspase 3 ⁄ 7 activity was calculated withrespect to samples incubated with 1.5 lMM staurosporine. Bars representthe standard deviation of three experiments.

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Figure 5. Intracellular accumulation and localization of P6. Thepictures report Jurkat cells 24 h after incubation with compounds P6(2 lMM) and the organelle probe LysoSensor-Green (75 nMM). Images oftransmitted light (a), P6 fluorescence (b) and LysoSensor-Greenfluorescence (c) were recovered with a confocal laser scanningmicroscope. Images of superimposed fluorescence (d) were created toillustrate the extent of co-localization. The diagram reports the amountof PS moieties in cell extracts after different incubation times withcompounds Pyro, FHP6 and P6 (1 lMM).


However, the higher intracellular uptake of FHP6 withrespect to FHP1 has also to be explained. In fact, FHP6 reachesin Jurkat cells five-fold higher molar concentrations thanFHP1. Again, the different chemical structures may explain the

dissimilar uptake of FHP1 and FHP6. The main structuraldifference between the two fullerene complexes is representedby the spacers connecting the PS to C60. In FHP1 the spacers

form ester bounds with 31,32-didehydrophytochlorin, while inFHP6 two branched first-generation Newkome dendrimers,containing seven amide groups, each carry a total of six

31,32-didehydrophytochlorin molecules. The 14 amide groupsin FHP6 give a strong contribution to its solubility in polarsolvents. They may hinder aggregation driven by the high

lipophilicity of C60 and increase the amphiphilicity of themolecule. The comparison between FHP6 and its fullerene-free analog P6 confirms the correlation between amphiphilicityand intracellular uptake. The fullerene hexaadduct FHP6

has a better ability than fullerene-free P6 to carry PS moleculesinto Jurkat cells. The only structural difference is representedby the C60 fullerene moiety. Also in this case, the asymmetry

and amphiphilicity achieved in FHP6 by coupling thePS-loaded Newkome dendron to fullerene determine a higherintracellular uptake.

Phototoxicity

The phototoxic activity of the studied compounds correlateswith their intracellular accumulation. The compounds havingthe higher intracellular uptake, Pyro and FHP6, were found to

be the most phototoxic derivatives (Fig. 3). FHP1 exerts alower but significant phototoxicity, while compounds P6 andFHP12 had, at the studied conditions, a very low phototoxic

activity. All studied compounds induce apoptosis after irradi-ation, with activation of the effector proteases caspase 3 and 7(Fig. 3a). However, a different dependency on the incubation

concentration was measured for Pyro with respect to fullerenehexaadducts. After photosensitization with Pyro concentra-tions higher than 0.3 lMM, a high number of necrotic cells were

observed, while apoptotic cell percentages decreased withincreasing Pyro concentrations. This is probably due to thehigh photosensitizing efficiency of free 31,32-didehydrophyto-chlorin. In general, an enhancement of necrotic cells to the

detriment of apoptotic ones is correlated with a high concen-tration of reactive oxygen species (ROS) (33). The highamount of ROS is thought to induce such an extensive

damage that cells undergo a rapid necrosis (34). Anotherhypothesis is that the high amount of ROS would damagecomponents of the apoptotic pathway preventing this process

(34). Finally, the preferential localization of fullerene hexaad-ducts in lysosomes and the generation of singlet oxygen mainlyin such cellular compartments might also influence the cell

death mechanism (35,36). Thus, the low apoptosis percentagesmeasured by Pyro at almost all used incubation concentrationswould be an evidence for the high oxidative stress. On theother hand, the high ratio of apoptosis observed in cells

photosensitized by the fullerene derivatives is indicative of alocalized production of ROS, high enough to start apoptoticcell death pathways.

Even if FHP6 gives PS intracellular concentrations similarto those of Pyro (Fig. 2b), its phototoxic activity is slightlylower than that of Pyro (Fig. 3). This can be explained taking

into consideration the singlet oxygen quantum yields of thestudied compounds. As already mentioned, investigation ofthe photophysical properties of C60 hexaadducts revealed thatenergy transfer processes occurring between neighboring 31,32-

didehydrophytochlorin moieties cause the dissipation ofabsorbed energy and consequently a decrease in singlet oxygenquantum yield (FD) with increasing number of carried PSs (18–

21). The lower FD (0.23) of FHP6 compared with that of Pyro(0.50) influences strongly its in vitro phototoxic activity.Nevertheless, the high intracellular uptake and FD of FHP6

result in an efficient phototoxic effect exerted mainly throughapoptosis induction. The value of FD for compound FHP1 is0.43, which is only slightly reduced with respect to Pyro. For

that reason, despite its low intracellular uptake, FHP1 exertsan appreciable phototoxicity. On the contrary, FHP12 has alow FD (0.13) and a low intracellular uptake. Consequently, itexerts a very low phototoxicity (Fig. 3b). For compound P6, a

FD of 0.15 was measured (19), which is strongly reduced withrespect to that of Pyro. As molecular modeling studies put inevidence, the average distances between PS units in FHP6 are

longer than P6 (19). In the fullerene hexaadduct FHP6, theoctahedral substitution pattern influences the three-dimen-sional organization of the complex and slightly hinders the

excitonic interactions that are responsible for the low FD of P6(19). Thus, the higher intracellular uptake and FD of FHP6

with respect to P6 determine FHP6 higher phototoxic activity.Ideally, a carrier system for PDT should influence as less as

possible the photophysical properties of the carried PS whileimproving biological properties like selectivity, intracellularuptake and efficacy. The use of fullerene hexaadducts as

building blocks for tumor-selective modular carrier systemswas proposed with the rationale that the octahedral substitu-tion pattern typical of these adducts should hinder interactions

between the components of the complex. Our previousinvestigations of the photophysical properties of the studiedfullerene hexaadducts revealed that excitonic interactions

occur between the vicinal Pyro moieties leading to a reductionin the singlet oxygen quantum yield. However, thanks to

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ion

%DMF P6 FHP6

Figure 6. Jurkat cell vitality and proliferation 24 h after irradiationwith different doses of red light (668 nm, 2.12 mW cm)2). Cells werepreviously incubated (24 h) with the investigated compounds at aconcentration of 0.33 lMM. The percentages of living cells afterirradiation were determined with the XTT test. Bars represent thestandard deviation of three experiments.


fullerene octahedral substitution pattern, the reduction in FDoccurring in FHP6 turned to be lower than that occurring in itsanalogous P6.

In this work, we intended to investigate how far the

coupling of PSs to fullerene hexaadducts affects the PS in vitrointracellular uptake and phototoxicity. We can conclude thatthe coupling of PSs to a supramolecular carrier system

strongly alters their photobiological properties. The molecularsize and the grade of asymmetry ⁄ amphiphilicity of theseconjugates determine their intracellular uptake mechanism and

subcellular localization. The energy transfer processes betweenvicinal PS molecules reduce their singlet oxygen quantum yieldand consequently influence the in vitro phototoxicity of the

complex. On the other hand, it has been found that the use offullerene can partially hinder the interactions between theloaded PSs and favor the complex intracellular uptake bymeans of a special endocytic mechanism. These results indicate

that asymmetric fullerene hexa-kisadducts might be efficientmultiplying units in MDDS for PDT. In our future work weplan to couple a PS-loaded asymmetrical complex to a

monoclonal antibody. Furthermore, the introduction of aphoto-instable bond between antibody and fullerene or at thePS site will be pursued. Thus, once the antibody conjugate has

reached the target cells the drug could be released byirradiation and exert its maximal phototoxic activity.

Acknowledgements—This work was supported by the Deutsche Fors-

chungsgemeinschaft (DFG, BO 1352 ⁄ 2-1; RO 142 ⁄ 11-1; HI 468 ⁄ 11-1).The authors thank Mrs. Wohlecke of the Department of Physics,

Humboldt-Universitat zu Berlin, Germany for technical support and

Dr. Schumacher of the Department of Pneumology, Universitatsklini-

kum Charite for helpful advice.

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Intracellular Uptake and Phototoxicity of 31,32-Didehydrophytochlorin-fullerene Hexaadducts