Photochemistry and Photobiology, 2013, 89: 1011–1019

Rapid Communication

Protoporphyrin IX–b-Cyclodextrin Bimodal Conjugate: Nanosized DrugTransporter and Potent PhototoxinChrysie Aggelidou, Theodossis A. Theodossiou* and Konstantina Yannakopoulou*Institute of Advanced Materials, Physicochemical Processes, Nanotechnology & Microsystems, National Center for ScientificResearch “Demokritos”, Aghia Paraskevi, Attiki, GreeceReceived 1 April 2013, accepted 27 June 2013, DOI: 10.1111/php.12127

ABSTRACT

Topical or systemic administration of 5-aminolevulinic acid(ALA) and its esters results in increased production and accu-mulation of protoporphyrin IX (PpIX) in cancerous lesionsallowing effective application of photodynamic therapy (PDT).The large concentrations of exogenous ALA practicallyrequired to bypass the negative feedback control exerted byheme on enzymatic ALA synthesis and the strong dimerizationpropensity of ALA are shortcomings of the otherwise attrac-tive PpIX biosynthesis. To circumvent these limitations andpossibly enhance the phototoxicity of PpIX by adjuvant che-motherapy, covalent bonding of PpIX with a drug carrier,b-cyclodextrin (bCD) was implemented. The resultingPpIX + bCD product had both carboxylic termini of PpIXconnected to the CD. PpIX + bCD was water soluble, wasfound to preferentially localize in mitochondria rather than inlysosomes both in MCF7 and DU145 cell lines while its photo-toxiciy was comparable to that of PpIX. Moreover,PpIX + bCD effectively solubilized the breast cancer drugtamoxifen metabolite N-desmethyltamoxifen (NDMTAM) inwater. The PpIX + bCD/NDMTAM complex was readilyinternalized by both cell lines employed. Furthermore, themultimodal action of PpIX + bCD was demonstrated inMCF7 cells: while it retains the phototoxic profile of PpIX andits fluorescence for imaging purposes, PpIX + bCD can effi-ciently transport tamoxifen citrate intracellularly and confercell death through a synergy of photo- and chemotoxicity.

INTRODUCTIONPhotodynamic therapy of cancer (PDT) (1,2) provides cancertreatment through the synergy of three essential, yet individuallynonchemotoxic components: (1) The photosensitizer (PS), a lightactivated drug, (2) light of the appropriate wavelength to activatethe PS and (3) oxygen which is the terminal generator of toxicspecies (3,4). Most of the PSs used in PDT are porphyrins, tetra-pyrrole derivatives that stimulate production of cytotoxic reactiveoxygen species in tissues illuminated with visible light. Porphy-rins are known for their insolubility in aqueous media due tostrong aggregation phenomena, the driving force being the over-

all thermodynamic gain due to p–p-stacking and hydrophobicinteractions (5,6). Aggregation is manifested by widening andsplitting of the intense Soret band around ca 400 nm in theabsorption spectra, as well as deviations from Beer’s law and thequenching of fluorescence with increase in concentration. Forexample, cationic porphyrins (7) show concentration-, tempera-ture-, pH- and ionic strength–dependent aggregation. Protoporphy-rin IX (PpIX) is an endogenous porphyrin in heme biosynthesisfrom 5-aminolevulinic acid (ALA). PpIX is the penultimate stepprior to heme production following complexation with Fe(II).Exogenous administration of ALA and its esters (8) practicallybypasses the negative feedback control exerted by heme on enzy-matic ALA synthesis, thus making ALA a very efficient PpIX pro-drug widely used for clinical PDT, following either topical orsystemic application (9). Nevertheless it was previously shownthat exogenous application of PpIX to cells resulted in very effi-cient photocytotoxicity (10). One serious limitation in the exoge-nous use of PpIX much alike many other PSs is its low watersolubility, and consequently the difficulty in preparing pharmaceu-tical formulations. Alternative strategies have been sought forimproving the delivery characteristics of the PSs including liposo-mal formulations, oil dispersions/micelles, polymeric nanoparti-cles, or polymer-PS conjugates (11). Moreover, it seems that PSamphiphilicity could be an advantage: molecules should be hydro-philic enough to travel through the blood stream but also lipophilicenough to bind to the receptors on cancer cell membranes (12).PpIX (Scheme 1) is a characteristic example of an amphiphilic,water insoluble porphyrin (13). At pH 0–3 PpIX appears in mono-meric form due to repulsion between charged units. At pH > 8PpIX aggregates mostly as dimers via axial p–p interactions andlateral hydrophobic forces. At intermediate pH (3–7) aggregationto larger entities is favored due to additionally formed H-bondsamong the propionic acid tails (13). In the crystalline state the unitcell of PpIX dimethyl ester is composed of two molecules, thevinyl groups being nearly coplanar with the pyrrole rings (14).

Cyclodextrins (CDs) are an extensively studied family ofcyclic oligosaccharides composed of a-D-glucopyranose units(a-, b- jaι c-CDs with 6, 7 or 8 units respectively) (15). Inaqueous media CDs act as hosts that encapsulate hydrophobicmolecules such as drugs in their cavities (16). Inclusion complexformation is usually associated with increase in aqueous solubil-ity, stability and bioavailability of the drug guest. CDs addition-ally are known to facilitate the crossing of biological barriers ofvarious bioactive molecules (17).

*Corresponding authors email: dyanna@chem.demokritos.gr (Konstantina Yannako-poulou); theo@chem.demokritos.gr (Theodossis A. Theodossiou)© 2013 The American Society of Photobiology

1011

To enhance the aqueous solubility of porphyrins, cyclodex-trin–porphyrin conjugates have been prepared, either as enzymemimics (18,19) or as PSs (20,21) and only in few examples asdrug delivery systems (22). Whereas multisubstituted CD–porphyrin conjugates are reportedly water soluble, mono-CD-porphyrin conjugates display limited aqueous solubility (21,23).

The aim of the present work was to demonstrate successfulconjugation of PpIX to b-CD via a simple and reproducible pro-cedure affording a bimodal product, PpIX + bCD, endowed withsatisfactory water solubility, ability to host drugs and transportthem through cell membranes, and capacity to target subcellularcompartments. The drug selected herein as a paradigm ofPpIX + bCD-mediated intracellular transport was N-desmethyl-tamoxifen (NDMTAM), a chemotherapeutic agent mainly appli-cable to estrogen receptor–positive breast cancer.

MATERIALS AND METHODSNDMTAM hydrochloride, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), paraformaldehyde, dimethyl sulfoxide(DMSO), O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexa-fluorophosphate (HATU), dry dimethylformamide (DMF) and the dialy-sis membranes (cellulose tubing, benzoylated, MWCO 1.2 kD) werepurchased from Sigma-Aldrich. Deuterium oxide (D2O), deuterated dim-ethylsulfoxide (DMSO-d6), deuterated chloroform (CDCl3) and deuter-ated methanol (CD3OD) were purchased from Deutero GmbH. bCDwas a product of CycloLab. bCD and derivatives were dried by heatingat 70°C under vacuum for 20 h before reaction. N,N-diisopropylethyl-amine (DIPEA) was used without purification. PpIX was purchasedfrom Porphyrin Systems (Appen, Germany). 1D and 2D NMR spectrawere performed on a Bruker Avance 500 MHz spectrometer. RPMI1640 without phenol red, fetal bovine serum (FBS), penicillin/streptomy-cin, L-glutamine, PBS, Mitotracker® Green FM, LysoTracker® GreenDND-26 and trypsin/versene were purchased from Invitrogen Ltd.,(Paisley, UK).

Fluorescein-N-NDMTAM (NDMTAM-FITC) was prepared as describedpreviously (24). Mono(6-p-toluenesulfonyl)-b-cyclodextrin (bCD) (25) wasconverted into mono(6-azido-6-deoxy)-bCD (26) and subsequently to mono(6-amino-6-deoxy)-bCD (27) according to previously published methods.Synthesis. PpIX (PpIX, 0.020 g, 0.0355 mmol) was dissolved in dryDMF (1 mL) and allowed to stir at 0°C for 30 min under an argon atmo-sphere. HATU (0.103 g, 0.272 mmol) was slowly added at 0°C and stir-red for 1 h. The mixture was allowed to attain room temperature andthen DIPEA (33 lL, 0.191 mmol) was added, followed by gradual addi-tion of mono(6-amino-6-deoxy)-bCD (0.040 mg, 0.0355 mmol) during1 h with more DMF (1 mL). The reaction mixture was allowed to stir at20–23°C for 3 days while protected from ambient light. Subsequently thesolvent was evaporated under vacuum to dryness and washed exhaus-tively with methanol to remove unreacted PpIX (checked by TLC on alu-minum-backed silica gel plates eluted with isopropanol/ethyl acetate/water/ammonia, 5:3:3:1, v/v, visualized under a UV lamp and subsequentheating following spraying with 10% sulfuric acid in ethanol). The mate-rial was then dialyzed against deionized water (2 days) and then chroma-tographed using size exclusion chromatography (Sephadex G50) anddistilled water as eluent. The first fraction collected gave a dark purplesolid (30%) that contained PpIX-CD (90%) and PpIX-2CD (10%) in 9:1ratio, as indicated by mass spectra (vide infra). This solid, namedPpIX + bCD, was used for the cell experiments described subsequently.1H ΝΜR (DMSO, 298 K, 500 ΜHz): d 10.40-10.31 (4H, H5, H10, H15,H20), 8.55 (m, 2H, H3a, H8a), 6.49-6.24 (2d, 4H, H3b, H8b), 5.71 (br s,OH2, OH3), 4.84 (br s, 9H, CD-H1), 4.60-4,17 (m, OH6, 13a, 17a),4.03-2.46 (2 br s, CD-H6, 12a, 18a, 2a, 7a, CD-H3, -H5, -H6A), 3.31(br s, CD-H4, CD-H2), 2.81 (br s, 13b, 17b), �3.85 (m, 2H, NH) ppm.13C NMR (DMSO, 298 K, 125 ΜHz): d 129.44 (C3a, C8a), 120.70(C3b, C8b), 100.8 (CD-C1), 96.21 (5,10,15,20), 80.22 (CD-C4), 72.34,71.46, 71.84 (CD-C3, CD-C5, CD-C2), 59.29 (CD-C6), 50.74 (CD-C6A),36.2 (C13b, C17b), 21.99 (C13a, C17a), 12.32, 10.83 (C2a, C7a, C12a,C18a) ppm. UV/Vis (DMSO): 405, 504, 540, 571, 630 nm; emission spec-trum (kex = 405 nm): 632, 680, 698 nm; UV/Vis (H2O): 388, 510, 545,575 nm; emission spectrum (kex = 388nm): 626, 691 nm. Mass Spectrum

(MALDI-TOF) m/q: Found, 1660.7 (100%, base peak, [M + Η]+),2794.2 (11%, [PpIX-2CD + H]+). Calculated for C76H101N5O36 (PpIX-CD), 1659.62; calculated for C118H172N6O69 (PpIX-2CD): 2793.01.

Binding studies of NDMTAM. HCl with parent bCD by NMRspectroscopy. Initially, the stoichiometry of the complex bCD/NDM-TAM.HCl was determined with the continuous variation plots method bymixing variable concentrations (0–2 mM) of the substances to a constantfinal volume and recording of the 1H NMR spectral changes of bCD cav-ity protons. Plots of DdΗ3Χ[bCD] vs mole fraction (Figure S8) gave astoichiometry of 1:1. Furthermore, titration of a 2 mM solution of bCD indeuterium oxide with portions of solid NDMTAM.HCl was performed;following 10–15 min sonication, the 1H NMR spectra were acquired andthe chemical shift changes (strong shielding) of bCD cavity protons, H3and H5, were recorded vs molar ratio [NDMTAM]/[bCD]. After additionof ca 0.5 equiv. solid NDMTAM.HCl the onset of cloudiness could beobserved. The ratio of solubilized [NDMTAM.HCl]/[bCD] was obtainedfrom integration. The data (Dd Η3 vs molar ratio) were plotted and fittedto an equation suitable for 1:1 stoichiometry (28) using GraphPad Prismfrom which the binding constant was estimated.

Cell culture. Cells used in this study were the DU145 human prostatecarcinoma and MCF7 human breast adenocarcinoma cell lines. All cellswere grown at 37°C in a humidified, 5% CO2 atmosphere, in RPMI1640 media supplemented with 10% FBS and penicillin (50 IU mL�1)/streptomycin (50 lg mL�1). Cells were inoculated into 96-well plates(2 9 104 cells per 100 lL media per well) 24 h prior to phototoxicityexperiments or onto coverslips housed in 35 mm Petri dishes (1 9 105

cells per 2 mL media per dish) 24 h prior to confocal microscopy imag-ing. For cell experiments 15 lL of 10 mM PpIX + bCD (stock) was ini-tially diluted into 360 lL of PBS (400 lM PpIX + bCD—4% DMSO).This was further diluted with cell media to a final PpIX + bCD concen-tration of 7 lM (or 20 lM for imaging) resulting in DMSO content addedto cells <0.3%. Accordingly, 15 lL of 10 mM PpIX in DMSO wasdiluted with cell media down to 7 lM (or 20 lM for imaging), againyielding a final DMSO concentration <0.3%.

Confocal microscopy. Cells seeded onto coverslips were incubatedwith: (1) PpIX (stock in DMSO, final concentration 20 lM) orPpIX + bCD (stock in PBS-4% DMSO, final concentration 20 lM) for3 h. Both the parent (PpIX) and the daughter (PpIX + bCD) moleculeswere ultrasonicated for 1 h and then syringe filtered (0.22 lm) before theadministration to cells. Half an hour prior to imaging, the subcellularorganelle probes (Mitotracker� Green FM 200 nM, Lysotracker� GreenDND-26 200 nM) were added to the appropriate cell groups. (2)PpIX + bCD complexed with NDMTAM-FITC (24): A 2 mM solution ofPpIX + bCD (4% DMSO—96% PBS, [stock in PBS-4% DMSO]) andNDMTAM-FITC at equimolar ratio was left under stirring overnight.Upon stirring the solution lost all turbidity and became clear, indicatingthat NDMTAM-FITC had been entirely taken up by the PpIX + bCDhost. The solution immediately before being introduced to cells was sub-jected to ultrasonication for 1 h and cells were subsequently incubatedfor 3 h at a final PpIX + bCD concentration 20 lM.

In all cases, the coverslips were next washed twice with PBS andplaced over the 639 oil immersion quartz objective (NA 1.3) of a BioradMRC 1024 scanning confocal microscope, in physiological saline. Intra-cellular PpIX fluorescence was excited using the 568 nm line of anargon–krypton laser (3–10% of total laser power) whereas the internal-ized probes or NDMTAM-FITC were excited by the 488 nm line of thesame laser (3% of total laser power). PpIX fluorescence was collectedwith the use of a long-pass filter at ≥585 nm, whereas FITC-type fluores-cence was collected through a bandpass filter centered at 522 (�35) nm.During image acquisition a Kalman level 3 (three iterations per image)smoothing routine was applied each time to eliminate spurious signal.The contribution of cell autofluorescence was checked each time inuntreated cells and was, in all cases, found to be negligible.

Light irradiation protocol. In 96-well plates the 16 (4 9 4) centralwells were used for irradiation. Cells were incubated half with PpIX andhalf with PpIX + bCD (7 lM) in complete media for 3 h, then immedi-ately prior to irradiation washed twice with PBS and complete media(without phenol red) restored. Light-positive controls without PpIX orPpIX + bCD, as well as dark PpIX and PpIX + bCD controls wereincluded in each experiment. Light was provided by a halogen lightsource (50 W) through a Fresnel lens. The light transversed a long-passfilter (Schott RG610; Schott UK Ltd., Stafford, UK) and irradiated thecells through the plate underside. RG610 has a cut off at 610 nm, allow-ing irradiation above 610 nm i.e. at the 635 nm Q-band of PpIX; the

1012 Chrysie Aggelidou et al.

average light fluence rate at the cell level was maintained at15 mW cm�2 throughout the experiments as determined by a LI-185AQuantum /Radiometer/ Photometer (LI-COR Instruments, Lincoln, NE).The light dose was each time determined by irradiation duration. Thelight source power jitter was in all cases less than 5%.

Cell viability assessment. Twenty-four hours following irradiation, themitochondrial redox function (relating to cytotoxicity at that time frame)of all cell groups, including nonirradiated controls was assessed by theMTT assay. This was carried out by adding 100 lL of complete mediacontaining 1 mg mL�1 MTT to cells and incubating at 37°C in a 5%CO2 humidified atmosphere for 2 h. MTT media were subsequentlyremoved from all cells and the resulting formazan was solubilized with100 lL DMSO per well. The plates were then shaken for 10 min at100 rpm in a Stuart SI500 orbital shaker, and the endpoint absorbancemeasurement at 562 nm was performed in an Infinite M200 plate reader(Tecan group Ltd., M€annedorf, Switzerland). Blank values measured inwells with DMSO and no cells were in all cases subtracted.

Synergistic effect of PpIX + bCD and tamoxifen citrate in vitro. Opti-mal PpIX complexation with tamoxifen citrate (TAM-CIT, prepared fromTAM; Sigma) was performed in an analogous fashion to NDMTAM.Briefly, 15 lL of 10 mM PpIX + bCD (stock) was initially diluted into360 lL of cell media (400 lM PpIX + bCD—4% DMSO). EquimolarTAM-CIT was added and the solution was left stirring overnight. Uponcomplete complexation the resulting solution was further diluted with cellmedia to a final PpIX + bCD concentration of 7 lM and added to theappropriate MCF7 cell groups for 3 h. Half the cells were incubated with7 lM empty PpIX + bCD vector prepared in the same experimental con-ditions as the complex. The cells were then irradiated as per the irradia-tion regimen described above at 4 and 8 J cm�2. The toxicity wasassessed in all cell groups including dark and light controls at 48 h fol-lowing irradiation by standard MTT assays.

Statistics. All experiments were repeated at least three times indepen-dently. The graph error bars represent one standard deviation for at leastfour independent values.

RESULTS AND DISCUSSIONThe PpIX to bCD conjugation, performed under HATU-mediatedcoupling conditions for amide bond formation (Scheme 1), repro-ducibly afforded a dark purple solid product; conversely, tradi-tional N-hydroxybenzotriazole and dicyclohexylcarbodiimecoupling reagents, instead of HATU, could not facilitate conjuga-tion. The product was subjected to size exclusion chromatographyto yield the water soluble fraction that had both porphyrin andcyclodextrin content, according to thin layer chromatography.

The 1H NMR spectrum in DMSO-d6 (Figs. S1, S2) allowedsatisfactory characterization of the product: the carboxyl groupprotons of the parent PpIX (Figure S3) were now absent, sug-gesting that reaction had occurred at the carboxyl sites. Parts ofthe PpIX signals due to the exocyclic double bonds (ca 6.5 and8.6 ppm) were observed along with those of the meso-positions(ca 10.3 ppm) and of the pyrrole endocyclic NH (ca �3.8 ppm).On the other hand, the anomeric proton signals of bCD (ca5.0 ppm) were reasonably separated from those of the bCD pri-mary (ca 4.5 ppm) and secondary (ca 5.8 ppm) hydroxyl groups.The nearly 1 ppm spread of the signals due to the CD-H6,6′,revealed by the HSQC spectrum (Figure S2), indicated that thecyclodextrin macrocycle had been modified at its primary sidethus its molecular symmetry had been reduced. Integration of theporphyrin and CD regions gave a ratio PpIX/bCD �1.3. The IRspectrum of the product verified coexistence of the two macrocy-clic molecules and the shift of the PpIX carbonyl stretchingvibrations from ca 1695– ca 1730 cm�1 (Figure S4). TheMALDI-TOF mass spectrum confirmed PpIX conjugation tobCD by a two-point attachment, yielding the rigid amido ester ofPpIX-bCD (Scheme 1) (base peak, 100%) but also revealed pres-ence of a doubly CD-bearing product, PpIX-2bCD (11%). The

ratio of the two conjugates, as inferred from the MS spectrumwas ca 9:1 while the corresponding value obtained from 1HNMR integration is ca 8:2. It is interesting to note that the prox-imity of the hydroxyl groups of the bCD primary side to thepoint of initial amide bond formation probably facilitated the sec-ond linking via the ester bond to the same bCD, thus favoringthe intramolecular over the intermolecular reaction, i.e. attach-ment of a second bCD to PpIX. As a result, the PpIX-2bCDconjugate is the minor component. The product will be referredas PpIX + bCD in the following sections.

The 1H NMR spectrum in D2O exhibited very broad signalsindicating extensive aggregation in aqueous solution presumablydue to the high concentration used (mM), required by the method(Figure S5). Aqueous solubility was, however, achieved at bio-logically relevant concentrations (lM range) as UV–Vis and fluo-rescence spectra of PpIX + bCD were successfully obtained inboth aqueous and DMSO solutions, while PpIX alone was solu-ble only in DMSO. The absorbance intensity of the product inH2O (Fig. 1A) despite the nearly double concentration was halfof that in DMSO (Fig. 1B).

This suggested that some aggregation occurred even at thelow lM range. In addition, broadening of the Soret band in Η2Οrelative to DMSO also indicated a degree of aggregation. Conse-quently, the emission of the product in Η2Ο (Fig. 1C,D) wasnearly five times lower than in DMSO (Fig. 1E,F) On the otherhand, comparison of the absorption spectrum of PpIX + bCD inDMSO with that of parent PpIX (Fig. 1B) revealed a small butnoticeable increase in the Soret bandwidth in the product eventhough the extinction coefficient was similar. Finally, comparison

PPIX-CD PPIX-2CD

N

NH N

HN

HO OOHO

N

NH N

HN

OO

NH2

OH OH

HO6

7

NH

OH OH

HO6

7

OH

OH OH

HN6

NH

OH OH

HO

7

N

NH N

HN

OO

7

O 5

H6A

H6A

ΗΑΤU, DIPEA, DMF, 25 0C

Major Minor

Scheme 1. Preparation of the PpIX + bCD product.

Photochemistry and Photobiology, 2013, 89 1013

of the emission spectra of PpIX + bCD with that of parent PpIXin DMSO (Fig. 1E,F) revealed a nearly 20% quenching of thefluorescence at ca 632 nm in the product, and the appearance ofenhanced intensity of the band at ca 680 nm, absent in the emis-sion spectra of PpIX + bCD in water.

The product structure (both of major and minor components)suggests that it retains the amphiphilic character of the parentPpIX: the CD region is apparently a heavily hydrated part, whereasthe porphyrin region with its vinyl groups is highly lipophilic. Thisfeature evidently promotes some residual aggregation of the conju-gate in water despite the aqueous solubility. DLS experiments at20 lM concentration in RPMI 1640 medium with 4% DMSO (asused for cell experiments, vide infra) indicated formation of nano-particles sized ca 6.5 nm on average, corresponding to aggregationof a few molecules based on molecular size (estimated ca 2.5 nmfor PpIX-bCD and ca 2.9 nm for PpIX-2bCD, Chem3D modelmeasurements). The presence of larger aggregates in very smallamounts could be observed in the size vs intensity diagrams (Fig-ure S6). The inclusion capability of the CD moiety in the product,that is, the accessibility of the cavity by a potential guest moleculewas subsequently investigated.

N-Desmethyltamoxifen, one of the primary active metabolitesof tamoxifen, was chosen as a suitable and strategic guest to besolubilized by the conjugate PpIX + bCD in water. When oneequivalent of NDMTAM.HCl was added into a 2.5 mM solutionof the conjugate in D2O-4% DMSO-d6 gradual solubilizationwas observed suggesting possible formation of a water solubleinclusion complex. Indeed the 1Η NMR spectrum of the resultingsolution showed broad peaks for PpIX + bCD and NDMTAMalike, indicating that NDMTAM had been incorporated in theaggregate formation revealing a final envelope of peaks insteadof discrete resonances (Figure S7). The process was indirectlyquantified by studying the complex formation between the parentbCD and NDMTAM.HCl in water. The stoichiometry of thecomplex, as determined by continuous variation plots (FigureS8A) was found to be clearly 1:1. On the other hand, 1H NMRtitrations of a bCD solution with portions of solid NDM-TAM.HCl confirmed inclusion complex formation in the fastexchange regime in the NMR time scale by the very strongshielding (Dd = dfree � dcomplx) of only the cavity protons(Figure S8B) suggesting strong binding. The onset of slightcloudiness observed after addition of ca 0.5 equivalents of

Figure 1. Absorption spectra of (A) PpIX + bCD in Η2Ο (1.7 9 10�6M) and (B) in DMSO (black solid trace, 10�6

M) in comparison with PpIX alone(red dashed trace, 10�6

M). Fluorescence spectra of (C, D) PpIX + bCD in water (1.7 9 10�6M), kex = 388 and 510 nm respectively; (E, F) of PpIX +

bCD in DMSO (black solid trace, 10�6M), kex = 406 and 505 nm, respectively, in comparison with PpIX alone (red dashed trace, 10�6

M), kex = 407and 507 nm respectively.

1014 Chrysie Aggelidou et al.

NDMTAM.HCl indicated that the solubility of the resulting com-plex should be below 2 mM. Treatment of the Dd observed witha suitable equation for 1:1 complexation in fast exchange (28)

gave an estimate of the binding constant. The best fit valueswere found around 7000 (�1500) M

�1 (R2 = 0.98, evidently dueto use of unavoidably approximate concentrations), nevertheless

Figure 2. Representative confocal microscopy images following DU145 and MCF7 cell 3 h incubation with 20 lM concentration of PpIX or PpIX + bCD.The cells were in all cases coincubated with Mitotracker� Green FM (200 nM) for 30 min. PpIX fluorescence was excited at 568 nm and collected at≥585 nm, whereas Mitotracker� Green FM fluorescence was excited at 488 nm and collected at 522 (�35) nm. In the overlay images yellow denotes goodgreen and red fluorescence colocalization.

Figure 3. Representative confocal microscopy images following DU145 and MCF7 cell 3 h incubation with 20 lM concentration of PpIX or PpIX + bCD.The cells were in all cases coincubated with LysoTracker� Green DND-26 (200 nM) for 30 min. PpIX fluorescence was excited at 568 nm and collected at≥585 nm, whereas LysoTracker� Green DND-26 fluorescence was excited at 488 nm and collected at 522 (�35) nm. In the overlay images yellow denotesgood green and red fluorescence colocalization.

Photochemistry and Photobiology, 2013, 89 1015

indicating strong binding indeed. Furthermore, 2D ROESY spec-tra revealed intense intermolecular dipolar interactions betweenthe bCD cavity protons with all NDMTAM phenyl groups in theaqueous environment (Figure S9). NDMTAM is a tritopic guestmolecule, thus on statistical grounds it is expected to displayenhanced binding to bCD: the data above confirm that the partic-ipation of all aromatic rings in the inclusion process results inbinding considerably higher of that of a typical phenyl-monosub-stituted guest (16) and indicate that even in the low concentra-tions used for the cell experiments (vide infra) NTMTAM couldbe efficiently transported by the prepared PpIX + bCD conju-gate. Few data on tamoxifen and CDs are found in the literature:complexation between tamoxifen citrate and bCD or 2,3-di-O-hexanoyl-bCD (29) was previously studied with solid-state meth-ods. The complex with the amphiphilic 2,3-di-O-hexanoyl-bCDwas found to display augmented anticancer activity tested on theMCF7 breast cancer cells. Elsewhere, the pharmacokinetics oftamoxifen and tamoxifen citrate following oral or intravenousadministration were improved upon formulation with hydroxub-utenyl-bCD (30).

This study showed that the bCD cavity can host the phenylgroups of tamoxifen and thus the PpIX + bCD conjugate couldefficaciously solubilize and transport NDMTAM via a similaraction.

Cell experiments

PpIX and PpIX + bCD subcellular localization in confocalmicroscopy. As incubation was in all cases carried out in serum-containing media, there was a concern that partitioning of thecompounds between the cells and media could become a limitingfactor in the cellular uptake, especially in the case ofPpIX + bCD that is profoundly more water soluble than PpIX,leading to a lower intracellular PpIX + bCD uptake. Both PpIXand PpIX + bCD were, however, internalized by both cell linesquite efficiently, without apparent quantitative discrepancies,judging by their corresponding fluorescence intensities (Figs. 2and 3). This can be ascribed to the amphiphilic nature of thePpIX + bCD product that preserves some hydrophobicity in partof the molecule, although the actual structure of the in situformed aggregates is not known. The subcellular distributions ofPpIX and PpIX + bCD are shown in Figs. 2 and 3.

In all cases PpIX-type (red) fluorescence partly localized incell membranes and cell synapses. Moreover, a rather punctuatecytosolic component is evident, which upon prolonged and moreintense irradiation became diffuse presumably upon photoin-duced changes to organelle structures and concomitant dyerelease; in that context imaging durations as brief as possibleand low laser intensities were kept in all occasions. No notablenuclear fluorescence was observed for PpIX or for PpIX + bCD.These results are in good accordance with our previous PpIXfindings (10). Coincubation with Mitotracker� Green FM(Fig. 2) revealed a significant mitochondrial accumulation forboth the parent and daughter species and in general there was nonotable change of that trend following PpIX conjugation withbCD. Although mitochondrial localization was evident for bothcompounds in accordance with previous reports on PpIX (31),we could not verify a notable lysosomal component expectedupon exogenous PpIX administration (31) for either PpIX orPpIX + bCD and in either of the cell lines studied (Fig. 3). Infact, in all the occasions of Fig. 3 the LysoTracker� Green

DND-26 green fluorescence was quite limited in comparisonwith the PpIX-associated red fluorescence. In these conditions itis very difficult and thus precarious to assign lysosomal localiza-tion; however, if there were any, it would not be extensive andcertainly not comparable to the corresponding plasmalemmal ormitochondrial deposits.

PpIX and PpIX + b-CD photocytotoxicity. The dark toxicity ofPpIX and PpIX + bCD is shown in Fig. 4. It can be seen thatcell incubation with 7 lM PpIX for 3 h confers a residual toxic-ity of around 25%, whereas the corresponding dark toxicity ofPpIX + bCD is 15%. Although these values are not considerablydiverse within experimental errors, there is a certain trend ofdark toxicity reduction for PpIX + bCD in both lines. Heuristi-cally, from our extended experience from handling the com-pounds we believe that the PpIX dark toxicity is mainly derivedfrom the formation of aggregates. In the case of PpIX + bCDthe possibility of aggregate formation (especially p-stacking) islargely diminished due to steric hindrance by the bCD. Inany case the selected concentrations of both compounds and

Figure 4. Toxicity and phototoxicity of PpIX and PpIX + bCD: (A). Darktoxicity 24 h following 3 h cell incubation with 7 lM PpIX and PpIX +bCD. (B). Phototoxicity 24 h following 3 h cell incubation with 7 lMPpIX and PpIX + bCD and irradiation through a Schott RG610 long-passfilter. Spheres and open circles: MCF7 cells; open and filled squares:DU145 cells. Solid lines: PpIX; dashed lines: PpIX + bCD.

1016 Chrysie Aggelidou et al.

especially PpIX + bCD exhibit minimal dark toxicity. In ourprevious study on exogenously administered PpIX (10), we dis-covered a higher concentration tolerance (20 lM); however, ithas to be noted that we then used the dimethyl ester instead ofthe free acid of PpIX used herein, while also the cell lineemployed was different (PAM 212 keratinocytes).

In Fig. 4B, the photocytotoxicities of PpIX and PpIX + bCDare shown for both cell lines studied. Both compounds werefound to be phototoxic, albeit perhaps to a different extend inthe two cell lines. In that context the LD50 values for PpIXwere 10 and 7 J cm�2 for MCF7 and DU145 cells, respec-tively, whereas the corresponding values for PpIX + bCD were10 and 6 J cm�2. It can also be concluded from Fig. 4B thatessentially there was no discrepancy between the photocytotoxic

capacities of PpIX and PpIX + bCD; in fact, if there is a slightdivergence, this is in favor of PpIX + bCD, i.e. PpIX + bCDis less chemotoxic and slightly more phototoxic. These resultsshow that PpIX conjugation to bCD had no adverse affects onits phototoxicity while it reduced its chemical toxicity and pro-vided the possibility of drug transportation in its bCD cavity. Itadditionally has to be noted that at 15 J cm�2, the cell viabilitywas close to zero in all cases and certainly below 10%. In ourprevious study of exogenously administered PpIX to PAM 212keratinocytes (10) the photodynamic action was more profoundagain in that case, the incubation concentration was consider-ably higher (20 lM), the incubation time longer (5 h) but alsodifferent cell type and PpIX moiety (dimethyl ester) wasemployed.

Cell internalization of the PpIX + bCD/NDMTAM-FITCcomplex

The main incentive for the implementation of PpIX + bCD wasthe possibility of inclusion of a guest molecule in the CD cavityto endow the parent PpIX with a bimodal action. In this contextwe complexed FITC-labeled NDMTAM (24) (NDMTAM-FITC)with PpIX + bCD, and incubated our cell lines with the watersoluble complex to study the intracellular fate of the two moie-ties. The results of this study are shown in the representativeimages of Fig. 5.

In both cell lines the two fluorescent species are shown tolargely colocalize subcellularly. The minor discrepancies inunmatched red fluorescence especially observed in cell mem-branes could be attributed to empty PpIX + bCD vector(Figs. 2 and 3). Conversely, all NDMTAM-FITC carried byPpIX + bCD seems not to profoundly localize at cell mem-branes, perhaps suggesting that the guest molecule could possi-bly determine the intracellular fate of the complex. Theseresults nevertheless, unambiguously demonstrate that therobustly bound constituents of the complex (as shown by theROESY spectra, Figure S9) do not take separate subcellularroutes; in that context PpIX + bCD apart from its photody-namic capacity is an efficient intracellular transporter of suitablebioactive moieties.

Bimodal photo- and chemotoxic action of PpIX + bCDcomplex with TAM-CIT in MCF7 cells

The synergistic effect of PpIX + bCD phototoxicity and TAM-CIT guest chemotoxicity at 48 h following irradiation isshown in Fig. 6. The dark toxicity of PpIX + bCD is ca 86%as compared with media controls which is consistent with thedark toxicity values in Fig. 4A. The dark toxicity of the com-plex was found to be ca 50% which is a combination of thedark toxicity of PpIX + bCD and the chemotoxic effect ofTAM-CIT. Irradiation of the PpIX + bCD only MCF7 cellgroups with 4 J cm�2 red light conferred a 30% phototoxicity,however, irradiation of the complex with the same light doseyielded a 70% synergistic cytotoxicity. The corresponding val-ues for 8 J cm�2 irradiation were 67% phototoxicity and 85%bimodal toxicity. The above results clearly demonstrate a pro-found synergy between the phototoxicity and the adjuvantchemotoxicity of the PpIX + bCD-TAM complex, substantiallyenhancing the PDT effect of the parent PpIX or the emptyPpIX + bCD molecule.

Figure 5. Representative confocal microscopy images following DU145and MCF7 cell 3 h incubation with 20 ll PpIX + bCD in complex withNDΤΑΜ-FITC: PpIX + bCD fluorescence was excited at 568 nm andcollected at ≥585 nm, whereas NDΤΑΜ-FITC fluorescence was excitedat 488 nm and collected at 522 (�35) nm. In the overlay images yellowdenotes good green and red fluorescence colocalization.

Figure 6. Bimodal action of PpIX+bCD complexed with TAM-CIT inMCF7 cells. Toxicity 48 h following 3 h cell incubation with 7 lM PpIX +bCD and PpIX + bCD complexed with TAM-CIT and irradiation through aSchott RG610 long-pass filter at 4 and 8 J cm�2.

Photochemistry and Photobiology, 2013, 89 1017

CONCLUSIONA novel PpIX + bCD product has been developed with a repro-ducible procedure. The product is a bicomponent composition,with one (major) and two (minor) bCD cavities connected to thePpIX carboxyl groups. The product was shown to display goodaqueous solubility and the ability to traverse cell membranes,localizing primarily in the mitochondria and also transporting guestmolecules (NDMTAM) intracellularly. PpIX + bCD displayedsatisfactory dark toxicity and phototoxicity equivalent of that ofthe parent PpIX. The demonstrated product properties along withits ease and reproducibility of preparation and potential for upscaling represent an important bimodal system combiningdelivery of a chemotherapeutic agent with a well-documentedphotodynamic action. Furthermore, the multimodal action ofPpIX + bCD was demonstrated: While it retains the ptototoxicprofile of PpIX and its fluorescence for imaging purposes,PpIX + bCD can efficiently transport chemotoxins (or otherdrugs) into cells and confer cell death through a synergy ofphoto- and chemotoxicity.

Acknowledgements—Funding by the Marie Curie Initial Training Networks(FP7-People-ITN-2008 CYCLON), Project no. 237962 “CYCLON” isgratefully acknowledged. A scholarship to C. A. by NCSR “Demokritos” isgratefully acknowledged. We also thank Mr. A. R. Goncalves of our groupfor preparing tamoxifen citrate and NDMTAM.HCl. Istituto di RicercheChimiche e Biochimiche “G. Ronzoni” Milano, Italy, is also thanked forthe MALDI-TOF measurements.

SUPPORTING INFORMATIONAdditional Supporting Information may be found in the onlineversion of this article:

Figure S1. 1Η ΝΜR (500 MHz, DMSO-d6, 25°C) of PpIX +bCD product.

Figure S2. 2D HSQC NMR spectrum of PpIX + bCD product(DMSO-d6, 25�C).

Figure S3. 1H NMR spectra of PpIX in DMSO-d6, 25�C.Figure S4. Overlay of IR spectra: PpIX (blue trace) and PpIX +

bCD (red trace).Figure S5. 1Η ΝΜR spectrum of PpIX + bCD in D2O.Figure S6. DLS diagrams showing particle size distribution in

RPMI 1640 solution-4% DMSO (20 lM, 22°C) of PpIX + bCD (a)before and (b) after filtration through a 0.22 lm disk.

Figure S7. Overlay of 1Η ΝΜR NMR spectra of PpIX + bCD/NDMTAM.HCl in D2O, PpIX + bCD in D2O and NDMTAM.HClin DMSO-d6.

Figure S8. A. Continuous variation (Job) plots ofNDTAM.HCl/bCD complex in D2O. B. Dual display of 1HNMR spectra of bCD alone and in the presence of NDM-TAM.HCl.

Figure S9. 2D ROESY spectra NDMTAM.HCl/bCD (1:1)complex in D2O.

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