Study of the stabilization of zinc phthalocyanine in …...Study of the stabilization of zinc...

Study of the stabilization of zinc phthalocyanine in sol-gel TiO2 forphotodynamic therapy applications

Tessy Lopez, PhDa,b,c, Ema Ortiz, PhDb, Mayra Alvarez, PhDb, Juan Navarrete, PhDd,Jose A. Odriozola, PhDe, Fernando Martinez-Ortega, PhDg, Edgar A. Páez-Mozo, PhDg,

Patricia Escobar, PhDf, Karla A. Espinoza, PhDb, Ignacio A. Rivero, PhDh,i,⁎aUniversidad Autónoma Metropolitana, Departamento de Atención a la Salud, México, DF

bInstituto Nacional de Neurología Y Neurocirugía “MVS” Laboratorio de Nanotecnología, Tlalpan, México, DFcDepartment of Chemical and Biomolecules Engineering, Tulane University, New Orleans, Louisiana, USA

dInstituto Mexicano del Petróleo, México, DFeInstituto de Materiales de Sevilla, Centro Mixto CSIC-US, Isla de la Cartuja, Sevilla, España

fCentro de Investigación en Enfermedades Tropicales (CINTROP), Escuela de Medicina,Universidad Industrial de Santander, Bucaramanga, Colombia

gCentro de investigacion de catalisis (CICAT), Depto de Quimica, Universidad Industrial de Santander, Bucaramanga, ColombiahCentro de Graduados e Investigación, Instituto Tecnológico de Tijuana, Tijuana, México, DF

iInstituto Nacional de Investigaciones Nucleares, Departamento de Química, Ocoyoacac, México, DF

Received 26 March 2009; accepted 14 April 2010

Abstract

Photodynamic therapy (PDT) has emerged as an alternative and promising noninvasive treatment for cancer. It is a two-step procedurethat uses a combination of molecular oxygen, visible light, and photosensitizer (PS) agents; phthalocyanine (Pc) was supported over titaniumoxide but has not yet been used for cell inactivation. Zinc phthalocyanine (ZnPc) molecules were incorporated into the porous network oftitanium dioxide (TiO2) using the sol-gel method. It was prepared from stock solutions of ZnPc and TiO2. ZnPc-TiO2 was tested with fourcancer cell lines. The characterization of supported ZnPc showed that phthalocyanine is linked by the N-pyrrole to the support and is stableup to 250°C, leading to testing for PDT. The preferential localization in target organelles such as mitochondria or lysosomes could determinethe cell death mechanism after PDT. The results suggest that nanoparticulated TiO2 sensitized with ZnPc is an excellent candidate assensitizer in PDT against cancer and infectious diseases.

From the Clinical Editor: Photodynamic therapy is a two-step procedure that uses a combination of molecular oxygen, visible light andphotosensitizer agents as an alternative and promising non-invasive treatment for cancer. The results of this study suggest thatnanoparticulated TiO2 sensitized with ZnPc is an excellent photosensitizer candidate against cancer and infectious diseases.© 2010 Elsevier Inc. All rights reserved.

Key words: Titania; Phthalocyanine; Cancer; Photodynamic therapies

Photodynamic therapy (PDT) is a promising noninvasivetreatment for cancer1; additionally, it has been studied in a varietyof nononcologic applications.2-4 The process is a two-stepmethod, in which a combination of a photosensitizer (PS) agent

and UV-visible (UV-Vis) light are used in the presence ofmolecular oxygen to obtain a therapeutic effect. The PDT effectoccurs when the PS absorbs photons (Figure 1), and the groundsinglet state is an excited single state. A fraction of the excitedsingle-state molecules is transformed via intersystem crossinginto excited triplet state, forming free radicals or ions. Then, thehydrogen atom is removed and an electron is transferred tobiological substrates such as membrane lipids, solvent, ormolecular oxygen. These radicals interact with molecular oxygenin two steps: (1) A free electron or hydrogen atom is transferredto membrane and/or (2) the excited triplet state transfers itsenergy to molecular oxygen in singlet state (1O2) to form a highlyreactive nonradical in triplet state (3O2). Both processes can be

POTENTIAL CLINICAL RELEVANCE

Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 777–785

Original Articlewww.nanomedjournal.com

This work was supported by the Instituto Colombiano para el Desarrollo dela Ciencia y la Tecnologia “Francisco Jose de Caldas” COLCIENCIAS (Grant1102-04-14130; RC No 480-2003), the Universidad Industrial de Santander,Bucaramanga, Colombia, and the program “Apoyo a los doctoradosnacionales”, from COLCIENCIAS. Thanks to CONACYT-FONCICYT(Grant 96095).

⁎Corresponding author: Centro de Graduados e Investigación, InstitutoTecnológico de Tijuana, Apartado Postal 1166, 22000, Tijuana, BC, México.

E-mail address: [email protected] (I.A. Rivero).

1549-9634/$ – see front matter © 2010 Elsevier Inc. All rights reserved.doi:10.1016/j.nano.2010.04.007

Please cite this article as: T., Lopez, et al, Study of the stabilization of zinc phthalocyanine in sol-gel TiO2 for photodynamic therapy applications.Nanomedicine: NBM 2010;6:777-785, doi:10.1016/j.nano.2010.04.007

occurring simultaneously, and the ratio between them dependson the nature of the PS as well as substrate properties.2,5-9

The phthalocyanines (Pc) are a type of PS; these moleculeshave been studied for PDT applications,10 because they areefficiently accumulated into target cells and nontoxic for healthycells. In addition, Pc exhibit excellent photochemical propertiescharacterized by high singlet-oxygen quantum yields and strongabsorption of far-red region wavelengths (600–850 nm).11 Zincphthalocyanines (ZnPc) have proved to be highly promising asPSs because of their intense absorption in the red region of thevisible spectrum. High triplet-state lifetimes and quantum yieldsare required for efficient sensitization, and these criteria may befulfilled by the incorporation of diamagnetic metal. These kindsof molecules are lipophilic and selectively cytotoxic for tumortargets.5-7,11 This tendency suggested the study of ZnPcencapsulated in nanostructured titania to increase their photo-chemical properties and enhance their performance in killingcancer cells and pathogen microorganisms.3,8,9,12,13 The study ofthe photochemical and photophysical properties of such com-plexes is still very limited. In vitro studies have demonstrated thephototoxicity of ZnPc or aluminum phthalocyanine in Leish-mania parasites, responsible for millions of deaths in tropicalcountries, especially in poor areas of the world.13-15

The use of nanoparticles as PS carriers is a very promisingapproach, because these nanomaterials can satisfy all therequirements for an ideal PDT agent. Most prominent amongthe features of successful agents is their photosensitizing ability,wherein targeted mitochondria absorb visible light; however, thisfeature often has been found serendipitously or empirically. Inthe present work nanostructured titania with ZnPc was prepared.Titanium dioxide (TiO2), is a wide–band gap (EG = 3.2 eV)semiconductor photoactive agent with strong oxidizing power,chemical inertness, and nontoxicity.16 UV-Vis light–irradiatedtitania generates an electron hole pair on the surface17; pure TiO2

is photoactive against microorganisms and cancer cells whenexposed to UV light, and it photocatalyzes a number offunctional changes in cells.18 However, its use as a PS forPDT is restricted, because the UV wavelengths (320–400 nm)that excite TiO2 are harmful to the human body. Recently,sensitization of wide–band gap semiconductors with organic andinorganic dyes have been demonstrated.19 In this system, a PSadsorbed at the support surface is excited by visible light, and anintercomponent electron transfer is realized in the couplemolecular semiconductor–semiconductor oxide, extending theuseful wavelength of TiO2 from the UV to the visible region. Theaim of this work is to investigate the possible synergy of zinc

Figure 1. Mechanism of PDT cytotoxicity photosensitizer (PS) administration (step I) leading to photophysic reactions represented by modified Jablonskidiagram (step II) (vibrational levels omitted).

778 T. Lopez et al / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 777–785

phthalocyanines supported on nanostructured titanium oxide andprobe their “in vitro” photoactivity using visible light, on cancercells and Leishmania parasites.

Methods

Synthesis of nanoparticles

The TiO2 nanoparticles were synthesized by the sol-gelmethod, using two different conditions.

(1) Acetic acid medium (TiO2-ZnPc-Ace). ZnPc (AldrichChemical Co., Milwaukee, Wisconsin) (4.2 g, 12.36mmol) was dissolved in acetic acid (50 mL), and then,titanium n-butoxide (85 mL) (Sigma-Aldrich, St Louis,Missouri) solution was added dropwise (4 hours). Thesolution was maintained under stirring until the gel wasformed. Finally, the excess of water was removed atreduced pressure and the gel dried at 30°C for 8 days.

(2) A similar process was used to prepare the oxalic acidmedium (TiO2-ZnPc-Oxa). The PS was prepared in thedark to avoid photobleaching.

Fourier transform infrared (FT-IR) technique:infrared spectroscopy

TiO2-ZnPc nanoparticles were analyzed by FT-IR spectros-copy using Nicolet-710 equipment (Madison, Wisconsin).Powder samples were mixed with KBr (5 wt%) (Reasol, México,México) and pressed into thin disks mounted on a Pyrex cell withCsI windows coupled to a vacuum line. The samples were heatedat 400°C under an air flux.

UV-Vis absorption spectra

The absorption spectra were obtained in the 200- to 800-nmrange using a Varian (Palo Alto, California); Cary-III spectropho-tometer, equipped with an integrator sphere for diffuse reflectancestudies. MgO (100%) was used as reflectance reference.

Thermogravimetric analysis (TGA)–differential scanningcalorimetry (DSC) technique

TGA and DSC curves were made with a Perkin-Elmer(Waltham, Massachusetts) TG-7 apparatus using a ramp from20°C up to 800°C at 10° per minute under air flux.

Raman spectroscopy

Measurements were carried out at room temperature on acomputerized Spex 1043 double monochromator (Edison, NewJersey), with the 514.5-nm line of the argon laser (lexel Laser) ata power level of 40 mW. Raman spectra were taken directly fromsample powders in backscattering geometry.

Stock solutions of ZnPc, TiO2, ZnPc-TiO2, and the “in vitro”culture were prepared on dimethylformamide (0.1%, vol/vol).

Figure 2. UV-Vis spectra of TiO2 and TiO2-ZnPc.

Figure 3. FT-IR spectra of TiO2-ZnPc samples evacuated at differenttemperatures.

779T. Lopez et al / Nanomedicine: Nanotechnology, Biology, and Medicine 6 (2010) 777–785

Dimethylformamide was not toxic for the cells at the useddilution. The concentration of ZnPc on ZnPc-TiO2 was calculatedconsidering the final weight relation of ZnPc on TiO2-ZnPc.The average particle size of each sample was measured bydynamic light scattering using a Nicomp 380/ZLS analyzer (PortRichey, Florida).

Mammalian cells lines and parasites

Cells and parasites were obtained from the African greenmonkey epithelial cells (Vero cells; American Type CultureCollection [ATCC], Manassas, Virginia); human hepatocellularliver carcinoma cells (HepG2; ATCC), human acute monocyticleukemia cell line THP-1 (ATCC), and a primary culture ofhuman-derived fibroblasts (HDFs) were used (MolecularProbes, Eugene, Oregon). The cells were maintained bycontinuous culture using RPMI 1640 medium supplementedwith 10% (vol/vol) heat-inactivated fetal calf serum (Gibco,Grand Island, New York) (hFCS) at 37°C, in a humidifiedatmosphere of 5% CO2–95% air mixture. Adherent cell weredetached with 0.25% trypsin-EDTA treatment (Gibco). TheTHP-1 cells were transformed to adherent cell phenotype bytreatment with propylene glycol monomethyl ether acetate(10 ng/mL) for 72 hours. Leishmania chagasi promastigotes(MHOM/BR/74/PP75) and L. panamensis (MHOM/PA/71/LS94) were donated by the Centro Internacional de Entrena-miento e Investigaciones Médicas (CIDEIM), Cali, Colombia.Parasites promastigotes were cultured in RPMI 1640 mediumsupplemented with 20% hFCS, 0.1 mM adenosine, 0.04 MHEPES, and 0.25 mg/mL of hemin, at 28°C. All experimentswere carried out using the cells at exponential growth phase.

Phototoxic assays on mammalian cells

Cell lines were prepared using mammalian cells at 2 × 106

cells/mL, which were incubated with increased concentrations ofTiO2, ZnPc-TiO2, and ZnPc in a threefold dilution series (0.05 to50 μM) at 5%CO2–95% air mixture and 37°C for 24 hours. Afterthe incubation, cells were washed with fresh culture medium andexposed to red-light doses of 2,5 J/cm2 using a nonionic laser

light system (BFW; Edmund Industrial Optics, Barrington, NewJersey) at 670 nm (abbreviated as LS670nm) or a photoreactorsystem equipped with four lamps (50W, 120 V), 0.33 mV power,and red filters (Edmund Industrial Optics) with a spectral range of597 to 752 nm (abbreviated as PRS597-752nm). Control cells weremaintained in the dark. The cell viability was calculated using thecolorimetric MTT reduction assay. Twenty-four hours afterirradiation, 20μL ofMTT (5mg/mL)were added into the cells for4 hours, and blue formazan crystals were dissolved with dimethylsulfoxide. The optical density (OD) was measured using amicroplate reader (Sensident Scan Merck, Darmstadt, Germany)at a wavelength of 580 nm. The percentage of cytotoxicity wascalculated using the following equation: Cytotoxicity (%) = 1 –(OD treatment group/OD control group) × 100. The compoundactivity was expressed in 50% and 90% lethal concentration LC50

and LC90 values calculated by sigmoidal regression analysis(Msxlƒit; ID Business Solution, Guildford, United Kingdom). Aspecific phototoxic index (PI) was calculated by dividing LC50

from nonirradiated cells/LC50 for irradiated cells. Values ofcompound activity from nonirradiated cells was referred as celltoxicity, whereas those from irradiated cells were referred as cellphototoxicity.20,21 The PI indicates how many times thecompound was specifically active in the presence of light, soPI = 1 or N1 indicates no specific phototoxic activity.

Figure 4. Thermogravimetric curves of different TiO2-ZnPc samples.

Table 1Weight loss of TiO2-ZnPc samples during the thermogravimetric analysis

Sample Temperatureinterval (°C)

Weightloss (%)

Total weightloss (%)

TiO2-ZnPc-Oxa 25–100 3.31100–205 9.47205–387 40.36

53.14TiO2-ZnPc-Ace 25–134 10.45

134–244 6.05244–370 16.27

32.77

Similar TGA curves were obtained for TiO2-ZnPc sample, with slightchanges of the temperature and weight loss values reported in Table 1.


Phototoxic assays on Leishmania

The performance was carried out with incubated parasitesand the reference drug or the compounds by 24 hours andilluminated at light intensities of 2.5 J/cm2 (ref. 14). Twenty-four hours after the treatment, inhibition of promastigotesgrowth was microscopically determined by counting parasitenumbers in a hemocytometer. Inhibition of parasite growth wasdetermined by comparison to untreated controls. The phototoxiceffect was demonstrated by comparing the activity of thecompounds with and without illumination. Each experiment wasdone in triplicate. Inhibitory concentration (IC50) and IC90

values were calculated by sigmoidal regression analysis. A PIwas calculated as described above.

Cellular localization

Mammalian cells were seeded into 12-well fluorescence glassslides and incubated with 15 μM ZnPc and 100 μM ZnPc-TiO2

in the dark at 37°C, 5% CO2 for 24 hours. Cells were washedtwice with culture medium and incubated with specific cellorganelle probes. Mitochondria were stained with MitoTrackerGreen FM (200 nM, 1 hour, 37°C), lysosomes with LysoTrackerGreen DND-26 (200 nM, 1 hour, 37°C), and the nucleus withHoechst 33342 (0.1 μg/mL, 5 minutes, 37°C) (Molecular Probes,Eugene, Oregon). After washing twice with phosphate-bufferedsaline, cells were examined under a fluorescence microscope(Nikon Eclipse E4000, Tokyo, Japan) using 40× magnification.Images were recorded using a charge-coupled device colordigital camera (Nikon Coolpix 5000). Two independent filterswere used: UV-2A filter (Ex = 330–380, DM = 400, BA = 420)for compounds and nucleus stain, and B-2A filter (Ex = 450–490, DM = 500, BA = 515) for the other probes. Red dotsrepresented the fluorescence images of photosensitizers, the bluefluorescence represented the DNA of the cells, and the greenfluorescence patterns represented mitochondrial or lysosomalorganelles. When dyes and probes localize in the same organelle,the fluorescence appears as yellow-orange.

Results

UV-Vis spectroscopy

TiO2 and TiO2-ZnPc samples were characterized, and thephotodynamic activities of phthalocyanines and TiO2-ZnPc in thebiological tests were analyzed. UV-Vis diffuse reflectance spectraof TiO2 and TiO2-ZnPc samples are shown in Figure 2. A well-known electronic transition at 390 nm (band gap 3.2 eV) due toTiO2 charge transfer was observed, indicating that at this energyTiO2 generates electron-hole pair and hydroxyl radicals. In theTiO2-ZnPc-Ace spectrum, additional transitions were observed.

In the ZnPc spectrum two bands at 557 and 598 nm appeardue to interactions between the zinc atom and the heterocyclicof ZnPc; an electron is transferred from the macrocycle to thed-orbital of the zinc atom. In TiO2; two bands at 256 and326 nm are assigned to electron charge transfer from the 2poxygen orbital to the 3d titanium orbital. Nevertheless, in thevisible region of the TiO2-ZnPc spectrum an electronictransition d-d at 750–600 nm, at 409 nm can be attributedto interactions between ZnPc and TiO2 molecules, displayingthe sensitization of the TiO2. In the region 400–600 nm, acharacteristic valley of the metallophthalocyanines wasobserved. The band gap values evaluated from the UV spectrawere 3.42 eV for TiO2-ZnPc-Oxa, 3.32 eV for TiO2-ZnPc-Ace, and 2.87 eV for TiO2.

The infrared spectra

TiO2-ZnPc samples spectra as a function of temperature areshown in Figure 3. The broad band centered at 3267 cm–1 in theTiO2-ZnPc-Ace is assigned to OH stretching vibration due tothe presence of water and Ti-OH bonds. The peak at 1621 cm–1

is related with symmetrical flexion vibrations of adsorbedwater. This band together with the OH band disappearedwhen the temperature was raised to 200°C. The smallpeak located at 2930 cm–1 is assigned to C-H asymmetricaland symmetrical stretching vibrations in the ring. Normally

Table 2Activity of TiO2-ZnPc and ZnPc on Leishmania promastigotes

Fluency(J/cm2)

ZnPc μM)

Leishmania chagasi Leishmania panamensis

IC50⁎ PI† IC50 PI

ZnPc-TiO2 0 N10 N102.5║ N10 N102.5‡ N10 N1010 N10 N10

ZnPc 0 N15 14.76 12.5║ 12.86 ± 2.14 N1.165 6.63 ± 1.60 2.232.5‡ 0.19 ± 0.03 N78.60 0.39 ± 0.03 38.0410 5.63 ±0.67 N2.66 5.63 ± 0.80 2.62

AmB 0 0.046 ± 0.003 1 0.148 ± 0.006 12.5‡ 0.073 ± 0.009 0.63 0.128 ± 0.005 1.15

AmB, amphotericin B.⁎ Concentration that induces 50% of parasite inhibition.† PI, phototoxic index ( = IC50 value at 0 J/cm2/IC50 value after irradiation).‡ Irradiation system using a laser light LS670nm.║ Irradiation using a biological photoreactor PRS597-752nm. Values were expressed as means ± SD. The result shown is representative of three different experiments.


metallophthalocyanines show strong peaks in the 1000–1800 cm–1 region due to vibrations of the isoindole and pyrrolegroups. The spectral pattern in this region strongly depends on themolecular structure of the complexes and of the central metal.According to Seoudi et al22 the bands appearing at 1345, 1050,and 1027 cm–1 are assigned to the C-N in isoindole, in plane bandin pyrrole, and stretching vibration, respectively. The other twobands at 1541 and 1441 cm–1 are attributed to the C-H in planebending vibration. The vibrations to C-C, C-H, and C-N bondswere maintained without change up to 250°C, and decreasedgradually as the temperature was raised, disappearing totally at400°C. The spectrum measured at 400°C shows only onebroadband center at 500 cm–1 corresponding to Ti-O vibrations.The spectrum of the TiO2-ZnPc-Oxa sample exhibited differentbands: a sharp OH vibrations band at 3606 cm–1; at 3311 cm–1

N-H assigned to stretching vibrations, at 2973 cm–1 due to C-H;at 1693 cm–1 a strong band C = O from oxalic moiety and at

1607 cm–1 is assigned to C-C stretching vibrations in pyrrole. At1356 cm–1 and 1306 cm–1 are two stretching vibrations ofisoindole ring; two peaks at 916 cm–1 and 815 cm–1 are assignedto C-H bending out of plane. When the organic matter wasremoved at 200°C, only the broadband centered at 550 cm–1 wasobserved and is assigned to Ti-O vibrations.

Thermogravimetric analysis–differential scanning calorimetry

The TGA-DSC technique was used to verify the thermalstability of ZnPc in sol-gel nanostructured titania. In Figure 4and Table 1, the percentage weight loss for the TiO2-ZnPcsamples is presented. The initial weight loss (3.31%) for TiO2-ZnPc-Oxa occurring between 25° and 100°C is caused bywater evaporation and alcohol used during the preparationstep. The second weight loss (9.74%) between 100° and210°C corresponds to the elimination of residual alkoxide and

Figure 5. Cellular internalization of TiO2-ZnPc and ZnPc in Vero, HepG2 cells, and human-derived fibroblasts (HDFs). After 24 hours of incubation with 15 μMZnPc or 100 μg/mL of TiO2-ZnPc, live cells were stained with Hoechst 3342 to identify the nucleus. The compounds were observed in the cytoplasm but not inthe nucleus. The fluorescence signal of TiO2-ZnPc was not observed in HDFs.


hydroxyl groups. Above 200°C a gradual weight loss wasobserved up to 400°C, representing 40.36% of total weightloss. This corresponds to the full dehydroxylation of the solidsand the decomposition of organic matter, i.e., phthalocyaninesand oxalates. The residual mass (44.4%) corresponds to TiO2.

The DSC graphs of TiO2-ZnPc-Oxa showed two endothermicpeaks at 144° and 280°C attributed to desorption of water andalkoxide; and one exothermal process at 382°C attributed tooxidation of organic matter. Similar DSC curves were obtainedfor TiO2-ZnPc sample, with slight changes of the temperatureand weight loss values (reported in Table 1).

Raman spectroscopy

Samples (oxalic and acetic acids titanias–ZnPc system) showgreat differences in Raman spectra of the original compoundslike Zn-Pc, TiO2, oxalic and acetic acids. The Raman shiftfrequency is characteristic of materials structure; however, insupported organometallic complexes it is common to observelattice defects, oxygen vacancies, and effects of the particle size.Raman spectrum of TiO2-ZnPc-Oxa shows an intense and sharpsignal at 1500 cm–1 assigned to pyrrole stretching vibrations.Only intense signals below 1000 cm–1 that can be assigned to C-H out of plane of aromatic rings. These observations couldsuggest that the interaction Pc-TiO2 takes place via the nitrogenatoms. In the sample TiO2-ZnPc-Ace is observed a noisy linewithout the bands of the medium range, showing bands at higher

energy (3000–3500 cm–1) that must be related to O-H, N-H, andC-H vibrations.

Photosensitivity of L. chagasi and L. panamensis promastigotesto TiO2, TiO2-ZnPc, and ZnPc

Leishmania spp. is the causative agent of leishmaniasis, oneof the major problems of public health in several tropicalcountries. One of the earliest events after promastigotes haveentered the mammalian host is their contact with plasmaproteins. It has been shown that fresh normal human serumcan cause the lysis of Leishmania spp.

We propose an important alternative to treat leishmaniasis. Itconsists of using photoactive nanostructured TiO2 pure or doped.Treatment with TiO2 pure or with ZnPc-TiO2 was not phototoxicto either species of Leishmania used (Table 2). In contrast,treatment with ZnPc was photoactive in a dose-response range,and as in the mammalian model, a higher phototoxic effect wasobserved after using PRS597-752nm. As was also describedpreviously by Escobar et al14 a similar range of photoactivitywas observed on both species of Leishmania. No photoactivitywas induced by the reference drug amphotericin B (AmB) afterillumination at 2.5 J/cm2 using PRS597-752nm (Table 2).

Intracellular distribution

Upon evaluation after 24 hours' incubation, most of thephthalocyanine fluorescence (red color) was localized in the cell

Figure 6. Comparative subcellular localization of MitoTracker Green FM and LysoTracker Green DND 26 with 15 μM of ZnPc and 100 μg/mL of TiO2-ZnPc indifferent cell lines at 24 hours postincubation. (A) HepG2 cells stained with mitochondria probe, ZnPc, and colocalization, respectively. The overlay pictureclearly indicates that both MitoTracker Green FM and compound localize in mitochondria. Similar patterns were observed in Vero and HDFs with ZnPc, andHepG2 and Vero cells with TiO2-ZnPc. (B) No colocalization of lysosome probe incubated with ZnPc in HDFs. Lysosomal colocalization was observed only inHepG2 cells with ZnPc.


cytoplasm. No fluorescence was detected in the nucleus. Theoptimal concentration of ZnPc and ZnPc-TiO2 used was 15 μMand 100 μg/mL, respectively, as was determined by dose-response experiments. No detectable autofluorescence waspresent in control untreated cells. Some qualitative differencesin the intensity of fluorescence signals were microscopicallyobserved between compounds and cells. Cells treated with ZnPc-TiO2 showed signals of lower intensity than those treated withZnPc at equivalent concentrations. This was true for all studiedcells (Figure 5). No detectable fluorescence signal was observedin HDFs after incubation with ZnPc-TiO2 (Figure 5).

Cytotoxicity

Specific molecular probes were used to determine mito-chondria or lysosome (green color) colocalization of ZnPc-TiO2

and ZnPc. An orange-yellow color was observed when thefluorescence originated from the same organelle. Mitochondriallocalization was observed in Vero, HepG2, and HDFs whenusing ZnPc, and in Vero and HepG2 with TiO2-ZnPc(see Figure 6). ZnPc-TiO2 localization on HDFs was notdetermined due its low fluorescence signal. Lysosomal localiza-tion of ZnPc was observed in HepG2 cells after treatment.

Cytotoxicity in HepG2 culture

The aim was to evaluate the intracellular distribution of TiO2-ZnPc and free ZnPc in both, the tumors and normal derivedhuman cells, to determine the activity of photosensitizers andtheir efficiency as a potential PDT treatment for cancer.

The photodynamic activities of phthalocyanines and TiO2-ZnPc were investigated against four different cell lines, namelyhuman macrophage THP-1 and human hepatocellular carcinomaHepG2, Vero cells, and normal HDFs. The cells were incubatedwith the compounds in the dark, and the migration was analyzedby fluorescence microscopy and registered in a digital camera.Tumor and normal cells were stained additionally with specificmitochondria, lysosomal, and nuclear fluorescent markers. Theefficiency of diffusion of TiO2-ZnPc and free ZnPc into twodifferent types of cells (tumor and normal cells) and the cell

localization are discussed. The preferential localization in targetorganelles, as mitochondria or lysosomes, could determine thecell death mechanism after PDT.

Evaluation of photoactivity of ZnPc-TiO2 versus ZnPc

This evaluation was carried out with THP-1, HepG2, andHDFs; these cells were 17 to 23 times more photosensitive toZnPc than to ZnPc-TiO2, and high PIs were observed (Table 3).Vero cells were statistically (P N 0.05) more photosensitive toZnPc-TiO2 than ZnPc, however, those cells showed the lowestspecific PI values.

Photosensitivity of mammalian cells to TiO2, ZnPc-TiO2,and ZnPc

The cells were evaluated with treatment of TiO2 alone, whichwas not phototoxic to the cells until the higher concentration of50 μg/mL was reached (data not shown). In contrast, ZnPc-TiO2

or ZnPc were photoactive at a small dose (Table 3). Thephototoxic effect was associated with the irradiation system usedand with the type of compound or cell tested. A higher phototoxiceffect was observed using PRS597-752nm than LS670nm. In allexperiments the maximal concentration assayed was not able tokill 90% of the cells except treatment with ZnPc on Vero cellsusing LS670nm. No toxicity was induced on nontreated mamma-lian cells after illumination using PRS597-752nm or LS670nm.Treatment with ZnPc-TiO2 after irradiation at light intensities of2.5 J/cm2 using PRS597-752 was photoactive on all the cells (LC50

0.025 to 3.54 μM). Epithelial Vero cells and the HDFs were themore photosensitive cell line (Table 2). In the absence of light,ZnPc-TiO2 compound was toxic on Vero cells; however, PIvalues higher than 2 were observed (Table 2). Treatment withZnPc-TiO2 on HepG2 cells after LS670nm irradiation was notphotoactive (Table 2). Treatment with ZnPc was photoactive forall cells using LS670nm or PRS597-752nm irradiation system with arange of activities from LC50 0.136 to 1.076 μM and LC50 0.021to 0.152 μM, respectively (Table 1). In the absence of light, ZnPcwas cytotoxic; however, specific PI values in the rank order of

Table 3Photoactivity of TiO2-ZnPc and ZnPc on different mammalian cell lines

FluencyJ/cm2

μM ± SD of ZnPc

Human-derivedfibroblasts

THP-1 macrophages HepG2 cells Vero cells

CC50⁎ PI† CC50 PI CC50 PI CC50 PI

ZnPc-TiO2 0 N10 N1 N10 N1 N10 N1 0.087 ± 0.006 12.5‡ 5.51 ± .63 N1.81 7.45 ± .0.15 N1.34 N10 N1 0.079 ± .0.02 1.12.5║ 3.54 ± .0.49 N2.82 0.28 ± .0.01 N35.64 0.43 ± .0.04 N23.45 0.023 ± .0.003 3.82

10‡ ND‡ ND 2.00 ± 0.22 N5.02 5.50 ± 0.84 N1.81 0.001 ± 0.0007 8.82ZnPc 0 9.21 ± 1.22 1 7.74 ± 3.06 1 10.70 ± 1.17 1 0.78 ± 0.04 1

2.5‡ 1.08 ± 0.05 8.56 0.14 ± 0.04 57.10 0.28 ± 0.07 38.62 0.24 ± 0.02 3.242.5║ 0.15 ± 0.015 60.70 0.02 ± 0.002 369.76 0.035 ± 0.004 303.95 0.09 ± 0.005 8.63

10‡ 0.05 ± 0.04 8.79 0.17 ± 0.01 46.33 0.086 ± 0.015 124.04 0.038 ± 0.002 20.48

⁎ Cytotoxic concentration that induces 50% of cell death.† Phototoxic index ( = CC50 value at 0 J/cm2/CC50 value after irradiation).‡ Irradiation system using a laser light LS670nm.║ Irradiation using a biological photoreactor PRS597-752nm.


THP-1 (PI: 387.25) N HepG2 (PI: 305.94) N HDFs (PI: 61.01) NVero cells (PI: 5.15) were observed.

Discussion

We have studied the photosensitizing effect of ZnPc, nano-TiO2, and ZnPc-TiO2 conjugate, against a panel of tumor andnontumor mammalian cells and on promastigote forms ofLeishmania parasites. As was expected, nano-TiO2 alone undervisible light irradiation was not phototoxic for the cells; incontrast, ZnPc treatment at the same condition was photoactivefor all the studied cells and parasites. The composite ZnPc-TiO2

was not phototoxic for L. chagasi or L. panamensis promasti-gotes; however, it was active against tumor and nontumormammalian cells but less than the pure ZnPc PS. ZnPc-TiO2 wasinternalized by the cells at a lower level than ZnPc. Thelocalization of ZnPc-TiO2 and ZnPc were observed in mito-chondrial cytoplasm. No fluorescence signal was observed inHDFs exposed to ZnPc-TiO2.

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