Photophysicochemical properties and in vitro cytotoxicity of zinc tetracarboxyphenoxy phthalocyanine – quantum dot nanocomposites

Accepted Manuscript Photophysicochemical properties and in vitro cytotoxicity of zinc tetracarboxyphenoxy phthalocyanine – quantum dot nanocomposites David O. Oluwole, Chelsea M. Tilbury, Earl Prinsloo, Janice Limson, Tebello Nyokong PII: DOI: Reference:

S0277-5387(15)00810-4 http://dx.doi.org/10.1016/j.poly.2015.12.060 POLY 11759

To appear in:

Polyhedron

Received Date: Accepted Date:

29 July 2015 30 December 2015

Please cite this article as: D.O. Oluwole, C.M. Tilbury, E. Prinsloo, J. Limson, T. Nyokong, Photophysicochemical properties and in vitro cytotoxicity of zinc tetracarboxyphenoxy phthalocyanine – quantum dot nanocomposites, Polyhedron (2016), doi: http://dx.doi.org/10.1016/j.poly.2015.12.060

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1

Photophysicochemical properties and in vitro cytotoxicity of zinc tetracarboxyphenoxy phthalocyanine – quantum dot nanocomposites David O. Oluwole,a Chelsea M. Tilbury,a Earl Prinsloo,b Janice Limson,b Tebello Nyokonga* a

Department of Chemistry, and b Biotechnology Innovation Centre, Rhodes University, Grahamstown 6140, South Africa Abstract Cadmium based quantum dots (QDs) capped with two different ligands (thioglycolic acid, TGA, and glutathione, GSH) were synthesized. The QDs are: CdTe, CdTe/ZnO, CdTeSe, CdTeSe/ZnO and CdSe/ZnS (the last one for TGA only). Cytotoxicity of the QDs against MCF-7 epithelial breast cancer was evaluated. The TGA capped core QDs were found to be highly cytotoxic to the cell lines when compared to GSH capped ones. The glutathione capped QDs were covalently linked to zinc tetracarboxyphenoxy phthalocyanine (ZnTCPPc). Cytotoxicity and photophysicochemical properties of the conjugates were investigated. The toxicity of the core QDs was reduced in the presence of ZnTCPPc. Enhanced triplet quantum yields and long triplet lifetimes were obtained for ZnTCPPc in the presence of all QDs.

Key words: quantum dots, phthalocyanine, fluorescence quantum yield, cytotoxicity *

Corresponding author. Tel: + 27 46 6038260; Fax: + 27 46 6225109. E-mail: [email protected]. (T. Nyokong)

2 1. Introduction Quantum dots (QDs) have long being explored in biomedical imaging due to their excellent photoluminescence properties [1-4]. The unique optical properties of quantum dots include size dependent emission, photostability, broad absorption, and narrow emission spectra [5]. QDs may consist of a core which is sometimes surrounded by a “shell”, or in some cases, two shells, collectively known as a double shell [6,7]. In this work, we synthesized cadmium based quantum dots containing different shells and two different capping ligands. The QDs employed in this work are: thioglycolic acid (TGA), and glutathione (GSH) capped CdTe, CdTe/ZnO, CdTeSe, CdTeSe/ZnO and CdSe/ZnS (the latter capped with TGA only). This work focuses on cadmium containing quantum dots, which are most commonly used due to their bright fluorescence [6]. Unfortunately, health risks associated with these quantum dots is still under investigation, due to the leaching of Cd2+ ions from the core [8]. The cytotoxicity of CdSe and CdTe QDs has been explored in bacteria [9], yeast [10], on cell lines [11-13] and in vivo (using monkeys) [14]. It has been reported that QDs that contain cadmium, selenium and zinc are not toxic to monkeys for periods of up to 90 days [14]. It is also known that the presence of a shell on QDs reduces toxicity [15]. In this work, cytotoxicity (against MCF-7 breast cancer cell line) behavior of QDs are evaluated when alone or conjugated to zinc tetracarboxy phenoxy (ZnTCPPc) (conjugation for GSH capped QDs only).

3 A metallophthalocyanine (MPc) is employed in this work since there complexes have been reported to have the capacity to generate cytotoxic singlet oxygen that kills tumor cells through photodynamic therapy (PDT) of cancer [16-19]. The amine moiety of the GSH-QDs was linked to the carboxylic acid moiety of the ZnTCPPc (complex 1, Scheme 1) via a carbonamide bond formation to form 1-QDs. MPcs have been linked to QDs for Förster resonance energy transfer (FRET) studies [20-22]. This work reports for the first time on the in vitro cytotoxicity of QDs when linked to MPcs. We show that the cytotoxicity of QDs is improved in the presence of the MPc. 2. Experimental 2.1. Materials Ultra-pure water was obtained from a Milli-Q Water System, selenium powder, sodium borohydride, tellurium granules, cadmium chloride, zinc acetate dihydrate, thioglycolic acid (TGA), glutathione (GSH), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS) and trypan blue were obtained from Sigma Aldrich®.

Methanol (MeOH), dimethylformamide

(DMF) and absolute ethanol (EtOH) were obtained from SAARCHEM®. Cultures of MCF-7 breast cancer cell lines were obtained from Cellonex®. Dulbecco's phosphate-buffered saline (DPBS) and Dulbecco's modified Eagle's medium (DMEM) were obtained from Lonza®, 10 % (v/v) heat-inactivated fetal calf serum (FCS) and 100 unit/mL penicillin-100 µg/mL streptomycin-amphotericin B were obtained from Biowest®. The TGA capped QDs (CdTe and CdTe/ZnO [23],

4 CdSe/ZnS [24]), GSH capped CdTe QDs [25] and zinc tetracarboxyphenoxy phthalocyanines (ZnTCPPc, complex 1) [26] were synthesized as reported in literature.

2.2. Equipment Ground state electronic absorption was measured on Shimadzu® UV-2550 spectrophotometer. Fluorescence excitation and emission spectra were measured on a Varian Eclipse® spectrofluorimeter using a 360 - 1100 nm filter. Excitation spectra were recorded using the Q band of the emission spectra. Fluorescence lifetimes were measured using a time correlated single photon counting setup (TCSPC) (FluoTime 300, Picoquant® GmbH) with a diode laser (LDH-P-485 or LDH-P-670, Picoquant ® GmbH, 20 MHz repetition rate, 44 ps pulse width). LDH-P-485 was used for exciting at a wavelength where QDs absorb, while LDH-P-670 was used at the wavelength for ZnTCPPc (complex 1) absorption. A monochromator with a spectra width of about 8 nm was used to select the required emission wavelength band. The response function of the system, which was measured with a scattering Ludox solution (DuPont®), had a full width at half-maximum (FWHM) of about 300 ps. The ratio of stop to start pulses was kept low (below 0.05) to ensure good statistics. All luminescence decay curves were measured at the maximum of the emission peak. The data were analyzed with the FluoFit software (Picoquant®). The support plane approach was used to estimate the errors of the decay times.

5 FT-IR spectra were recorded on a Perkin-Elmer spectrum 100 with universal attenuated total reflectance (ATR) sampling accessory. X-ray powder diffraction patterns were recorded using a Cu K radiation (1.5405˚A, nickel filter), on a Bruker® D8 Discover equipped with a proportional counter and the X-ray diffraction data were processed using the Eva® (evaluation curve fitting) software. The morphology of the nanoparticles was assessed using transmission electron microscope (TEM) ZEISS LIBRA® model 120 operated at 90 kV.

Triplet quantum yields were determined using laser flash photolysis system. The excitation pulses were produced using a tunable laser system consisting of a Nd:YAG laser (355 nm, 135 mJ/4–6 ns) pumping an optical parametric oscillator (OPO, 30 mJ/3–5 ns) with a wavelength range of 420–2300 nm (NT342B, Ekspla). The analyzing beam source was from a Thermo Oriel® xenon arc lamp, and a photomultiplier tube was used as detector and signals were recorded with a digital real-time oscilloscope (Tektronix® TDS 360) [27]. The detailed procedure of the flash photolysis experiment is as follows: The absorbance of the sample and the ZnPc standard solution were ~1.5 at Q band. The solution was introduced into a 1 cm path length UV-visible spectrophotometric cell and de-aerated using argon for 15 min. Thereafter the solution was sealed and irradiated using an appropriate excitation wavelength (the cross-over wavelength of complex 1 and its nanocomposites with the ZnPc standard). The maximum triplet absorption wavelength (490 nm) was determined from the transient curve.

The triplet lifetimes were

6 determined by exponential fitting of the kinetic curves using OriginPro® 8 software. The time resolved phosphorescence of singlet oxygen at 1270 nm was used to measure the singlet oxygen quantum yield using an ultrasensitive germanium detector (Edinburgh® Instruments, EI-P) combined with a 1000 nm long pass filter (Omega®, 3RD 1000 CP) and a 1270 nm band pass filter (Omega®, C1275, BP50) [28]. Signals were recorded with a digital real-time oscilloscope (Tektronix TDS® 360). The singlet oxygen phosphorescence signals for complex 1 and its nanocomposites were compared with that in the presence of ZnPc standard. Illumination for photodegradation was performed using a general electric quartz lamp (300 W). A 600 nm glass cut off filter (Schott®) and a water filter were employed to filter off ultra-violet and infrared radiation, respectively. An interference filter, 700 nm with a band of 40 nm, was placed in the light path between the sample chamber and 600 nm glass cut off filter for photodegradation quantum yields studies. Light intensities were measured with a POWER MAX 5100 (Molelectron® detector incorporated) power meter and were found to be 4.3 × 10 photons cm -2 s-1 for photodegradation studies. The MCF-7 carcinoma cells were cultured in 75 cm 2 vented flasks (Porvair®) in a humidified atmosphere incubator with ~5 % CO 2 and physiological temprature at 37

o

C (HealForce®). The cells were viewed using a Zeiss®

AxioVert.A1 fluorescence LED (FL-LED) inverted microscope and the cell viability was measured using WST cell proliferation neutral red reagent

7 (Roche®) with Synergy 2 multi-mode microplate reader (BioTek®). Cells were examined using a Zeiss AxioVert.A1 FL-LED inverted microscope with phase contrast.

2.3.

Synthesis

2.3.1. Synthesis of TGA and GSH capped CdTeSe and CdTeSe/ZnO QDs, Scheme 1A TGA and GSH capped CdTeSe (or CdTeSe/ZnO) QDs were synthesized in this work following the procedure reported for CdTeSe/ZnS with some modifications [29]. Sodium hydrogen telluride (NaHTe) precursor was prepared as follows: 0.85 g (6.66 mmol) of tellurium granules and 0.53 g (14.01 mmol) of sodium borohydride were introduced into 50 mL round bottom flask and 20 mL of millipore water was added. The mixture was maintained under ice bath with argon gas bubbling for 10 h. After this time, a pink colouration was observed which indicated the successful formation of the precursor. The same mole ratio was applied to the formation of the NaHSe precursor using selenium powder instead of tellurium granules. Cadmium chloride (1.2 g, 6.55 mmol) was weighed into 250 mL 3-neck round bottom flask, 0.95 mL (0.013 M) of TGA (or GSH) and 200 mL of millipore water were added. The mixture was adjusted under stirring to pH 11 with 1 M sodium hydroxide, followed by deaeration for 30 min under argon. First, 3 mL of the inert NaHTe precursor was injected into the mixture followed by reflux at 100 0C for 40 min, after which 3 mL of the NaHSe precursor was equally added. The mixture was then maintained under argon gas for 3 h.

8 The fluorescence and absorption profiles were measured at predetermined time intervals using aliquots of the growing quantum dot solution. The QDs formed at this stage are the GSH-CdTeSe or TGA-CdTeSe QDs. For the addition of the ZnO shell, 0.44 g (2 mmol) of zinc acetate dihydrate was dissolved in 40 mL ultrapure millipore water (adjusted to pH 8 with 1 M NaOH solution) and this was added into the core (GSH-CdTeSe or TGA-CdTeSe) quantum dots in open air to form GSH-CdTeSe/ZnO or TGA-CdTeSe/ZnO QDs. The fluorescence and absorption profile where further monitored at predetermined time intervals using aliquots of the growing quantum dots. Once the desired emission maximum was observed, the reaction was stopped and cooled at room temperature. The fine crystals were precipitated out of solution using absolute ethanol under centrifugation and successively purified with methanol. The samples were dried under vacuum for 24 h and stored in the dark for further characterization. One size of the core TGA-CdTeSe (3.4) and GSH-CdTeSe (3.6) QDs and two different sizes of the core shell CdTeSe/ZnO (represented as GSH-CdTeSe/ZnO (5.1), GSHCdTeSe/ZnO (6.5), TGA-CdTeSe/ZnO (4.7) and TGA-CdTeSe/ZnO (6.6)) (Table 1), were prepared. The numbers in brackets refer to the QDs sizes as determined by XRD below. 2.3.2. Synthesis of GSH capped CdTe/ZnO QDs To prepare the GSH capped CdTe/ZnO QDs, the method used by Yuan et al. for TGA capped CdTe/ZnO QDs was employed [23]. TGA-CdTe QDs were synthesized as reported in literature [23]. GSH ligand (0.18 g, 0.6 mmol) was introduced into the growing TGA quantum dot core to achieve a ligand exchange [29]. It is

9 important to note that residue of TGA might be present in the formed GSH-CdTe core. In order to form the shell on the GSH-CdTe (to form GSH-CdTe/ZnO), the crude GSH-CdTe solution was heated at 100°C. Zinc acetate dihydrate (0.133 g, 0.61 mmol) in water (8 mL) was added to the solution at a rate of 0.5 mL/2 min. Aliquots of the QDs were collected at predetermined time intervals to monitor the emission and absorption profiles of the QDs. Absolute ethanol was added to the formed QDs and successively centrifuged at 3500 rpm. The GSH-CdTe/ZnO (4.6) QDs were dried under vacuum for 24 h. 2.3.3. Conjugation of complex 1 to glutathione QDs (Scheme 1 B) Complex 1 (0.01 g, 8.9 × 10-3 mmol) was dissolved in 2 mL of dry DMF. Then EDC (0.019 g, 0.1 mmol) and NHS (0.012 g, 0.1 mmol) were added into the solution to activate the carboxylic acid moiety of the ZnTCPPc (complex 1). The reaction was left stirring at ambient temperature for 24 h, after which 3 mL of a solution containing 20 mg GSH-QDs (CdTe (2.9), CdTe/ZnO (4.6), CdTeSe (3.6), CdTeSe/ZnO (5.1) or CdTeSe/ZnO (6.5)) were added and the mixture was left stirring for further 24 h leading to the formation of a carbonamide linked MPc-QD. The nanocomposites were precipitated out of solution with ethanol and were exhaustively purified with methanol. The nanocomposites (represented as 1-CdTe (2.9), 1-CdTe/ZnO (4.6), 1-CdTeSe (3.6), 1-CdTeSe/ZnO (5.1) and 1-CdTeSe/ZnO (6.5)) were dried under vacuum to obtain a powder form for characterization and further studies.

2.4. Photophysicochemical parameters

10 The fluorescence (ΦF) quantum yields of the quantum dots were assessed using comparative method reported in literature [30]. Rhodamine 6G dissolved in ethanol was used as reference standard for QDs in water (ΦF =0.94) [31]. The fluorescence quantum yields of the quantum dots in the conjugate with complex 1 (ɸ ) were calculated using equation (1):  

Φ Conjugate = Φ F(QD) F(QD)

Conjugate FQD

(1)

FQD

where ΦF(QD) represents the fluorescence quantum yield of the quantum dots alone and was used as a standard, FQD is the fluorescence intensity of the quantum dots alone,

 !"#$

F 

represent the fluorescence intensity of the

quantum dots in the conjugate. The triplet state quantum yields (ΦT) of complex 1 alone or when linked with QDs were also determined using comparative methods reported in literature using ZnPc in DMSO as a standard (ΦT(std) = 0.65) [32]. The time resolved phosphorescence decay curve of singlet oxygen at 1270 nm was used to determine the singlet oxygen quantum yields for ZnTCPPc and its conjugates with QDs. Equation 2 was employed [33]:

I (t ) = B

τD [ e −t / τ − e −t / τ ] τ T −τ D T

D

(2)

where, I(t) is the phosphorescence intensity of 1O2 at time t, τD is the lifetime of 1

O2 phosphorescence decay, τT is the triplet state lifetime of the standard or

sample and B is a coefficient involved in sensitizer concentration and 1O2 quantum yield.

11 The singlet oxygen quantum yield (ΦX) of complex 1 and it conjugates with QDs were then determined using Equation 3: *

Φ∆ = Φ '() ∆ . *'()

(3)

where Φ Std is the singlet oxygen quantum yield for the standard ZnPc ( Φ∆ = ∆ Std

0.67 in DMSO [34]), B and BStd are the coefficients of the sample and standard, respectively. The photodegradation quantum yields (Φ,- ), were determined using equation (4) [35]:

Φ,- =

. / 0 .1 2345 . #

(4)

where C0 and Ct in mol dm-3 are the concentrations of sample before and after irradiation, respectively, V is the solution volume, t is the irradiation time per cycle while Iabs is defined by equation (5).

I"78 =

α.9.2 :;

(5)

where α = 1-10-A(λ), A(λ) is the absorbance of the sensitizer at the irradiation wavelength, I is the intensity of light (4.3 × 10 cm-2 s-1), and S is the irradiated cell area (2.4 cm2), and NA is Avogadro's constant. The efficiency of energy transfer (S=  between triplet state and molecular oxygen was determined using equation (6):

>∆ =

Φ∆ Φ?

(6)

where ΦT is the triplet quantum yield and Φ∆ is the singlet quantum yield.

2.5. Cytotoxicity studies

12 The MCF-7 carcinoma cell line were cultured using Dulbecco's modified Eagle's medium (DMEM) containing 4.5 g/L glucose with L-glutamine and phenol red, supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS), and 100 unit/mL penicillin-100 µg/mL streptomycin-amphotericin B. The cells were grown in 75 cm2 vented flasks (Porvair®) and incubated at 37 oC and 5 % CO2 with humidified atmosphere and routinely subcultured by standard trypsinisation. Once 100 % cell confluence was achieved, viable trypsinised cells were counted via trypan blue dye exclusion assay (0.4 % trypan blue solution) using a hemocytometer. The cells were seeded at a cell density of 10,000 cells/well in supplemented DMEM containing phenol red in 96-well tissue culture plates (Porvair®). Afterwards, the cells were incubated at 37 oC and 5 % CO2 for 24 h to foster cell attachment to the wells. The attached cells were rinsed with 100 µl DPBS once, followed by administration of 100 µl supplemented DMEM containing 5 mg/mL of complex 1, QDs or 1-QDs. Four concentrations 2, 3, 5 and 10 mg/mL were studied, cytotoxicity increased with increase in concentration. However 5 mg/mL is used as an example for the cytotoxicity studies. The conjugates used are 1-CdTe (2.9), 1-CdTeSe/ZnO (5.1) and 1-CdTeSe/ZnO (6.5). The stock concentration of the ZnTCPPc and the nanocomposites were prepared by dissolving them in DMSO and making the volume up with supplemented DMEM. The effect of DMSO on the cells were investigated by incubation of the cells for 24 h with 0.1 % (v/v) DMSO in supplemented DMEM which represents the highest

13 percentage of DMSO used for preparing the highest concentration of the drugs. Control cells were incubated with or without DMSO in supplemented DMEM only. The 96-well plates containing cells and the drugs were incubated at 37 oC and 5 % CO2 in the dark for 24 h. After 24 h treatment, the wells were rinsed with 100

µl DPBS, supplemented DMEM with phenol red was added and the plates reincubated for 24 h. Cell survival was expressed as percentage of placebo cells (cells without drugs containing supplemented DMEM with phenol red). After 24 h re-incubation with supplemented DMEM with phenol red, cell proliferation neutral red reagent (WST-1 assay) was used to quantify the surviving cells. The WST-1 assay was used to assess the toxicity and cell proliferation in the monolayer of the cells treated with the drugs and the placebo cells respectively: this was carried out based on the manufacturer’s instruction using a Synergy 2 multi-mode microplate reader (BioTek®) at a wavelength of 450 nm. The percent cell viability was determined using the formula below: J78K7"L$ 8"M,N$ "# O P

% cell viability = J78K7"L$ L#KN "# O P × 100

(7)

where the absorbance of sample is the cells containing drugs while absorbance of control is the placebo cells containing only supplemented DMEM with phenol red. 2.5.2. Experimental data analysis The analytical data obtained from the experiments were statistically analysed using Microsoft Excel worksheet 2010 (Microsoft®) and Graphpad Prism6®. The mean obtained from the control and experimental conditions were analysed with

14 paired student T–test at 95% (α = 0.05) confidence interval and p values less than 0.05 (P < 0.05) was considered statistically significant. The data obtained from the three independent triplicate experiments (n=3) were analysed with one-way analysis of variance (ANOVA) for the in vitro dark cytotoxicity.

3. Results and discussion

Scheme 1A illustrates the synthetic route for the TGA-CdTeSe/ZnO (as an example). Scheme 1B illustrates the route for the covalent linkage of complex 1 to GSH-QDs. The linkage was via carbonamide bond formation. The size of the Pcs is approximately 1 nm while the sizes of the glutathione quantum dots range from 2.9 nm to 6.5 nm (as determined by XRD below). Hence, it is likely that more than one Pcs can be bond to each QD considering the size of Pc relative to QDs. The number of Pcs bonded to the QDs was estimated following literature methods, but using absorption instead of fluorescence [36]. The QDs:Pcs ratios are: 1-CdTe(2.9) (1:3). 1-CdTe/ZnO(4.6) (1:5). 1-CdTeSe(3.6) (1:4), 1CdTeSe/ZnO(5.1) (1.4), 1-CdTeSe/ZnO(6.5) (1:8). Thus, the number of ZnTCPPc bound to each QD are higher for the larger 1-CdTeSe/ZnO (6.5) compared to the smaller 1-CdTeSe/ZnO (5.1).

3.1. Characterization of the QDs and their conjugates with ZnTCPPc 3.1.1. UV/Vis absorption and emission spectra

15 Figure 1A depicts the normalised absorption and emission spectra of thioglycolic acid capped QDs (as examples), with broad absorption bands ranging from 493 nm to 589 nm and narrow emission bands ranging from 562 nm to 628 nm, Table 1.The glutathione capped QDs showed absorption bands ranging from 471 nm to 511 nm and emission band ranging from 523 nm to 612 nm, Table 1. Larger QDs of the same type (compare GSH-CdTeSe/ZnO (5.1) with GSH-CdTeSe/ZnO (6.5) and TGA-CdTeSe/ZnO (4.7) with TGA-CdTeSe/ZnO (6.6)) are red shifted compared to the smaller ones. Figure 1B shows the normalised absorption spectra of the complex 1 and its nanocomposites (1-QDs, using CdTeSe (3.6), CdTe/ZnO (4.6) and CdTeSe/ZnO (6.5) as examples). The observed enhancement at less than 610 nm in the absorption spectra could be attributed to the presence of GSH-QDs. No significant shift was observed in the Q bands of complex 1 in the presence of QDs. Fig. 1C shows the normalised absorption, emission and excitation at 610 nm in DMSO of 1-CdTeSe/ZnO (6.5) nanocomposite (as an example) with absorption maxima at 677 nm, emission maxima at 688 nm and excitation maxima at 679 nm. The emission spectrum was found to be a mirror image of the excitation and absorption spectra of the nanocomposites.

3.1.2. FT-IR spectra The FTIR spectrum of the thioglycolic acid ligand alone showed four distinct bands: 3440 cm-1 and 2565 cm-1 corresponding to hydroxyl stretch and sulfhydryl, respectively, while bands at 1703 cm-1 and 1396 cm-1 correspond to symmetrical

16 and asymmetrical carbonyl (Figure 2(a)). Similar bands were observed for the d QDs but with disappearance of sulfhydryl band. The disappearance of sulfhydryl band is due to bonding of TGA (or GSH) to QDs (Figure 2(b). The spectra of the GSH-QDs (using GSH-CdTeSe/ZnO (6.5) as an example) showed

four distinct

bands at 1225 cm-1 (C–N), 1378 cm-1, 1563 cm-1 (symmetrical and asymmetrical COO-, respectively) and 3334 cm-1 (NH2 stretch, Fig. 2(b)). 1-CdTeSe/ZnO(6.5) showed bands at 1596 and 1736 cm-1 corresponding to primary and secondary carbonamide bands (O=C-NH-) (Figure 2(c)), respectively. ZnTCPPc showed five bands: at 1225 cm-1 (C-O-C), 1470 cm-1 (C=N), 1597 cm-1 (C=C), 1687 cm-1 (C=O) and 3065 cm-1 (OH) (Figure 2(d)). It is known that shifts in IR bands confirm structural change [37-39]. Thus, the formation of the carbonamide band, the shift in the IR vibrations due to C-O-C from 1225 cm-1 to 1232 cm-1confirm the successful formation of the nanocomposites. 3.1.3. TEM The morphology of the QDs were assessed using the TEM (Fig. 3). The QDs were found to be monodispersed. The sizes determined by XRD are employed in this work. The sizes determined by TEM are 2.8 nm, 4.5 nm, 4.2 nm, 4.9 nm, 7.3 nm and 4.3 nm for TGA capped QDs: CdTe (2.8), CdTe/ZnO (4.0), CdTeSe (3.4), CdTeSe/ZnO (4.7), CdTeSe/ZnO (6.6), CdSe/ZnS (4.0), respectively. For the GSH capped QDs sizes are (2.7 nm, 4.7 nm, 5.8 nm, 5.9 nm, 6.1 nm) (Table 1). Some of the sizes obtained from TEM were in conformity with the sizes obtained from XRD while some were found to have slight deviation from XRD values and this

17 could be attributed to the effects of aggregation on the QDs in some cases for TEM.

3.1.4. XRD studies The QDs exhibited three prominent diffraction peaks at 2θ = 27°, 44°, 52°. The 2θ values obtained were in accordance with zinc blend crystal and cubic structure at planes 111, 220 and 311, respectively. The QDs sizes were calculated based on the Debye-Scherrer eq. (8) [40]:

d=

kλ β Cosθ

(8)

where λ is the wavelength of the X-ray source (1.5405 Å), k is an empirical constant equal to 0.9, β is the full width at half maximum of the diffraction peak and θ is the angular position. The sizes of the QDs are listed in Table 1 are used in this work. Upon functionalization with ZnTCPPc there were slight shifts in XRD peaks of the QDs (Fig. 4(b)). Fig. 4(c), shows a peak near 2θ = 25º, which is typical of phthalocyanines [41], showing its amorphous state.

3.2. Fluorescence (Φ F) quantum yields and lifetimes of QDs when alone or in conjugation with complex 1. These studies were performed in order to study the effects of phthalocyanines on QDs emission, since this is the most important property of the QDs. The largest ΦF value for the TGA capped QDs was observed in TGA-CdTe (2.8) with 0.60 while the least was seen in TGA-CdSe/ZnS (4.0) with 0.02. For the GSH

18 capped QDs, the highest ΦF value was found for CdTe/ZnO (4.6) at 0.36 and the least in CdTeSe (3.6) at 0.09, Table 1. Lower fluorescence quantum yields are observed for the larger QDs of the same type (compare GSH-CdTeSe/ZnO (5.1) with GSH-CdTeSe/ZnO (6.5) and TGA-CdTeSe/ZnO (4.7) with TGA-CdTeSe/ZnO (6.6)). The low ΦF value for the larger QDs could be due to high surface traps which create dangling bonds on the surface of the QDs as the size increases [42]. Literature reports have shown that CdTeSe core QDs have structural defects, resulting in non-radiative decay [43-45]. In Table 1, we observed an enhancement of fluorescence quantum yields (Table 1) for both core/shell GSHCdTeSe/ZnO (5.1) and GSH-CdTeSe/ZnO (6.5) QDs compared to core GSHCdTeSe. For TGA capped QDs, the ΦF increased only for core/shell TGACdTeSe/ZnO (4.7) compared to core TGA-CdTeSe. No significant increase in fluorescence was obtained for TGA CdTeSe/ZnO (6.6). For the TGA-CdSe/ZnS (which has the lowest ΦF value), the low ΦF value could be due the quenching effects associated with ligand exchange involving organic ligand capped QDs to form aqueous capped QDs [46,47] which leads to loss of fluorescence quantum yield. Three photoluminescence lifetimes were obtained as is typical of QDs [48]. Typical photoluminescence decay curves are shown in Fig. 5 for (a) GSHCdTe/ZnO (4.6) and (b) 1-CdTe/ZnO (4.6), on excitation where QDs aborb. Average lifetimes are provided on Table 1 and the discussion below is based on these values. Long fluorescence lifetimes were observed where there was high

19 fluorescence quantum yield (vice versa). Of the TGA capped QDs, TGA-CdTe (2.8) with the longest lifetime of τf = 22.8 ns had the largest fluorescence quantum yield (0.60). A similar trend was observed for the GSH capped QDs with GSH-CdTe/ZnO (4.6) accounting for the longest lifetime τf of 35.69 ns and a high fluorescence quantum yield of ΦF = 0.35. In the presence of complex 1, the data was recorded in DMSO due to aggregation in aqueous media. The fluorescence parameters for the QDs alone were thus repeated in DMSO in order to compare with the values for QDs in the conjugate. Thus the values for QDs alone in Table 1 differ only slightly to those in Table 2. We observed a decrease in the fluorescence quantum yields and lifetimes of the QDs upon linkage with ZnTCPPc, Table 2, and this observed phenomenon could be due to FRET and other process which deactivate the excited singlet state of the QDs [49-51]. In addition, MPcs may exert strain on QDs eventually creating new trap states, leading to decrease in the fluorescence emission [49]. 3.3. Photophysicochemical parameters for complex 1 alone and in conjugation with QDs The triplet quantum yields studies of the ZnTCPPc (complex 1) alone and the nanocomposites were carried out in DMSO. Figure 6 shows the triplet decay curve of ZnTCPPc (complex 1) (Figure 6a) alone and in the presence of QDs (1-CdTeSe/ZnO (6.5)) (Figure 6b). Figure 7 shows the singlet oxygen decay curve for ZnTCPPc in 1-CdTeSe/ZnO (6.5). Increases in triplet and singlet quantum yields were observed in the nanocomposites of the QDs and ZnTCPPc

20 as compared to complex 1 alone. These increases could be due to heavy atom effect associated with quantum dots which encourages intersystem crossing to the triplet state [52,53]. The efficiency of energy transfer S∆ between triplet state and molecular oxygen of the nanocomposites were found to be significantly higher for the nanocomposites compared to complex 1 alone. The nanocomposites were found to be highly stable upon exposure to irradiation for 90 minutes which is a good indication for a viable photosensitizing agent with photodegradation quantum yield of the order 10-7 (Table 2). Unstable phthalocyanines usually show photodegradation quantum yield in the order of 10-3 [54] which is not an ideal condition for a suitable photosensitizer.

3.4

Cytotoxicity

The percent cell viabilities of the DMSO (used as the solvent) at 0.1 %, was found to be similar with what was obtained in the supplemented DMEM alone control (supplementary information, Fig. 1).

The percentage cell viability (representing active cells) data for all the QDs alone and conjugates of GSH-QDs with complex 1 are listed in Table 3. The GSHQDs, Table 3, showed less cytotoxic effects (higher % viability) when compared to the corresponding TGA-QDs analogues. This observation could be due to the fact that glutathione is a bifunctional ligand which is also known to have the ability to serve as an essential co-factor in cellular metabolism [55].The

21 addition of a shell to the core QDs resulted in the increase in percent viability, hence the QDs are less toxic in the presence of the shell (with the exception of GSH-CdTe (2.9) and GSH-CdTe/ZnO(4.6)).

ZnTCPPc (1) and the nanocomposites: 1-CdTe (2.9), 1-CdTeSe/ZnO (5.1) and 1CdTeSe/ZnO (6.5) were tested for dark cytotoxicity.

ZnTCPPc alone or in its

conjugates had higher percent cell viability (> 70 % at the investigated concentrations) than QDs alone in the absence of illumination (Table 3). The percent cell viability (related to active cells) of the conjugates is higher than for QDs alone but lower than for ZnTCPPc alone, Table 3. Thus, the Pc complex appears to reduce the toxicity of QDs.

Figure 8, shows the photo-micrograph of the cell morphology (effect of cytotoxicity) in absence of drug (control) (Figure 8a), in the presence of 1CdTeSe/ZnO (5.1) or TGA-CdTe (2.8) (Figure 8b and 9c, respectively). We observed that the nanocomposites (using 1-CdTeSe/ZnO (5.1) as an example) showed little or no cytotoxic activities against the MCF-7 cell line. However, the TGA-CdTe (2.8) cell micrograph showed very few viable cells which further confirms the toxicity of core quantum dots (Figure 8c).

4. Conclusion

We evaluate for the first time the photophysicochemical properties and in vitro photodynamic therapy activity of ZnTCPPc and it nanocomposites with GSH-QDs using MCF-7 carcinoma cell lines. An enhancement in the triplet and singlet

22 quantum yield was observed for ZnTCPPc in the presence of GSH capped QDs when compared to ZnTCPPc alone. The TGA capped core QDs were found to be more cytotoxic to the cell lines than the GSH capped QDs. QDs were found to be less toxic in the presence of ZnTCPPc.

Acknowledgements This work was supported by the Department of Science and Technology (DST) Innovation and National Research Foundation (NRF), South Africa through DST/NRF South African Research Chairs Initiative for Professor of Medicinal Chemistry and Nanotechnology (UID 62620) as well as Rhodes University.

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28

A

O

S

O

S

ZnO

CdTe

o

100 C, Ar, 3 h

CdTeSe

zinc acetate o

100 C, open air, 2 h

O OH

OH

OH NaHSe

S

CdTeSe

Scheme 1: Synthetic route of (A) thioglycolic acid capped-CdTeSe/ZnO and (B) conjugate 1-QDs

29 1.0

Normalised Absorbance

(a) (b) (c)

2.0

0.8

1.5

0.6

1.0

0.4

0.5

0.2

0.0 300

400

500

600

700

Normalised Photoluminescene

2.5

A

0.0 800

B

1.0

Normalised Absorbance

Wavelength (nm)

0.8

0.6

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

0.4

0.2

0.0 300

400

500

600

Wavelength (nm)

700

800

Normalised Absorbance

C

1.0

1.0

0.8

0.8

0.6

0.6

0.4

0.4 (a) (b ) (c )

0.2

0.2

0.0

Normalised Intensity

30

0.0 300

400

500

600

700

800

Wavelength (nm)

Figure 1: (A) Normalised absorption (dotted line) and emission (solid line) spectra of TGA capped QDs. (a) CdTe (2.8), (b) CdTeSe/ZnO (4.7), and (c) CdTeSe/ZnO (6.6) in water at excitation wavelength of 400 nm. (B) Normalised absorption spectra of (a) ZnTCPPc (complex 1), (b) 1-CdTe/ZnO (4.6), (c) 1CdTeSe (3.6) and (d) 1-CdTeSe/ZnO (6.5) in DMSO. (C) Normalised absorption (a), emission (b) and excitation (c) spectra of 1-CdTeSe/ZnO (6.5) in DMSO at excitation wavelength of 610 nm. Excitation for (D) at the Q band maxima. All Pc spectra are normalized to the Q band absorption.

31

1396

2565

3440

1703

Transmittance (%)

(a)

1225 1563

3334

1378

(b)

1736 3251

1596 1391

(c)

3065

1687 1597

1440 1225

(d) 4000

1232

3500

3000

2500

2000

1500

-1 Wavenumber (cm )

1000

Figure 2: FT-IR spectra of (a) TGA, (b) GSH-CdTeSe/ZnO(6.5), (c) 1CdTeSe/ZnO (6.5), (d) ZnTCPPc (1).

32

Figure 3: TEM micrograph of thioglycolic acid capped-QDs: (a) TGA-CdTe/ZnO (4.0), (b) TGA-CdTeSe/ZnO (6.6) and (c) TGA-CdSe/ZnS (4.0).

33

111

a 220

311

Lin Counts

b

c

20

30

40

2-Theta

50

60

Figure 4: XRD diffractograms of (a) GSH-CdTeSe/ZnO (6.5), (b) 1-CdTeSe/ZnO (6.5) and (c) ZnTCPPc (1).

34

5000

Amplitude

4000 3000 (a)

2000

(b)

Residuals

1000 0 4 3 2 1 0 -1 -2 -3 -4 0

30

60

90

120

Time (ns)

Fig. 5: Fluorescence lifetime decay curve for (a) GSH-CdTe/ZnO (4.6) in water, and (b) 1-CdTe/ZnO (4.6) in DMSO at excitation wavelength of 527 nm.

Change of Absorbance

0.03

0.02

(a) 0.01

(b)

0.00 0

0.0005

0.001

0.0015

0.002

Time (µs)

Fig 6: Triplet lifetime decay curve of (a) ZnTCPPc 1, (b) 1-CdTeSe/ZnO (6.5) in argon deaerated DMSO excited at 490 nm.

35 0.03

Intensity

0.02

0.01

0.00 0

25

50 T im e

75

100

(µ s )

Fig 7: Singlet Oxygen Phosphorescence decay curve of 1-CdTeSe/ZnO (6.5) in DMSO.

36

(a)

(b)

(c)

(c)

Figure 8: Photo-micrograph for cytotoxicity of MCF-7 carcinoma cell lines at 200 µm magnification: (a) control cells (placebo cells), (b) 1-CdTeSe/ZnO (5.1) and (c) TGA-CdTe (2.8).

37 Table 1: Fluorescence parameters for cadmium based quantum dots in water. Abundances are shown in brackets for lifetimes Compound

a

λabs

(nm)

b

λems (nm)

d (nm) XRD

d (nm) TEM

(±0.02)

ΦF

c

τf(ns)

c

(±1.5)

(±0.4)

(±0.08)

Mean τf

τf(ns)

c

τf(ns)

Thioglycolic acid capped QDs CdTe(2.8)

539

581

2.8

2.8

0.60

31.62(61.55)

9.34(35.76)

0.19(2.69)

22.81

CdTe/ZnO(4.0)

589

628

4.0

4.5

0.07

49.0(12.14)

2.01(84.65)

18.05(3.21)

8.23

CdSe/ZnS(4.0)

568

597

4.0

4.3

0.02

12.04(39.94)

2.52(56.03)

1.43(4.03)

6.28

CdTeSe(3.4)

503

562

3.4

4.2

0.09

11.29(61.52)

0.19(24.07)

2.32(14.41)

7.33

CdTeSe/ZnO(4.7)

493

586

4.7

4.9

0.25

22.22(74.48)

6.20(23.76)

0.80(1.76)

18.04

CdTeSe/ZnO(6.6)

499

608

6.6

7.3

0.10

0.72(2.77)

3.77(16.19)

17.29(81.04)

14.64

Glutathione capped QDs CdTe(2.9)

471

523

2.9

2.7

0.12

7.63(29.68)

25.67(67.47)

1.48(2.85)

19.63

CdTe/ZnO(4.6)

479

527

4.6

4.7

0.36

73.47(29.07)

23.96(56.17)

5.94(14.76)

35.69

CdTeSe(3.6)

500

594

3.6

5.8

0.09

26.41(58.64)

6.58(34.50)

0.96(6.86)

17.82

CdTeSe/ZnO(5.1)

491

582

5.1

5.9

0.29

64.88(24.49)

23.30(61.86)

5.84(13.65)

31.10

CdTeSe/ZnO(6.5)

511

612

6.5

6.1

0.14

60.63(28.61)

14.73(50.89)

2.46(20.50)

25.35

a

λabs: Absorption maxima, bλems: Emission maxima, c abundances in brackets

38

Table 2: Photophysicochemical data of zinc tetracarboxyphenoxy phthalocyanine linked with GSH-QDs in DMSO a

a

τf(QDs) (ns) (±0.02)

S∆

ΦF(QDs) (±0.02)

0.17

0.35

-

-

7.32

279

0.23

0.45

0.009(0.12)

3.24(19.6)

2.04

0.57

276

0.25

0.44

0.014(0.36)

4.03(35.69)

1.84

1-CdTeSe(3.6)

0.54

370

0.30

0.56

0.005(0.09)

2.36(17.8)

1.82

1-CdTeSe/ZnO(5.1)

0.53

370

0.29

0.55

0.009(0.29)

2.76(31.5)

1.63

1-CdTeSe/ZnO(6.5)

0.56

266

0.27

0.48

0.006(0.14)

3.43(25.4)

1.46

Compound

ΦT

τt(µs)

Φ∆

(±0.01)

(±0.02)

(±0.03)

ZnTCPPc (1)

0.49

238

1-CdTe(2.9)

0.51

1-CdTe/ZnO(4.6)

a

Numbers in brackets are for QDs alone in the absence of Pc.

Φpd 10-7

39

Table 3: Cytotoxicity (as percent cell viability) of cadmium based QDs, ZnTCPPc and nanocomposites

Compound

5 (mg/mL)

Thioglycolic acid (TGA) capped QDs

CdTe(2.8)

36 ± 2 %

CdTe/ZnO(4.0)

48 ± 2 %

CdTeSe(3.4)

65 ± 2 %

CdTeSe/ZnO(4.7)

67 ± 2 %

CdTeSe/ZnO(6.6)

66 ± 2 %

CdSe/ZnS(4.0)

64 ± 2 %

Glutathione (GSH) capped QDs CdTe(2.9)

65 ± 0 %

CdTe/ZnO(4.6)

60 ± 1 %

CdTeSe(3.6)

65 ± 1 %

CdTeSe/ZnO(5.1)

69 ± 1 %

CdTeSe/ZnO(6.5)

66 ± 3 %

ZnTCPPc (1) and Conjugates

ZnTCPPc (1)

99 ± 15 %

1-CdTe(2.9)

76 ± 1 %

1-CdTeSe/ZnO(5.1) 72 ± 3 % 1-CdTeSe/ZnO(6.5) 77 ± 3 %

The ± values indicate standard error of the mean of each data presented in percentage. Asterisk (*) values indicate insignificant difference between the dark cytotoxicity data and the photodynamic effects data obtained due to pvalue above 0.05 ((p > 0.05)) while data without asterisk show significant difference with p-value less than 0.05 (p < 0.05). Student t-test was employed for the comparison between the dark and photo-toxicity.

41

Photophysicochemical properties and in vitro photodynamic activities of zinc tetracarboxyphenoxy phthalocyanine – quantum dot composites David O. Oluwole, Chelsea M. Tilbury, Earl Prinsloo, Janice Limson, Tebello Nyokong Cytotoxicity of the CdTe, CdTe/ZnO, CdTeSe, CdTeSe/ZnO and CdSe/ZnS QDs against MCF-7 epithelial breast cancer was evaluated when alone or when linked to zinc tetracarboxyphenoxy phthalocyanine. The toxicity of QDs improved in the presence of the phthalocyanine

O

ZnO

OH

ZnO

C dTeSe

O

S O HOOC

N

N

HO

O

N

Zn

O

O

N

N

N

O

HO

NH

N

O

NH NH

O

N

O HOOC