The effects of silica based nanoparticles on the photophysicochemical properties, in vitro dark viability and photodynamic therapy study of zinc monocarboxyphenoxy phthalocyanine

Accepted Manuscript Title: The effects of silica based nanoparticles on the photophysicochemical properties, in vitro dark viability and photodynamic therapy study of zinc monocarboxyphenoxy phthalocyanine Author: David O. Oluwole Imran Uddin Earl Prinsloo Tebello Nyokong PII: DOI: Reference:

S1010-6030(16)30118-6 http://dx.doi.org/doi:10.1016/j.jphotochem.2016.07.002 JPC 10278

To appear in:

Journal of Photochemistry and Photobiology A: Chemistry

Received date: Revised date: Accepted date:

18-2-2016 26-6-2016 3-7-2016

Please cite this article as: David O.Oluwole, Imran Uddin, Earl Prinsloo, Tebello Nyokong, The effects of silica based nanoparticles on the photophysicochemical properties, in vitro dark viability and photodynamic therapy study of zinc monocarboxyphenoxy phthalocyanine, Journal of Photochemistry and Photobiology A: Chemistry http://dx.doi.org/10.1016/j.jphotochem.2016.07.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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The effects of silica based nanoparticles on the photophysicochemical properties, in vitro dark viability and photodynamic therapy study of zinc monocarboxyphenoxy phthalocyanine David O. Oluwole,a Imran Uddin,a Earl Prinsloo,b and Tebello Nyokong*a a

Department of Chemistry, bBiotechnology Innovation Centre , Rhodes University,

Grahamstown 6140, South Africa *

Corresponding author. Tel: + 27 46 6038260; Fax: + 27 46 6225109.

E-mail: [email protected]. (T. Nyokong) Graphical abstract

David O. Oluwole, Imran Uddin, Earl Prinsloo, and Tebello Nyokong

SiO2 and ZnO/SiO2 nanoparticles were covalently linked to zinc monocarboxyphenoxy phthalocyanine resulting in enhanced photophysicochemical behavior and improved in vitro dark cytotoxicity against MCF-7  

cell line

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Research Highlights



SiO2 and core/shell ZnO/SiO2 nanoparticles were covalently linked to zinc monocarboxyphenoxy phthalocyanine



he

nanoconjugates

showed

enhanced

photophysicochemical

behavior

as

compared to phthalocyanine alone



in vitro dark cytotoxicity against MCF-7 cell line was less for the conjugates than for phthalocyanine alone

Abstract Aminopropyl triethoxysilane functionalized core SiO2 and core/shell ZnO/SiO2 nanoparticles (NP) were covalently linked to zinc monocarboxyphenoxy phthalocyanine (ZnMCPPc, complex 1) via amide bond formation. The investigation of their photophysicochemical behavior, in vitro dark viability and photodynamic therapy (PDT) activity against human breast adenocarcinoma cell line (MCF-7 cells) were studied. The nanoconjugates showed enhanced photophysicochemical behavior as compared to complex 1 alone. Complex 1 showed higher dark toxicity against MCF-7 cells when compared to the conjugates. In the dark, complex 1 accounted for less than 50 % viable cells at 28.6 μg/mL and 57.1 μg/mL compared to the conjugates which accounted for more than 50 % cell viability at these concentrations. The in vitro dark viability and PDT activity of complex 1 was reduced in the presence of these nanoparticles.

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Key words: Photodynamic therapy, phthalocyanines, in vitro dark viability

ZnO/SiO2

nanoparticles,

cancer

cells.

1. Introduction Photodynamic therapy (PDT) involves the combination of a photosensitizing drug, oxygen and light to cause selective damage to the target tissue. The overall effectiveness of

PDT

requires

careful

planning

of

both

drug

and

light

dosimetry

[1].

Metallophthalocyanines (MPcs) have been found to be suitable PDT photosensitizers for the treatment of cancerous cells due to their unique photophysicochemical properties which include intense and tunable light absorption in the near-infrared region as well as their ability to efficiently generate singlet oxygen, which is the chief cytotoxic species for PDT [2,3]. On the other hand nanoparticles effectively improve the photophysicochemical and PDT activities of photosensitizers [4,5]. The ease of functionalization of nanoparticles (NPs) such as silica (SiO2 NPs) and zinc oxide (ZnO NPs) with different materials such as fluorophores, drugs, bio-molecules, dendrimers and polymers, has led to the development of improved and multifunctional materials that have found applications in areas such as drug delivery, sensing, diagnosis, imaging and theranostics [6-12]. In this study, aminopropyl triethoxysilane (APTES) functionalized silica nanoparticles (SiO2 NPs) and ZnO nanoparticle core with SiO2 nanoparticle shell (ZnO/SiO2 NPs) were covalently linked to zinc monocarboxyphenoxy phthalocyanines (ZnMCPPc, complex 1, Scheme 1) via the carboxyl functional moiety of the latter and the amine functional moiety of the former. The photophysicochemical properties, the in vitro dark cytotoxic

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and PDT activity (against MCF-7 cells) of complex 1 and its conjugates with NPs are investigated in this work. Silica nanoparticles have been linked to phthalocyanines with improved photophysical behavior [13,14]. But there have been no studies on their activity on cancer cells, until this report. Also, this is the first time core shell ZnO/SiO2 NPs are linked to phthalocyanines. The presence of Zn in the ZnO/SiO2 NPs is expected to result in even more improved triplet state population of complex 1 due to the heavy atom effect of Zn in the NPs, which encourages intersystem crossing to the triplet state. A ZnPc derivative was chosen due to the heavy atom effect of Zn and a mono carboxy substituent allows for a single coordination for the NPs. 2. Experimental 2.1. Materials Ultra-pure water was obtained from a Milli-Q Water System, dimethylsulfoxide (DMSO),

N,N’-dicyclohexylcarbodiimide

(DCC),

4-(dimethylamino)pyridine

(DMAP),

zinc

phthalocyanine (ZnPc) 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), trypsin ethylenediaminetetra acetic acid (EDTA) and Dulbecco's modified Eagle's medium (DMEM) were obtained from Sigma Aldrich®, 10 % (v/v) heat-inactivated fetal calf serum (FCS), neutral red cell proliferation reagent (WST) and 100 unit/mL penicillin-100 μg/mL streptomycin-amphotericin B were obtained from Lonza®. SiO2 NPs and ZnO/SiO2 NPs coated with aminopropyl triethoxysilane

5    (APTES) were synthesized according to literature [15-17]. ZnMCPPc was also synthesized

as reported before [18].

2.2. Equipment The ground state electronic absorption was recorded on Shimadzu® UV-2550 spectrometer. FT-IR spectra were measured on Bruker® ALPHA FT-IR spectrometer with universal attenuated total reflectance (ATR) sampling accessory. Fluorescence emission and excitation spectra were acquired on Varian Eclipse® spectrofluorometer using a 360-1100 nm filter. Fluorescence lifetimes were measured using a time correlated single photon counting setup (TCSPC) (FluoTime 300, Picoquant® GmbH) with LDH-P-670, Picoquant® GmbH, 20 MHz repetition rate, 44 ps pulse width). Details have been provided before [19]. 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 Xray diffraction data were processed using the Eva® (evaluation curve fitting) software. The morphologies of the nanoparticles and nanoconjugates were assessed using transmission electron microscope (TEM) ZEISS LIBRA® model 120 operated at 90 kV and micrograph were processed with iTEM® software. Energy dispersive x-ray spectrometer (INCA PENTA FET coupled with VAGA TESCAM® operated at 20 kV) was used to qualitatively determine the elemental compositions of the NPs and the nanoconjugates. Micrometrics ASAP® 2020 Surface Area and Porosity Analyzer was used to acquire the nitrogen adsorption/desorption isotherms of the NPs operated at 77 K.

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X-ray photoelectron spectroscopy (XPS) data were collected using a Kratos Axis Ultra DLD with an Al (monochromatic) anode, equipped with charge neutralizer. The operational protocol has been reported before [20].

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 (NT-342B, Ekspla) as described before [19].

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) [21]. Signals were recorded with a digital real-time oscilloscope (Tektronix TDS® 360). The singlet oxygen phosphorescence signal of ZnMCPPc (complex 1) alone and in conjugates were compared with that of ZnPc standard. The MCF-7 carcinoma cells were cultured in 75 cm2 vented flasks (Porvair®) in a humidified atmosphere incubator with ~5 % CO2 and physiological temperature at 37 oC (HealForce®). The cells were viewed using a Zeiss® AxioVert A1 fluorescence LED (FL-LED) inverted microscope with phase contrast. Cell viability was measured using WST cell proliferation neutral red reagent (Roche®) with Synergy 2 multi-mode microplate reader (BioTek®).

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Illumination set–up for PDT was performed using a general electric quartz lamp (300 W) while 600 nm ( 3 nm) cut-off glass filter (Schott®) for ultra violet radiation and a water filter for infrared radiation. An interference filter, 700 nm with a band of 40 nm. Light intensities were measured with a POWER MAX 5100 (Molelectron® detector incorporated) power meter and were found to be 4.3  1015 photons cm-2 s-1.

2.3

Conjugation of ZnMCPPc (1) to NPs

ZnMCPPc (complex 1, 0.02 g, 2.80 × 10-2 mmol) was dissolved in 2 mL dry DMF and the carboxyl functional moiety was activated using 0.015 g (7.27 × 10-5 mol) of DCC. The solution was left stirring undisturbed for 24 h at ambient temperature. DMAP (0.0075 g, 6.14 × 10-5 mol) was then added, followed by addition of 0.05 g/mL solution of APTES functionalized ZnO/SiO2 NPs or SiO2 NPs and the solution was left stirring for 48 h to obtain the amide linkage between the MPc and the NPs to form 1-ZnO/SiO2 NPs and 1SiO2, respectively. The conjugates were precipitated out of solution using ethanol and successively washed with ethanol and methanol. The nanoconjugates were vacuum dried. 2.4. Photophysicochemical parameters The fluorescence (F) quantum yields of the ZnMCPPc and the nanoconjugates were assessed using comparative method reported in literature [22]. Unsubstituted ZnPc dissolved in DMSO was used as a standard for the MPc and the nanoconjugates in DMSO (F =0.2) [23].

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The triplet state quantum yields (T) of ZnMCPPc alone or when linked with SiO2 or ZnO/SiO2 NPs were determined using comparative methods reported in literature using unsubstituted ZnPc in DMSO as a standard (T = 0.65) [24]. The time resolved phosphorescence decay curve of singlet oxygen at 1270 nm was used to determine the singlet oxygen quantum yields for ZnMCPPc and it nanoconjugates with SiO2 or ZnO/SiO2 NPs using Equation 1 [21, 25]: I (t )  B

D [e  t /   e t /  ] T  D T

(1)

D

where, I(t) is the phosphorescence intensity of 1O2 at time t, D is the lifetime of 1O2 phosphorescence decay, T is the triplet state lifetime of the standard or sample and B is a coefficient involved in sensitizer concentration and 1O2 quantum yield. The singlet oxygen quantum yields (Δ) of complex 1 and it nanoconjugates with SiO2 or ZnO/SiO2 NPs were then determined using Equation 2:

Φ Δ  Φ ΔStd 

B B Std

(2) (2)

where ΦΔStd is the singlet oxygen quantum yield for the standard ZnPc ( ΦΔ = 0.67 in Std

DMSO [26]), B and BStd are the coefficients of the sample and standard, respectively.

2.5. Cell culture studies

2.5.1 In vitro dark viability studies

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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 l DPBS once, followed by administration of 100

l supplemented DMEM containing 14.3, 28.6 and 57.1 μg/mL of ZnMCPPc (1) alone, SiO2, ZnO/SiO2 NPs or the conjugates. The stock concentration was prepared 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 1.6 % (v/v) DMSO in supplemented DMEM which represents the highest percentage of DMSO used for preparing the highest concentration of the MPc alone and it nanoconjugates with SiO2 or ZnO/SiO2 NPs. Placebo 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 l DPBS once,

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supplemented DMEM with phenol red was added and the plates re-incubated 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 equation 3: (3) 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 Photodynamic therapy activity Photodynamic therapy activities of the drugs were assessed by incubation of attached cells seeded as earlier described in the previous section. Concentrations of ZnMCPPc 1, 1ZnO/SiO2 and ZnO/SiO2 of 14.3, 28.6 and 57.1 g/mL were administered in a 96-well plate containing 10,000 cells/well in a supplemented culture DMEM (100L). Plates were incubated at 37 oC in 5% CO2 in the dark for 24 h, and rinsed with 100 L PBS (phosphate buffer saline) once and the media was replaced with supplemented DMEM without phenol red. The cells were subsequently illuminated with light doses of 112 J/cm2 using the set-up described above. Light from a general electric quartz lamp was

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expanded to cover the plate resulting in a power density of 93 mW/cm2. The illumination time was fixed at 1200 seconds corresponding to 112 J/cm2. After illumination, the media was replaced with supplemented DMEM with phenol red. Cell survival was expressed as percentage of placebo cells (cells without drugs). Surviving cells were quantified after re-incubation with cultured DMEM with the use of WST-1 assay after 24 h.

2.5.3. Statistical analysis The analytical data obtained from the experiments were statistically analysed using Microsoft Excel worksheet 2010 (Microsoft®) and Graphpad Prism6®. Each experiment was repeated in its entirety at least three times each with triplicate replicates (n=3). Analysis of variance (ANOVA) for the in vitro dark viability and PDT data of the molecules against MCF-7 cells was evaluated at (14.3, 28.6 and 57.1) μg/mL. 3. Results and discussion

Scheme 1 illustrate the route for the covalent linkage of ZnMCPPc to SiO2 NPs and ZnO/SiO2 NPs. The linkage was formed via amide bond using the carboxyl functional moiety of the MPc (COOH) and the amine (NH2) functional moiety of the NPs.

3.1. Characterization of the MPc, NPs, and their conjugates. 3.1.1 UV/Vis absorption and emission spectra

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Fig. 1A shows the normalised absorption spectra of ZnMCPPc (1), 1-ZnO/SiO2, SiO2 NPs, ZnO/SiO2 NPs, and 1-SiO2. The ground state electronic absorption spectra of SiO2 NPs and ZnO/SiO2 NPs, showed significant absorption around the B band region of complex 1 and this was reflected in the spectra of the nanoconjugates of complex 1 with SiO2. There was with no pivotal change in the Q-band maxima of the MPc upon conjugation, Table 1. Fig. 1B shows the normalised absorption, emission, and excitation spectra of the 1ZnO/SiO2 (as an example) in DMSO. The ground state electronic absorption and excitation spectra of the molecules were found to be similar and mirror images of the emission spectra. The same applies to complex 1 and 1-SiO2. The size of the MPc is approximately 1 nm while the sizes of SiO2 NPs and ZnO/SiO2 NPs were determined by TEM below to be 20 nm and 13 nm, respectively. Thus it is unlikely for more than one NP to bind to a Pc. But it is possible for more than one Pcs to bind to a NP.

The mass of ZnMCPPc loaded to the NPs was estimated using UV-Vis

spectroscopic methods reported in literature [27,28]. The mass loading of ZnMCPPc was found to be 6 μg and 68 μg per mg of SiO2 NPs and ZnO/SiO2 NPs respectively. Thus the larger SiO2 NPs (20 nm) has a lower mass loading as compared to the smaller ZnO/SiO2 NPs (13 nm). Dye loading increases with surface area and pore volume [29] and the smaller NPs have large surface area as will be shown below using BET.

3.1.2. XRD studies

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The XRD diffractogram of the ZnMCPPc showed a broad peak between 2 = 11 and 31 showing amorphous state of the crystal lattice (Fig. 2c) which is typical of phthalocyanines [30]. The SiO2 NPs showed a single broad peak at 2 = 27 (Fig. 2e). The broadness is due to the amorphous nature of these nanoparticles as reported before [31]. A broad peak is also observed for 1-SiO2 (Fig. 2d) with a shift in the diffractogram peak to 2 = 25. ZnO/SiO2 NPs showed an XRD pattern typical of these NPs [32] (Fig. 2a) which was maintained in the conjugate, with an attenuation of the ZnMCPPc peak (Fig. 2b).

3.1.3. TEM

The morphologies of the nanoparticles and the nanoconjugates were assessed using transmission electron microscope (TEM).

SiO2 NPs and ZnO/ SiO2 NPs showed

aggregation (Fig. 3a and c, respectively) which increased but upon functionalization with the ZnMCPPc (Fig. 3b, using 1-SiO2 NP as an example). Aggregation occurs through π-π interaction of MPcs on adjacent NPs. The sizes of the NPs were found to be 20 nm for SiO2 NPs (Fig. 3c) and 13 nm for ZnO/SiO2 NPs (Fig. 3a). Since high aggregation was observed with the nanoconjugates (Fig. 3b), the sizes could not be estimated. 3.1.4. FT-IR spectra The functional moieties of the complex 1 and the nanoparticles as well as their conjugates were assessed using FT-IR. In the SiO2 NPs, three distinct bands were observed at 3274 cm-1 and 797 cm-1 corresponding to primary amine (NH2) from the capping ligand and

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1068 cm-1 corresponding to siloxane band (Si-O-Si) (Fig. 4a). The conjugates of complex 1 with SiO2 NPs also showed three distinct bands at 3282 cm-1 and 797 cm-1 corresponding to primary amine (NH2), 1072 cm-1 corresponding to siloxane band (Si-O-Si) and a partially split peak at 1640 cm-1 and 1527 cm-1 corresponding to primary and secondary amide vibrational band which was not observed in the MPc (Fig. 4c) and SiO2 NPs alone (Fig. 4b). The presence of NH2 vibration in 1-SiO2 confirms that not all of these groups (located on NPs) are used for binding to complex 1. The presence of the primary and secondary amide vibrational bands confirm the amide bond formation. The IR spectrum for 1-SiO2 is dominated by the Si-O-Si peak due to the smaller amount of MPc compared to the NPs. The same FT-IR behavior was observed for the nanoconjugates involving ZnO/SiO2 NPs and ZnMCPPc.

3.1.5. EDX Spectra The EDX of the APTES capped SiO2 NPs, Fig. 5A, showed a significant amount of carbon in addition to silicon, oxygen, and nitrogen: this is expected based on the composition of silica nanoparticle which has aminopropyl triethoxysilane as it capping ligand. The conjugates of the SiO2 NPs with ZnMCPPc (Fig. 5B) showed similar pattern but with the presence of zinc from the ZnMCPPc. 3.1.6. Surface areas and pore sizes Analysis The surface areas and pore sizes of the NPs (Fig 6) were obtained from the nitrogen adsorption/desorption isotherms using the Brunauer-Emmette-Teller (BET) and BarretJoyner-Halenda methods respectively. Prior to analysis, SiO2 and ZnO/SiO2 NPs were

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degassed for 24 h using Micrometrics ASAP® 2020 Surface Area and Porosity Analyzer operated at 77 K. The nitrogen adsorption–desorption isotherms of APTES capped SiO2 (Fig 6A) and ZnO/SiO2 NPs (Fig 6B) shows type IV adsorption isotherms. The surface areas of 23 m2/g (35 m2/g) and pore sizes of 306 Å (397 Å) for SiO2 (ZnO/SiO2 in brackets) NPs were obtained. The surface area and pore size of the ZnO/SiO2 NPs was found to be larger as compared to the SiO2 NPs. Thus the smaller ZnO/SiO2 NPs (13 nm) have a larger surface area than the larger SiO2 NPs (20 nm). 3.1.7. XPS Analysis The wide scan depicted all the expected elemental compositions of the NPs (Fig. 7A (a) using ZnO/SiO2 NPs as an example) were as follows: Zn 2P (1021 eV and 1045 eV), Zn 2p auger peak (499 eV), Zn 3s (139 eV), O 1s (531 eV), N 1s (399 eV), C 1s (285 eV and 290 eV), Si 2s (153 eV), Si 1s (102 eV). Complex 1 (Fig. 7A (b)) shows the following peaks: Zn 2P (1021 eV and 1044 eV), O 1s (532 eV), N 1s (399 eV), C 1s (285 eV). Thus, the peaks present in complex 1 are also present in the ZnO/SiO2 NPs. 1-ZnO/SiO2 showed peaks similar to those of ZnO/SiO2 NPs alone. Using 1-ZnO/SiO2 as an example, Table 2 shows an increase in C and N when compared to individual components, since both have C and N. There is a decrease in O for 1-ZnO/SiO2 compared to ZnO/SiO2 NPs but an increase compared to complex 1 alone. It is possible that the intensity of the peaks is affected by the orientations. The N 1s spectra of ZnO/SiO2 NPs (Fig. 7B (a)) exhibited three distinct deconvoluted sub peaks at 398.39 eV (NH2), 400.12 eV (C-N), 403.04 eV (C-C-N). ZnMCPPc (1, Fig. 7B(b)) depicted three sub peaks at 398.64 eV (-C=N), 400.33 eV (N), 401.84 eV (N-C).

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1-ZnO/SiO2 showed (Fig. 7B (c)) four distinct sub peaks at 395.79 eV (N-C), 398.06 eV (C=N), 399.98 eV (C-NH-C) and 401.65 eV (O=C-NH). The latter is an additional peak that resulted due to the linkage of the primary amine of APTES functionalized NP with activated carboxylic acid moiety of the MPc to form carbonamide (O=C-NH) bond linkage (Scheme 1) [33,34].

3.1.8. TGA Analysis The thermal stability of complex 1 and conjugates were assessed using thermogravimetric o

o

analyser at a temperature range of 30 to 900 C, heating rate of 10 C min-1, maintained under a steady flow of N2 gas. The percent weight loss was obtained from the successive decomposition obtained using the derivative plot (Fig 1S, supporting information). The total percent weight losses for conjugate with 1-SiO2 and 1-ZnO/SiO2 were found to be 1.6 % and 3.8 %, respectively. Thus, 1–ZnO/SiO2 had lower thermal stability compared to 1–SiO2. The total percent weight loss of SiO2 NPs alone was found to be 0.3 % while ZnO/SiO2 was found to be 0.1 %, suggesting 1.3 % of complex 1 in 1–SiO2 and 3.7 % in 1–ZnO/SiO2. These results confirm the UV-Vis results which showed the latter to have a larger mass loading of Pc in the NP. The observed higher total percent weight loss of 1ZnO/SiO2 NPs, could be due to more of complex 1 loaded to the ZnO/SiO2 NPs.

3.2. Photophysicochemical Parameters 3.2.1. Fluorescence (F) quantum yields and lifetimes (τf)

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The fluorescence quantum yields and average lifetimes of the ZnMCPPc (complex 1) alone and in conjugates are provided in Table 1. A typical fluorescence decay curve is depicted in Fig. 8 for 1-ZnO/SiO2 NPs as an example. A single fluorescence lifetime was obtained for complex 1 alone while we observed two fluorescence lifetimes (averages shown in Table 1) for the nanoconjugates, possibly due to different orientations of the phthalocyanines on the NPs. A slight increase was observed in the fluorescence quantum yield of complex 1 when linked to SiO2 NPs, but no change was observed for 1-ZnO/SiO2 NPs. The observed increase in the fluorescence quantum yield for 1-SiO2 NPs could also be due to the ability of the structure of the phthalocyanines to allow for close grafting of the MPc on the surface of the SiO2 NPs, thereby increasing the efficiency of the fluorescence process [35]. For 1-ZnO/SiO2 NPs, the heavy atom effect of Zn will result in intersystem crossing to the triplet state, hence no increase in fluorescence quantum yield was observed for 1-SiO2 NPs. The fluorescence lifetimes vary in a similar manner to the fluorescence quantum yields, Table 1. 3.2.2. Triplet (T), lifetimes (T), and singlet oxygen (∆) quantum yields Fig. 9 shows the triplet absorption decay curve of 1-ZnO/SiO2 NPs as an example. In addition to an increase in triplet quantum yield (only a slight increase for 1-SiO2 NPs) significant increase in the triplet lifetimes were obtained for all the nanoconjugates. It is pertinent to note that whenever T increases, triplet lifetimes are expected to decrease [36] but this is not the case in this study, since there is a general improvement in both T and T values for complex 1 in the presence of ZnO/SiO2 or SiO2 NPs The lengthening of T for the Pc in the presence of NPs, could be due to the protection

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afforded to the MPc by the NPs. For 1-ZnO/SiO2 NPs, the enhanced triplet state quantum yield is due to the heavy metal effect of Zn which promotes effective intersystem crossing, resulting in increased population of the triplet state. For 1-SiO2 NPs the insignificant increase in T is a result of the lack of a heavy atom. The higher mass loading for 1-ZnO/SiO2 NPs (68 μg) compared to 1-SiO2 (6 μg), could also result in larger

T values, Table 1.

Singlet oxygen is formed through an energy transfer process between excited triplet state (T1) of MPc (3MPc*) and ground state molecular oxygen (3O2). Figure 10 shows the oxygen phosphorescence curve using 1-ZnO/SiO2 NPs use as a typical example. Slight improvement was observed in the ∆ of the ZnMCPPc in the presence of NPs. The small increase in ∆ values does not match the large increase in T for 1-ZnO/SiO2 NPs. In addition to encouraging intersystem crossing to the triplet state, ZnO NPs may also form charged species in the presence of excited Pcs. Upon excitation with visible light, an electron hole pair is formed in the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of the phthalocyanine. The electron in the LUMO is then injected into the conduction band of ZnO [37]. This could reduce the population of the excited states. Hence, there is a mismatch between the T and ∆ values.

3.3

Cell studies

3.3.1. In vitro dark viability studies

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All the cytotoxicity studies were carried out in dark and are referred to as in vitro dark viability (Fig. 11A). In vitro dark viability is undesirable for photosensitizers aimed for use in

PDT. The NPs, ZnMCPPc and the conjugates were tested against the MCF-7 cells. The percent cell viabilities of the DMSO (used for making stock solution of complex 1, the conjugates and the NPs) at 1.6 % (w/v), was found to be similar with what was obtained in the supplemented DMEM alone control. Complex 1 showed percent cell viabilities of less than 50 % at 28.6 μg/mL and 57.1 μg/mL (hence more dark toxicity), while the conjugates gave values larger than 50% (hence less dark toxicity) at the same concentrations. This confirms reduced dark toxicity of complex 1 in the presence of nanoparticles. At the highest concentration of 57.1 μg/mL, both NPs alone were toxic, though less toxic than complex 1. We observed statistically significance difference between complex 1 alone, NPs alone (ZnO/SiO2 or SiO2 NPs) and their nanoconjugates. There was statistically significance difference in the percent viability of the cells as the drug concentration increases. Upon analysis of the triplicate replicate data of each concentration, we observed no statistically significant difference as the p-value was found to be greater than 0.05.

3.3.2. Photodynamic therapy activity

PDT activity of complex 1, ZnO/SiO2 NPs and conjugate were studied (as examples) at fixed light dosimetry and varied concentrations, Fig. 11B. Illumination for PDT studies was obtained using a quartz lamp which has been reported to be effective light source in the photo-irradiation of tumor cells [38]. The PDT activity of complex 1 was found to be

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higher compared to the ZnO/SiO2 NPs and their conjugate. The observed high PDT activity of complex 1 could be due to its high dark toxicity, discussed above. PDT activities of 1-

ZnO/SiO2 and complex 1 were relatively better than that of ZnO/SiO2 NPs alone. The observed PDT activity of the ZnO/SiO2 NPs could be due to the fact that ZnO NPs have capacity to exhibit anticancer activity as a result of reactive oxygen species produced upon excitation at a suitable wavelength [39].

At *P < 0.05, is considered to be

statistically significant difference. We observed statistically significant difference as the concentration increases. There was a significant difference between the dark cytotoxicity and PDT data of complex 1 and the nanoconjugate. 4. Conclusion In this work, we report on the synthesis and covalent linkage of ZnMCPPc with SiO2 or ZnO/SiO2 nanoparticles. The nanoconjugates were characterized using XRD, TEM, UV/vis absorption and emission spectrometer, FT-IR spectrometer, XPS and EDX. In addition, the photophysicochemical behaviour, in vitro dark viability and photodynamic therapy activity of the Pc and its conjugates were studied. Improvements were observed in the fluorescence quantum yields (ɸF), triplet and singlet state quantum yield as well as the in

vitro dark viability of the conjugates compared to the MPc alone. The photodynamic therapy activity alone was higher than for the conjugates due to its dark toxicity. Acknowledgements This work was supported by the Department of Science and Technology (DST) Innovation and National Research Foundation (NRF), South Africa through DST/NRF

21   

South African Research Chairs Initiative for Professor of Medicinal Chemistry and Nanotechnology

(UID

62620)

as

well

as

Rhodes

University.

22   

References

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Figure 1: (A) Ground state electronic absorption spectra of (a) ZnMCPPc (1), (b) 1ZnO/SiO2, (c) SiO2 NPs, (d) ZnO/SiO2 NPs and (e) 1-SiO2; (B) Absorption (a), Excitation (b), Emission (c) spectra of 1-ZnO/SiO2 in DMSO at excitation wavelength of 608 nm.  

 

27   

a

Lin (Counts)

b

c

d

e

10

20

30

40

2 Theta (Degree)

50

60  

Figure 2: XRD diffractograms (a) ZnO/SiO2 NPs (b) 1-ZnO/SiO2, (c) ZnMCPPc 1, (d) 1SiO2 and (e) SiO2 NPs.                  

28    (a) 

  (b) 

(c)

Figure 3: TEM micrographs (a) SiO2 NPs, (b) 1-SiO2, (c) ZnO/SiO2 NPs and (d) 1ZnO/SiO2   

29   

3274

797

1068

(a) Transmittance (%)

1640 1527 3282

1072

(b)

3190

2919

1087

1699 1552

(c) 4000

797

3500

3000

2500

2000

1500

1226

1000

-1 Wavenumber (cm )

Figure 4: FT-IR spectra (a) SiO2 NPs, (b) 1-SiO2 and (c) ZnMCPPc (1)      

500

 

30   

A

 

B

 

Figure 5: EDX spectra (A) SiO2 NPs and (B) 1-SiO2.

31   

Figure 6: Nitrogen adsorption-desorption isotherms (A) SiO2 NPs (B) ZnO/SiO2 NPs

32    (a) 30000

Zn2p Zn2p

N1s

C1s

Zn3s

Si2s

10000 Si2p

10000

O1s

Zn2p

20000

20000

N1s

30000

Intensity (a.u)

O1s

40000 Zn2p Auger

Intensity (a.u)

50000

(b)

C1s

Zn2p

A

0 200

400

600

800

1000

1200

0

200

400

Binding energy (eV)

600

   

Intensity (a.u)

   

   

20000

N1s

Si2s

10000

Si1s Zn3s

   

30000

C1s

 

Zn2p Auger O1s

40000

Zn2p

(c)

 

Zn2p

 

 

  0

200

800

Binding energy (eV)

400

600 800 Binding energy (eV)

1000

1200

1000

1200

 

33       

B

(a)

 

Intensity (a.u.)

 

Intensity (a.u.)

                                                                  

N1s

(b)

N1s

398.39 eV 403.04 eV 400.12 eV

398.64 eV 400.33 eV 401.84 eV

    396

 

399

402

393

405

Binding energy (eV)

396

399

402

405

408

Binding energy (eV)

 

Intensity (a.u.)

(c)

N1s

398.06 eV 395.79 eV

392

399.98 eV 401.65 eV

396

400

Binding energy (eV)

404

Figure 7: XPS spectra (A) wide scans for (a) ZnO/SiO2 NPs, (b) ZnMCPPc (1) (c) 1ZnO/SiO2 and (B) High resolution spectra N 1s for (a) ZnO/SiO2 NPs, (b) ZnMCPPc (1) and (c) 1-ZnO/SiO2.

34   

Amplitude (Counts)

10000 8000 6000 4000

Residual

2000 0 4 2 0 -2 -4

0

5

10

15

20

Time (ns)

25

30  

Figure 8: Photoluminescence decay curve of 1-ZnO/SiO2 in DMSO at excitation wavelength of 684 nm    

35   

Change in Absorbance

0.06 0.05 0.04 0.03 0.02 0.01 0.00 0

500

1000

Time (s)

1500

2000

Figure 9: Excited state triplet absorption curve of 1-ZnO/SiO2 in deaerated DMSO at excitation wavelength of 490 nm.

Change in Absorbance

0.12

0.10

0.08

0.06

0.04

0.02 0  

50

100

Time (s)

150

200

Figure 10: Singlet phosphorescence decay curve of 1-ZnO/SiO2 in deaerated DMSO.

36   

Fig. 11: Histogram depicting (A) in vitro dark viability of (a) ZnMCPPc (1), (b) 1– ZnO/SiO2, (c) 1–SiO2, (d) ZnO/SiO2 NPs, and (e) SiO2 NPs and (B) PDT activity (illumination at 112 J/cm2) of (a) ZnMCPPc (1), (b) ZnO/SiO2, and (c) 1–ZnO/SiO2.

37   

Scheme 1: Covalent linkage of ZnMCPPc to SiO2 NPs or ZnO/SiO2 NPs

Table 1: Photophysicochemical data of ZnMCPPc and conjugates in DMSO Complexes

λabs (nm)

λems (nm)

F

τf(ns)

T

τt(μs)

∆

(±0.02)

(±40)

(±0.01)

38   

a

ZnMCPPc (1)

674

680

0.16

3.27

0.41

161

0.15

1-SiO2NPs

675

684

0.23

3.63a

0.43

277

0.17

1-ZnO/SiO2NPs

675

686

0.17

3.11a

0.64

281

0.25

average lifetimes

Table 2: XPS apparent surface composition of ZnMCPPc, and it nanoconjugates Complex

Atomic Concentration (%) C 1s

O 1s

N 1s

Si 2P

Zn 2P

ZnO/SiO2

20.02

42.61

5.69

31.62

0.06

ZnMCPPc (1)

12.01

10.93

4.91

N/A

0.36

1-ZnO/SiO2

50.96

31.47

7.31

5.03

5.22

N/A = Not applicable