Amphiphilic Gemini Pyridinium-mediated incorporation of Zn(II)meso-tetrakis(4-carboxyphenyl)porphyrin into water-soluble gold nanoparticles for photodynamic therapy

a. Departament de Farmacologia, Toxicologia i Química Terapèutica, Universitat de Barcelona, Avda. Joan XXIII 27-31, 08028 Barcelona, Spain. E-mail: mlperez@ub.edu b. Institut de Nanociència i Nanotecnologia UB (IN2UB), Universitat de Barcelona, Avda. Joan XXIII 27-31, 08028 Barcelona, Spain. c. Departament de Biologia Cel·lular, Fisiologia i Immunologia. Universitat Autònoma de Barcelona, Spain. d. School of Chemistry, University of East Anglia, Norwich Research Park, Norwich, Norfolk, NR4 7TJ, UK.


Introduction
PDT is an approach of cancer treatment based on the use of specific drugs, called photosensitizers, which can induce cell death after irradiation, due to the formation of reactive oxygen species [1][2][3]. PDT has several advantages in the treatment of cancer, since it is less invasive, minimizes the secondary effects and allows more localized areas of the body to be treated. The major drawbacks of PDT are the non-specific distribution of the photosensitizer into the body, and the water-solubility of the photosensitizer, which can be low and thus requires a formulation to improve the administration. In particular, porphyrins are one of the most studied photosensitizers in the last years, to be applied in PDT [2,[4][5][6][7] but also in sensors as hosts for molecular recognition [8,9]. One of the main characteristics of the porphyrin's structure is the possibility to incorporate a metal into its core, in particular bivalent cations such as Zn 2+ , Mg 2+ , Co 2+ or Fe 2+ . These metalloporphyrins are intensely investigated for their ability to form Reactive Oxygen Species (ROS) and thereby their interest as potent photosensitizers for use in PDT [6,10].
Furthermore, metalloporphyrins (especially Zn-containing porphyrin) have shown to be more efficient as photosensitizer in PDT than the metal-free porphyrin [11]. However, they frequently present low water solubility, which results in low distribution and consequently low efficiency. One way to overcome this drawback is by conjugating the molecule with a system that is used as vehicle.
In the last years, nanostructured systems have raised huge interest in the biomedical field because of their biocompatibility and the potential application as delivery agents for therapy [1,12,13]. One example is the use of such vehicles to target cells in cancer therapy [14]. One of the most studied systems in drug delivery is GNP [15,16], and the use of nanoparticles incorporating photosensitizers to improve their specificity in PDT has been reported [5,6,17,18].
For the synthesis of organic and water soluble GNP, different types of ligands have been studied as stabilizers, like water-soluble polymers [19], amino acid based amphiphiles [20] or peptides [21]. The use of pyridinium salts as stabilizer agents of GNP has also been reported [22]. On the other hand, gemini surfactants display excellent properties in the preparation and stabilization of monodisperse GNP (organic and water soluble GNP) [13,23,24]. However, to the best of our knowledge, the synthesis and stabilization of GNP coated with pyridinium-based gemini amphiphiles and the incorporation of metalloporphyrins into such systems has not yet been reported. In this context, this study describes the methodology for the synthesis of pyridinium-coated GNP, based on a monophasic method, where the gemini-pyridinium amphiphile 1·2Br acts as a promoter, a stabilizer agent as well as a host for the subsequent incorporation of the anionic photosensitizer Na-ZnTCPP into the Na-ZnTCPP, 1·GNP ( Figure 1). The new watersoluble GNP were characterized using UV-visible Absorption Spectroscopy, Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS) and Fluorescence Spectroscopy. Furthermore, the production of singlet oxygen after irradiation was measured for the porphyrin Na-ZnTCPP, 1·GNP (a control which does not contain photosensitizer) and Na-ZnTCPP-1·GNP, and the cytotoxicity as well as the phototoxicity of the 1·GNP and Na-ZnTCPP-1·GNP were also analysed in two different Human Breast cell lines, one of tumoral origin (SKBR-3) and one of normal epithelium origin (MCF-10A).

Synthesis of compounds 1·2Br and Na-ZnTCPP
The synthesis and characterization of bis-pyridinium salt 1·2Br follows a previously reported procedure for imidazolium analogues [23], ; in the case of the porphyrin Na-ZnTCPP they are explained in detail in the Supplementary Material (Section 1).

Synthesis of water-soluble gold nanoparticles 1·GNP and Na-ZnTCPP-1·GNP
A solution of α-thio-ω-carboxy-polyethylene glycol (1.3 mg, 0.0024 mmol) in water (1 mL) and a solution of bis-pyridinium salt 1·2Br (5 mg, 0.0052 mmol) in EtOH (2 mL) were added to a stirred solution of HAuCl4·3H2O (6.7 mg, 0.017 mmol) in water (1 mL). NaBH4 (3.3 mg, 0.087 mmol) in water (1mL) was added dropwise to the mixture at room temperature. The stirring continued for 24 h in the dark at room temperature. After this time the solvent was removed in a rotary evaporator, and the red residue was purified by multiple cycles of washing with EtOH (3 x 1 mL) and water (3 x 1 mL) and centrifugation (14000 rpm, 17 min at 15 °C). The new water-soluble GNP were named 1·GNP. For the incorporation of the porphyrin, a solution of Na-ZnTCPP (2 mg, 0.0021 mmol) in water (2 mL) was added to a stirred solution of 10 ml of 1·GNP (3 x 10 -3 µM) in water. The stirring continued for 24 h in the dark at room temperature. The solvent was removed in a rotary evaporator, followed by multiple cycles of washing with water (5 x 1 mL) and centrifugation (14000 rpm, 17 min at 15 °C), in order to eliminate the unbound porphyrin Na-ZnTCPP. These gold nanoparticles, named Na-ZnTCPP-1·GNP) were obtained at the concentration of 2.9 x 10 -3 μM.
The GNP were characterized using the following techniques: UV-visible absorption spectra were recorded on a UV-1800 Shimadzu UV Spectrophotometer, using quartz cuvettes with a 1 cm path length. Fluorescence excitation and emission spectra were recorded on a Hitachi F-4500 Fluorescence Spectrometer, using quartz cuvettes with a 1 cm path length. TEM was performed at the Centres Científics i Tecnològics de la Universitat de Barcelona (CCiT-UB). The samples were prepared by drop casting a 2 x 10 -3 µM aqueous solution of 1·GNP or Na-ZnTCPP-1·GNP over a carbon-coated copper grid, and were observed using a Tecnai SPIRIT Microscope (FEI Co.) at 120 kV. The images were captured by a Megaview III camera and digitalized with the iTEM program.
The size of the GNP core was measured with ImageJ. DLS analysis was carried out using a Zetasizer Nano ZS series (Malvern Instruments) from Departament de Farmàcia, Tecnologia Farmacèutica i Fisicoquímica at the Universitat de Barcelona.

Singlet Oxygen production of Na-ZnTCPP and Na-ZnTCPP-1·GNP
In a quartz cuvette, 3 μL of a solution of ABMA (0.2 mg, 0.51 mM) in MeOH (1 mL) was added to either Na-ZnTCPP (4.34 µL, 3 µM) or Na-ZnTCPP-1·GNP (485 µL, 3 μM of incorporated porphyrin) in water. The final volume (1.5 mL) in the cuvettes was completed with water and the solutions were thoroughly stirred. A light source in the range between 400 and 500 nm was used to irradiate the mixture during 4 h, using a laser power of 0.16 mw/cm 2 . The laser was located 3 cm away from each cuvette. Fluorescence emission spectra were recorded every hour, in the range of 390-600 nm, and singlet oxygen production was determined by the decrease of the fluorescence intensity of ABMA at 431 nm.

Cell culture
All experiments were performed with two human mammary epithelial cell lines, one with For each experiment, cells were seeded in 24-well dishes, with or without coverslips, at a density of 50,000 cells/well. Treatments were performed 24 h after seeding.

Photodynamic treatments
Cells were incubated in serum-free medium with different concentrations of Na-ZnTCPP To evaluate the toxicity of Na-ZnTCPP and Na-ZnTCPP-1·GNP in absence of irradiation, cells were also incubated in the presence of both compounds as described above and were kept in dark conditions (Dark toxicity, DT).

In vitro cytotoxicity assay
Cell viability was evaluated 24 h after treatments by the 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich). The absorbance was recorded at 540 nm using a Victor 3 Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA). For each treatment, viability was calculated as the absorbance of treated cells normalized to control conditions. Three independent experiments were performed in each case.
All graphics and statistical analyses were performed using GraphPad Prism version 6.01 for Windows, (GraphPad Software, La Jolla, California, USA). Results were analysed through a two-way ANOVA with a minimal significance level set at P ≤ 0.05.

Synthesis and characterization of 1·2Br and Na-ZnTCPP
The bis-pyridinium salt 1·2Br was selected to be used as stabilizer agent of GNP and also acts as host in the subsequent incorporation of the photosensitizer Na-ZnTCPP.
According to previous reports by our group [12,13,23], GNP stabilized with gemini imidazolium based amphiphiles showed good ability to incorporate anionic molecules.
The gemini pyridinium analogue 1·2Br is expected to expand the range of non-covalent interaction with anionic species. Consequently, the anionic porphyrin Na-ZnTCPP was selected in this work to be incorporated on the synthesized pyridinium-based GNP. Na-ZnTCPP was synthesized according to modification of previously reported methods [25,26]. The metalation step was monitored by UV-visible Absorption Spectroscopy: the four Q bands from the free base porphyrin are replaced by two Q bands of the corresponding Zn(II) derivative, indicating the metalation process is complete in 24 h. Na-ZnTCPP was obtained with a 94% yield (synthesis and characterization are explained in detail in Supplementary Material Section 1, Scheme S1 and Figures S1-S4).

Synthesis of water-soluble gold nanoparticles 1·GNP and Na-ZnTCPP-1·GNP
In order to obtain nanoparticles with a high potential use in biomedical applications, the synthesized GNP should be water soluble. For this reason, we used a mixture of the gemini pyridinium-based amphiphilic ligand 1·2Br and the thiolated polyethyleneglycol derivative α-thio-ω-carboxy-polyethylene glycol for the formation of all the new GNP.
Briefly, the GNP were synthesized by preparing small amounts of α-thio-ω-carboxypolyethylene glycol in EtOH, to favour the solubility in water of synthesized GNP, and 1·2Br as stabilizer agent and anionic binder; then adding an aqueous solution of HAuCl4 and then the reducing agent NaBH4. The obtained GNP were purified by sequential washing and centrifugation, and were named 1·GNP. These new water-soluble 1·GNP were later used as a model colloid for the biological control experiments.
In this work, we selected the anionic porphyrin Na-ZnTCPP as photosensitizer to be incorporate into the gemini-pyridinium coated GNP. This porphyrin has already shown high potential for use in PDT [27,28] due to its water solubility, and its negative charges allows its noncovalent incorporation into cationic GNP, thus providing an alternative delivery strategy with the potential to avoid photosensitizer leakage and processing issues, which has been reported for different drugs [29,30]. The anionic porphyrin Na-ZnTCPP was incorporated on 1·GNP, and the Na-ZnTCPP containing GNP were named Na-ZnTCPP-1·GNP. The schematic representation of 1·GNP and Na-ZnTCPP-1·GNP can be seen in Figure 1.

Characterization of Na-ZnTCPP, 1·GNP and Na-ZnTCPP-1·GNP
The formation of 1·GNP and the incorporation of the porphyrin Na-ZnTCPP into the Na-ZnTCPP-1·GNP were confirmed by UV-visible Absorption Spectroscopy (Figure 2 a)). The UV-visible absorption spectra were recorded in water. The free porphyrin Na-ZnTCPP showed the typical Soret band at 423 nm and two Q bands at 557 and 593 nm.
In the case of 1·GNP, the typical Surface Plasmon Resonance (SPR) band of the GNP was observed near 520 nm, while the Na-ZnTCPP-1·GNP show a peak at 530 nm, and also a peak at ca. 430 nm that corresponds to the porphyrin Na-ZnTCPP Soret band. In addition, the two typical Zinc porphyrin Q bands can be identified in the Na-ZnTCPP- 1·GNP and Na-ZnTCPP-1·GNP were characterized using TEM to study their morphology and their size distribution for 1·GNP and Na-ZnTCPP-1·GNP as seen in  Fluorescence spectroscopy was also used to identify the incorporation of the porphyrin into the synthesized Na-ZnTCPP-1·GNP. Fluorescence emission spectra were recorded in water for the free porphyrin Na-ZnTCPP and Na-ZnTCPP-1·GNP (see Supplementary Material Section 2 Figure S6), and both spectra exhibit two peaks at ca.
λ 606 nm and λ 660 nm following excitation at λ 421 nm, which is consistent with reports for Zn-porphyrin derivatives [31]. These results confirm the incorporation of Na-ZnTCPP into the Na-ZnTCPP-1·GNP, and also demonstrate that the fluorescence emission of the photosensitizer is not affected significantly when the porphyrin is linked to the GNP.

Quantification of Na-ZnTCPP incorporated into Na-ZnTCPP-1·GNP
The quantification of the amount of porphyrin Na-ZnTCPP per Na-ZnTCPP-1·GNP was performed using UV-vis absorption spectroscopy and taking into account the diameter size of Na-ZnTCPP-1·GNP, as previously determined by TEM. The wavelength selected to determine the amount of Na-ZNTCPP incorporated into Na-ZnTCPP-1·GNP was that corresponding to the Soret band (430 nm) because it was the most intense peak corresponding to the porphyrin. First, a calibration curve of Na-ZnTCPP was obtained using a range of concentrations between 0.5 µM and 10 µM (see Supplementary Material Section 3 Figure S7), in order to calculate its extinction coefficient (ε), that was found to be (ε423) = 355600 M -1 cm -1 . The Na-ZnTCPP-1·GNP UV-Visible absorption spectrum shows quite broad absorption bands and in order to normalize the Soret band absorbance value, a subtraction between the Soret band peak and the absorbance of the porphyrin into Na-ZnTCPP-1·GNP sloping background at 470 nm was calculated (see Supplementary Material Section 3 Figure S8). Accordingly, we  Table S1).

Singlet oxygen production of Na-ZnTCPP and Na-ZnTCPP-1·GNP
Singlet oxygen ( 1 O2) production was examined using water soluble ABMA as a probe.
Upon reaction with 1 O2, ABMA forms a non-fluorescent 9,10-endoperoxide product [32], was 30% and 49%, respectively, indicating that the porphyrin incorporated into Na-ZnTCPP-1·GNP is more efficient to produce the 1 O2 than the free porphyrin in solution.
To further compare the ability to produce singlet oxygen by Na-ZnTCPP both free in solution and incorporated in the Na-ZnTCPP-1·GNP, the maximum rate of ABMA photobleaching was normalized with the concentration of the photosensitizer Na-ZnTCPP  Figure S11). These results demonstrate that the porphyrin Na-ZnTCPP resulted more effective when immobilized on GNP rather than free in solution, with an increased singlet oxygen production, a feature previously reported for similar systems [2,33]. This fact is even more remarkable considering that the photobleaching of the porphyrin incorporated into GNP was measured in aqueous solution, where oxygen is much less soluble and usually leads to a less significant effect for this type of measurement because of the shorter lifetime of singlet oxygen in water [34].

Photodynamic effect of Na-ZnTCPP on cell cultures
Cell viability 24 h after treatments with Na-ZnTCPP was evaluated by MTT assay (see Na-ZnTCPP, showed a significant decrease in cell survival, but without significant differences between both cell lines, in accordance to preliminary data [38]. Actin microfilaments and nuclear morphology were observed by Alexa-Fluor®594conjugated Phalloidin and H-33258 staining. In absence of irradiation, both cell lines treated either with 1 or 3 µM Na-ZnTCPP did not present actin microfilaments or nuclear alterations (Figure 3 a) and c)). In contrast, after 10 min of irradiation, and at both concentrations of Na-ZnTCPP, MCF-10A cells showed a high disorganization of actin microfilaments and no stress fibres were observed, although nuclei remained unaltered (Figure 3 b)). SKBR-3 cells after irradiation showed a similar disorganization of the actin cytoskeleton but some apoptotic or necrotic nuclei were observed (Figure 3 d)).

Photodynamic effect of Na-ZnTCPP-1·GNP on cell cultures
Prior to the phototoxicity study of Na-ZnTCPP-1·GNP, the uptake and cytotoxicity of As observed for 1·GNP, MCF-10A showed a higher uptake of Na-ZnTCPP-1·GNP than SKBR-3 cells (Figure 4 a) and c)). It has been reported that MCF-10A cells can internalize both positively and negatively charged particles, whereas in SKBR-3 cells the uptake of negative charged particles is low [39,40]. The differences in cell uptake can be explained because Na-ZnTCPP-1·GNP are negatively charged. After irradiation, most of MCF-10A cells treated with 1µM Na-ZnTCPP-1·GNP remained unaltered, but some detached and contracted cells were observed (Figure 4 b)). On the contrary, most of the SKBR-3 cells subjected to the same treatments were floating in the medium and showed blebs in their plasma membrane (Figure 4 d)). Nuclear staining with H-33258 confirms these results: MCF-10A cells treated 1µM Na-ZnTCPP-1·GNP showed most of the nuclei unaltered, but with some apoptotic or necrotic nuclei (Figure 4 e)). In contrast, the same cells treated with 3µM Na-ZnTCPP-1·GNP showed a predominant necrotic morphology (Figure 4 f)

Conclusion
In this work, we successfully prepared new water-soluble 1·GNP based on bispyridinium amphiphiles 1·2Br following a monophasic method using as stabilizer agents α-thio-ω-carboxy-polyethylene glycol, to make the nanoparticles water soluble, and the pyridinium salt 1·2Br, which also acted as host to incorporate Na-ZnTCPP in the Na-ZnTCPP-1·GNP. The obtained porphyrin-loaded GNP are spherical and monodisperse, and the incorporation of the photosensitizer did not cause aggregation, thus suggesting they can be used as essentially single particle delivery system. The incorporation of the Na-ZnTCPP into the Na-ZnTCPP-1·GNP notably increased the capacity of the photosensitizer to generate singlet oxygen, which may be due to an enhancement effect of the GNP gold core on the porphyrin activity. SKBR-3 tumoral cells showed more sensitivity to Na-ZnTCPP-1·GNP, in dark conditions or after irradiation, than MCF-10A non-tumoral cells.
These findings suggest that the synthesized Na-ZnTCPP-1·GNP are a promising nanosystem for PDT. Future work includes the incorporation of antibodies through immobilization using the α-thio-ω-carboxy-polyethylene glycol present on the Na-ZnTCPP-1·GNP, to actively target cancer cells.