Photophysics of tetracarboxy-zinc phthalocyanine photosensitizers

Zinc-tetracarboxy-phthalocyanine (ZnPc(COOH)4) was synthesized by a melting method and basic hydrolysis. A ZnPc(COOH)4/Fe3O4/Ch composite was prepared by immobilization of ZnPc(COOH)4 onto Fe3O4/chitosan nanoparticles by a simple immersion method. The photophysical properties were studied using UV-vis spectrophotometry, fluorescence spectroscopy and time-correlated single photon counting (TCSPC) in different aqueous solutions. The UV-vis spectra of the ZnPc(COOH)4/Fe3O4/Ch composite displays absorption by the aromatic rings, with a Q band exhibited at λmax = 702 nm. Moreover, the ZnPc(COOH)4/Fe3O4/Ch composite exhibits long triplet-state lifetimes of 1.6 μs and 12.3 μs, crucial for application as a photosensitizer. A triplet quantum yield of 0.56 for the ZnPc(COOH)4/Fe3O4/Ch composite in DMSO/H2O was achieved. FTIR showed that the conjugation of ZnPc(COOH)4 with Fe3O4/chitosan nanoparticles was achieved by electrostatic interaction.


Introduction
Metallophthalocyanine (MPc) derivatives are popular photodynamic therapy (PDT) photosensitizers (PSs). Research on a novel PS requires extensive human effort and a high cost investment over decades before clinical application. Nevertheless, some MPc derivatives, such as: aluminium phthalocyanine (Photosens®, Russia), used against skin, breast and lung malignancies, and cancers of the gastrointestinal tract; 1 silicon Pc (Pc 4, USA), for the sterilization of blood components against human colon, breast and ovarian cancers, and gliomas; 2 and a liposomal zinc phthalocyanine formulation, using a controlled organic solvent dilution against squamous cell carcinomas of the upper aerodigestive tract, 3 have undergone clinical trials.
Current efforts are being made in the development of new photosensitizers (PSs) with improved solubility in body uids and injectable solvents, photostability, enhanced permeability and retention effect, elimination and cumulative systemic toxicity. [4][5][6][7][8][9] In the eld of organic photosensitizers, metallophthalocyanines (MPcs) play an important role due to their excellent photo-and electro-chemical stability and exclusive light-harvesting capability in the red/NIR spectral regions. [10][11][12][13] The main disadvantages of MPcs in PDT are the lack of solubility and selectivity; therefore, the combination of magnetic iron oxide nanoparticles with a photosensitizer is a new and promising approach in PDT. Fe 3 O 4 nanoparticles have been successfully applied in tumor therapy by inducing hyperthermia and oxidative stress that lead to tumor cell damage. [14][15][16] For application in PDT, magnetic nanoparticles (NPs) are usually coated with polymers, bound to the particle through organic linkers. 17 Functionalization of Fe 3 O 4 nanoparticles may lead to enhancement of their biocompatibility, colloidal stability, and an increase in the number of groups, through which the required antitumor effect can be obtained.
The major goal of this paper is to create a new photosensitizer with adequate solubility, especially in body uids and injectable solvents, with greater tumor selectivity, enhanced hydrophilicity, and strong absorption in the NIR spectral region. Therefore, conjugation of an MPc derivative to a magnetic NP functionalized with a polymer is the rst part of our research aimed at delivering PSs to tumor cells. The magnetic iron oxide nanoparticles will be used as the carrier of the photosensitizer because of: their ability to carry and deliver therapeutic photosensitizers into deep-seated tumours; the enhanced solubility of the hydrophobic PS with an appropriate size to accumulate in the tumour tissues via enhanced permeability and retention effect; and the ability to attack cancer cells selectively without harming other healthy cells. The Fe 3 O 4 NPs will be functionalized with chitosan, which is a biodegradable, biocompatible polysaccharide and, in comparison with many other polymers, has many free -OH and -NH 2 groups that can serve as anchors for conjugation of therapeutics and targeting ligands.
Considering the above mentioned information, we focused our research on attaching functionalized ZnPc with carboxylic groups (-COOH) to an Fe 3 O 4 /chitosan system hoping to get a synergistic effect in the photodynamic parameters of the resulting composite.

Equipment
The UV-vis spectra of the solutions were measured using a UVvis spectrophotometer (Lambda 25, PerkinElmer, Inc., Shelton, CT, USA) from 200 nm to 1200 nm in 10 mm quartz cuvettes. The steady-state uorescence spectroscopy was performed using a spectrometer (LS-55, PerkinElmer, Inc., Shelton, CT, USA) equipped with double-grating excitation and emission monochromators. Time-correlated single photon counting (TCSPC) was used to determine the uorescence lifetime. The time-resolved uorescence spectra were recorded on a spectrometer (FLS980, Edinburgh Instruments, Livingston EH54 7DQ, Oxford, UK). All the measurements were made at room temperature (295 ± 1 K). A Bruker D8 ADVANCE X-ray diffractometer (using Cu K a radiation with l = 1.5406Å) was used for structural investigation of the magnetic nanoparticles. A Bruker FTIR spectrometer was used to provide information about the chemical composition.

Synthesis
The synthetic pathway of ZnPc(COOH) 4 A mixture consisting of 4.35 g (0.022 mol) of trimellitic anhydride, 2.52 g of Zn(CH 3 COOH) 2 $2H 2 O, 0.3 g of ((NH 4 ) 6 MO 7 O 24 )$ 4H 2 O, 0.5 g of Na 2 SO 4 , 13.51 g (0.225 mol) of urea and 5 ml of 1bromonaphthalene was heated at 200-205°C for 8 h with continuous stirring. Aer 8 hours, the reaction mixture was cooled to room temperature and treated with methanol. The obtained suspension was ltered. The solid reaction product was washed on the lter with methanol, chloroform and, nally, with acetone. Aer drying, the product was crumbled and then reuxed for one hour in 5% hydrochloric acid (HCl) solution. Aer drying, the same procedure was carried out with 5% sodium hydroxide (NaOH) solution for one hour at 90°C. Finally, the solution was acidied with HCl until the pH was equal to 2, and the precipitated nal product was ltered and dried in the open air. 0.68 g of ZnPc(COOH) 4 was obtained with a yield of 70% (Fig. 1).

Preparation of chitosan-functionalized magnetic nanoparticles
Chitosan and Fe 3 O 4 were mixed in an appropriate proportion to form the chitosan-magnetic nanoparticles composite with amine groups by the reverse-phase suspension cross-linking method. 18 Aqueous acetic acid solution was used as a solvent for the chitosan polymer and H 2 O 2 was used as the cross-linker. In this specic procedure, a chitosan solution was prepared using a mixture of 2% acetic acid and 10% H 2 O 2 solutions. Then 0.2 g Fe 3 O 4 was added and stirred with strong ultrasonic agitation at room temperature for 4 h. At the end of this period, some of the chitosan-Fe 3 O 4 nanocomposite particles were collected from the reaction mixture by using a permanent magnet. The product was washed with ethanol and dried in vacuum at 60°C for 5 hours and used for XRD analysis (Fig. 2).
Chitosan is able to interact with negatively charged molecules, 19 such as the hydroxyl (Fe-OH) groups on the surface of magnetite nanoparticles. The presence of -OH groups on the surface of the Fe 3 O 4 nanoparticles was conrmed by the strong broad band with a maximum at 3431 cm −1 in the IR spectrum ( Fig. 4), corresponding to n(O-H) oscillations. We suppose that ionic interactions occur between the negatively charged CH 3 COO − species and the positively charged (NH 3+ ) groups of the chitosan molecules dissolved in the aqueous acetic acid solution.

to chitosan, Fe 3 O 4 and Fe 3 O 4 /Ch nanoparticles
Acetic acid is a weak acid and is a very common solvent for chitosan. A sample of 0.3 g of chitosan was dissolved in 50 ml of 2% concentrated acetic acid. Then 0.5 ml of 10% hydrogen peroxide was added to the solution for the destruction of intermacromolecular hydrogen bonds and interchain hydrogen bonds to make water-soluble chitosan. The appropriate ratio of chitosan to acetic acid in the chitosan-acetic acid solution was  1 : 0.5, and then ZnPc(COOH) 4 was dissolved in a 1 : 1 DMSO/ H 2 O solution. Aer that, both solutions were mixed, heated at 40°C and stirred continuously for 40 min.
In a separate experiment, ZnPc(COOH) 4 solution was mixed with a dispersion medium containing chitosan-functionalized magnetic nanoparticles at room temperature and stirred for 2 h using a mechanical stirrer.
Experiments where ZnPc(COOH) 4 was dissolved in 1 : 1 DMSO/H 2 O solution and simply mixed with Fe 3 O 4 were also performed.

Structural analysis of the Fe 3 O 4 and Fe 3 O 4 /chitosan magnetic nanoparticles
The X-ray diffraction patterns of the Fe 3 O 4 and Fe 3 O 4 /chitosan nanoparticles, along with the standard pattern of Fe 3 O 4 (JCPDS #75-0033), are shown in Fig. 3 and details of the peaks are given in Table 1. The similar XRD patterns reveal that Fe 3 O 4 does not undergo any phase changes following functionalization with chitosan, a situation also conrmed by other reports. 20,21 XRD diffraction analysis revealed a broad nature of the diffraction maxima, indicating that Fe 3 O 4 has small crystallite sizes. The crystallite sizes were evaluated using the Debye-Scherrer formula: where l is the wavelength of the X-rays (1.5406Å), b is the FWHM (full width at half maximum), q is the diffraction angle, k = 0.94 and D is the crystallite size. The metal oxide nanoparticles have a mean crystallite size of 13.95 nm. During the coating process with chitosan, the crystallite size slightly increases, as the size of the individual crystallite is related to the thickness of the chitosan layer. The mean crystallite size of the nanoparticles with chitosan increases up to 14.80 nm.

FTIR analysis
The FTIR spectra of chitosan, the      (Fig. 4). The result is consistent with similar investigations. 22,23 The chemical interaction of ZnPc(COOH) 4 with the Fe 3 O 4 /chitosan system is conrmed by the shi of the signal from 1702 cm −1 (n(C]O)) of the protonated COOH groups in the IR spectrum of ZnPc(COOH) 4 , associated with splitting, to 1660 cm −1 (n sym (COO)), and 1436 and 1406 cm −1 (n asym (COOO)) that correspond to deprotonated carboxylic groups. This can be explained by the dissociation of carboxylic groups and formation of electrostatic interactions between NH 3 + and COO − fragments (Fig. 16a).

UV-vis and uorescence analysis
Usually, MPcs give rise to electronic spectra with two strong absorption bands, one around 300 nm, called the "B" or Soret band, due to electronic transitions from the deeper p-HOMO to n*-LUMO energy levels, while the other at 600-650 nm, called the "Q" band, due to electronic transitions from the p-HOMO to p*-LUMO energy levels. 24 The UV-vis spectra of ZnPc(COOH) 4 and ZnPc(COOH) 4 /Ch in DMSO/H 2 O are presented in Fig. 6. The absorption spectra of the synthesized materials display absorption peaks in the visible region at around 700 nm. In the case of ZnPc(COOH) 4 and ZnPc(COOH) 4 Fig. 8, 2% acid acetic and 10% hydrogen peroxide were used. The Q band extends into the 580-800 nm region and exhibited two peaks at l max = 645 nm and 702 nm in the case of the Fe 3 O 4 nanoparticles linked to chitosan (Fig. 8), almost the same values as when Fe 3 O 4 is not bound to chitosan (Fig. 7). Both the ZnPc(COOH) 4 /Fe 3 O 4 /chitosan and ZnPc(COOH) 4 /Fe 3 O 4 spectra (Fig. 9) show similar specic absorption peaks of the phthalocyanine aromatic ring. The chitosan had no obvious absorption peak in the visible region, but leads to an increased intensity of the 702 nm peak and a narrower Q band. The comparison in Fig. 9 allows us to suppose that the Q absorption band could be assigned to the p-   p* transition on the ZnPc macrocycle. Introducing the peripheral -COOH substituent onto the macrocycle of ZnPc led to a signicant bathochromic shi of the absorption spectra due to an increased destabilization of the HOMO electron state versus the LUMO state.
The low energy peak is due to the monomer, while the high energy peak is caused by the aggregation. The aggregation species persisted more when the Fe 3 O 4 nanoparticles were not bound to chitosan.
The uorescence emission spectrum of ZnPc(COOH) 4 Fig. 10. The uorescence spectrum aer excitation at 615 nm shows two emission bands situated at 695 nm and 765 nm. The uorescence spectrum of the ZnPc(COOH) 4 /chitosan system (Fig. 11) aer excitation at 638 nm also shows two bands, as in Fig. 10, but they are both shied 10 nm into the near-infrared region. The uorescence      spectrum of ZnPc(COOH) 4 immobilized on the Fe 3 O 4 magnetic nanoparticles shows broad and structured uorescence at 702 nm, 764 nm, 789 nm and 826 nm, and shows an increase in intensity at 850 nm, when excited at 645 nm (Fig. 12). The limits of the measurement equipment did not allow us to record uorescence above 850 nm. The spectrum of ZnPc(COOH) 4 immobilized on the Fe 3 O 4 /chitosan magnetic nanoparticles shown in Fig. 13 displayed less structured uorescence. Only two broad bands situated at 713 nm and 784 nm shied to the nearinfrared region are revealed. The resultant red-shi was associated with the electrostatic interaction between ZnPc(COOH) 4 and the chitosan-functionalized Fe 3 O 4 nanoparticles.

in DMSO/H 2 O is shown in
The uorescence lifetimes of ZnPc(COOH) 4 and ZnPc(COOH) 4 /chitosan in DMSO/H 2 O solution are presented in Fig. 14.
The uorescence decays of ZnPc(COOH) 4 and ZnPc(COOH) 4 Table 2. So, we suppose that the surface interaction between the amino groups of the chitosan/Fe 3 O 4 and the carboxylic groups of ZnPc(COOH) 4 most probably forms an electrostatic interaction. In addition to the electrostatic interaction between charged surfaces of ZnPc(COOH) 4 and chitosan/Fe 3 O 4 , coordination bonds between the Zn 2+ ions of phthalocyanine and the oxygen atoms of chitosan/Fe 3 O 4 can be formed. 26,27 Also, hydrogen bonds between the nitrogen atoms of phthalocyanine and the hydrogen atoms of chitosan/Fe 3 O 4 are also possible, as shown in the scheme presented in Fig. 16.
So, signicant efforts have been made to develop the ZnPc(COOH) 4 /Fe 3 O 4 /chitosan composite that has strong absorption of long-wavelength light and a triplet quantum yield of 0.56 that can be promising for PDT. But further studies will continue to improve the triplet-state lifetime and the triplet quantum yield, and elucidate the physiochemical processes in this composite. Moreover, in vitro and in vivo studies are required to elucidate the PDT effects.
(2) Fe 3 O 4 /chitosan magnetic nanoparticles with a mean crystallite size of the nanoparticles up 14.80 nm using the suspension cross-linking technique.
(3) ZnPc(COOH) 4 immobilized on chitosan-functionalized Fe 3 O 4 nanoparticles through an immersion method with the aid of DMSO/H 2 O 2 /Ac.ac solution, exhibits higher triplet lifetimes of 1.6 ms and 12.3 ms.
The values of the triplet quantum yield (0.56) and the tripletstate lifetimes of ZnPc(COOH) 4 /Fe 3 O 4 /Ch make this composite a promising candidate for PDT.