Size-Dependent Photodynamic Activity of Gold Nanoparticles Conjugate of Water Soluble Purpurin-18-N-Methyl-D-Glucamine

Gold nanoparticles (GNPs) conjugates of water soluble ionic photosensitizer (PS), purpurin-18-N-methyl-D-glucamine (Pu-18-NMGA), were synthesized using various molar ratios between HAuCl4 and Pu-18-NMGA without adding any particular reducing agents and surfactants. The PS-GNPs conjugates showed long wavelength absorption of range 702–762 nm, and their different shapes and diameters depend on the molar ratios used in the synthesis. In vitro anticancer efficacy of the PS-GNPs conjugates was investigated by MTT assay against A549 cells, resulting in higher photodynamic activity than that of the free Pu-18-NMGA. Among the PS-GNPs conjugates, the GNPs conjugate from the molar ratio of 1 : 2 (Au(III): Pu-18-NMGA) exhibits the highest photodynamic activity corresponding to bigger size (~60 nm) of the GNPs conjugate which could efficiently transport the PS into the cells than that of smaller size of the GNPs conjugate.


Introduction
Photodynamic therapy (PDT) is a promising noninvasive cancer treatment by using a combination of photosensitizer (PS), light, and oxygen [1][2][3]. For excellent photodynamic activity, PS should be penetrated into the tumor cells suf-�ciently [4]. Most of PSs are hydrophobic and thus locate preferentially in the lipid bilayers of organelle membranes in cancer cells. However, the hydrophobic nature of PSs makes them insoluble under physiological conditions and hinders to reach the accumulation in the tumor sites [5]. erefore, for both hydrophobic and hydrophilic (amphiphilic) environments of PSs, introduction of water soluble PS with suitable carrier is a one potential method [6][7][8]. On the other hand, highly water soluble (hydrophilic) PSs allow poor cellular uptake based on a short pharmacological half-life which may have limit to penetrate through the tissue and cell membranes [9][10][11].
Nanoparticles (NPs) [12][13][14] are promising carrier system of PSs that could be protected from being uptaken by the reticuloendothelial system and extended the circulation time of NPs in the blood, and, �nally, preferentially accumulated in tumor sites through the so-called "enhanced permeability and retention (EPR)" effect [15][16][17]. Among the NPs, gold nanoparticles (GNPs) are highly efficient PDT drug delivery platform with good advantages based on their chemical inertness and minimum toxicity that has potential applications in biomedicine such as photothermal therapy (PTT) [18][19][20][21] of cancer, gene and drug delivery, biological imaging, and biosensing [22][23][24][25][26]. In addition, GNPs have large surface-to-volume ratios and easy tuning of the NPs size, resulting in penetration into tumor cells and intracellular localization at endosomes/lysosomes of the cells, and �nally targeting at mitochondrial of cancer cells induces apoptosis to destroy the cancer cells [27][28][29][30][31]. It is noted that the size of the GNPs plays a big role in their uptake at the cellular level leading to different PDT activity. However, to the best of our knowledge, there are few reports for relationship between GNPs and its size effect on photodynamic activity [28].
Previously, we developed new synthesis of PS-GNPs conjugate using water soluble ionic purpurin-18-N-methyl-Dglucamine (Pu-18-NMGA, PS1, Figure 1) and this conjugate showed better in vitro anticancer efficacy than that of free PS1 against A549 lung cancer cells [32].   In this paper, we have synthesized various sizes of PS-GNPs conjugates using a simple single-step synthesis from different molar ratios of HAuCl 4 /PS1 without adding any particular reducing agents and surfactants, and showed size effect allowed different photodynamic activity results of the conjugates as an important factor for PDT. We evaluated in vitro anticancer efficacy of the PS-GNPs conjugates against A549 cells using MTT assay.

Materials.
All reagents were purchased from Aldrich and used without further puri�cation. All aqueous solutions were made using triply distilled water. All reactions were monitored by thin-layer chromatography (TLC) using Merck 60 silica gel F254 precoated (0.2 mm thickness) glass-backed sheets. Silica gel 60A (230-400 mesh, Merck) was used for column chromatography. e 1 H NMR spectra were obtained using a Varian spectrometer (500 MHz) at Biohealth Products Research Center (BPRC) at Inje University. e chemical shis ( ) are given in parts per million (ppm) relative to tetramethylsilane (TMS, 0 ppm). High-resolution fast atom bombardment mass (HRFABMS) spectra were obtained with a Jeol JMS700 high-resolution mass spectrometer at the Daegu center of KBSI, Kyungpook National University, Korea.
e PS1 and PS-GNPs conjugates 2a-2e were characterized by a combination analysis of 1 H-NMR and UV-vis spectroscopies, transmission electron microscopy (TEM), and infrared (IR) spectroscopy. UV-vis absorption spectra were recorded using a SCINCO S-3100 UV-vis spectrophotometer using 1 cm quartz cuvette. TEM images were performed on a JEOL, JEM 2011. A typical sample for TEM was prepared by drying of a drop of the solution at room temperature on a carbon-coated copper grid. IR spectra were measured on a Varian-640 FT-IR spectrometer.

Preparation of Gold Nanoparticles
Conjugates. e commonly used synthetic way of GNPs is a reduction method of Au(III) salt (usually from HAuCl 4 ) using sodium citrate in water [35]. In this method, sodium citrate has a double role as a weak reducing agent as well as a capping agent that stabilizes the NPs. e particle size is controlled by a ratio between citrate and AuCl 4 − ions. Higher concentration of citrate afforded smaller particle size [35].
However, in this work, we used hydrophilic PS1 and Au(III) without any additional reducing agents and surfactants [32]. e hydroxyl groups of NMGA in PS1 have important roles as a reducing agent as well as a stabilizer through the electrically charged functional groups (i.e., carboxylate and amine groups) in forming the PS-GNPs conjugates [9]. PS1 was obtained from the carboxyl group of purpurin-18 (Pu-18) and the amine group of NMGA by simple and effective method ( Figure 1). Pu-18 was synthesized from a conversion of methyl pheophorbide a (MPa) by air oxidation in npropanol with KOH [34]. MPa was obtained from Spirulina paci�ca algae by the procedure reported by Smith et al. [33]. PS-GNPs conjugates were prepared from the reaction of different molar ratios between Au(III) and PS1 (2a, 1 : 2; 2b, 1 : 4; 2c, 1 : 6; 2d, 1 : 8; 2e, 1 : 10) in water to afford different particle sizes (Figure 2). e structures of the water soluble PS1 and the PS-GNPs conjugates were con�rmed by 1 H-NMR spectroscopy, mass spectrometry, and UV-vis spectroscopy (Figure 3). e water soluble PS1 acts not only as a reducing agent, but also as a capping agent in the reduction of HAuCl 4 for synthesis of PS-GNPs conjugates. e formation of PS-GNPs conjugates is stable in the aqueous solution due to the adsorption of oxidized PS1 on the surface of the GNPs through a strong coordinate-covalent bond between carboxylate on PS1 and gold metal. So the binding strength of PS1 on the GNPs surface is enough to allow accumulation of PS1 in culture medium or in vivo [8,24,36,37]. erefore, a large amount of water soluble PS was generally used in order to get stable GNPs. Hence, we have used �ve different concentration ratios between Au(III) and PS1 in order to �nd suitable concentration ratio that gives optimal size of the PS-GNPs conjugates for best photodynamic activity result. Figure 3 shows the UV-vis absorption spectra of the PS-GNPs conjugates 2a-2e in water. In each conjugate, typical plasmon resonance band of the GNPs was appeared at 506-525 nm, respectively [24]. In 2a-2c, the longest wavelength absorption ( max ) is longer (719-762 nm) than that of PS1 (702 nm), while max of 2d-2e is the same with that of PS1 (Table 1). Among the conjugates, 2b showed the longest wavelength absorption at 762 nm. In 2a-2b, the Soret band at about 330-450 nm was broadened, indicating the formation of stacking structure of the chlorin ring on the gold surfaces [25]. Figure 4 shows the typical TEM images of the PS-GNPs conjugates 2a-2e prepared by using different concentration ratios between Au(III) and PS1. e images of the conjugates are different from each other in size and shape corresponding to the different molar ratios used in the preparation of the conjugates ( Table 2). In 2a, when molar ratio was 1 : 2 for Au(III) : PS1, the GNPs are mainly peanut-shaped nanocrystals in water. And some spheres have diameters around 60 nm and are well dispersed with no aggregation between the GNPs in water. In 2b, when molar ratio was 1 : 4, the GNPs are nanospheres have diameters around 5-11 nm. And some GNPs are closely placed each other and have a chainlike appearance with branching. In 2c, when molar ratio was 1 : 6, the GNPs are nanospheres have diameters around 5-10 nm. However, some GNPs are aggregated together to form many bundles of GNPs, resulting in bigger diameters around 27-44 nm. In 2d, when molar ratio was 1 : 8, the GNPs are mainly aggregated bundles and shape was not spheres with length around 50-90 nm and width around 25-50 nm. And yield of the GNPs was low and some aggregated GNPs have size around 200 nm. In 2e, when molar ratio was 1 : 10, the GNPs are mainly aggregated and yield of the GNPs was very low, and some aggregated bundles of GNPs were around 50-70 nm size. When relatively lower molar ratio of PS1 (2 or 4) made stable GNPs conjugate, however, higher molar ratio allowed unstable GNPs conjugate and remains continuous aggregation [35]. Consequently, the molar ratio between Au(III) and PS1 is an important driving force to control GNPs size, shape, and aggregation degree of the GNPs in aqueous media.

UV-Vis Spectroscopic Investigation and Size Analysis by TEM Images.
Based on the UV-vis spectra and TEM images, there is a good relationship between absorption intensity and particle size. Higher absorption intensity of the conjugate corresponds to bigger particle size. In 2a, absorption intensity at over than 450 nm ranges is the highest among all the conjugates, which corresponds to the biggest size (about 60 nm) in the conjugates.
Compound 2b shows the longest wavelength absorption at 762 nm which is included in NIR wavelength region (PTT therapeutic window, 750-1100 nm), so there is a potential for using PTT. We are considering that the GNPs conjugate T 2: Summary of TEM images of the PS-GNPs conjugates 2a-2e.

Compound
Shape Diameter (nm) Dispersion 2a Sphere, peanut ∼60 GNPs are well dispersed and showed no aggregation. 2b Sphere, chain-like appearance with branching 5-11 GNPs are well dispersed and showed no aggregation. 2c Sphere 5-11 Some aggregated GNPs have diameter sizes of 27-44 nm. 2d Not sphere -GNPs have a lot of aggregation. 2e Not sphere -Very few aggregated GNPs have sizes of 50-70 nm. for a combination (synergy effect) therapy of PDT and PTT [38,39]. In all the compounds, upon photo irradiation, the cell viability was decreased corresponding to the increased incubation time aer PDT as well as increased concentration ( Figure 5), for example, at 48 h incubation and 3.2 g/mL, 80% at 3 h, 75% at 24 h, and 69% for PS1, and 66% at 3 h, 50% at 24 h, and 47% for 2a (Figure 6), respectively. Dark cytotoxicity of PS1 and conjugates 2a-2e is shown in Figure 7. At highest concentration (25 g/mL) with 48 h incubation time, all compounds showed high dark cytotoxicity (cell viability 32-61%).

Photodynamic Activity and
PS1 showed slightly higher photocytotoxicity (IC 50 , 10.5 g/mL = 14 M at 24 h incubation time) than that of the purpurin-18-choline derivative (IC 50 , 15 M at 24 h incubation time) that has been previously reported by us [25]. Conjugates 2a and 2b showed higher photocytotoxicity than that of PS1. At high concentration (e.g., at 25 g/mL), 2a showed higher dark cytotoxicity (69% at 3 h, 49% at 24 h, and 48% at 48 h incubation time, Figure 7) as compared to PS1 (62% at 3 h, 60% at 24 h, and 56% at 48 h incubation time), which might be attributed to large amount of PS1 molecules on the GNPs surface in 2a. However, conjugates 2c-2e showed lower photocytotoxicity than that of PS1. is result demonstrates that photodynamic activity signi�cantly depends on size and aggregation degree of the GNPs. For example, in 2a and 2b there is no aggregation between each other and 2a has about 60 nm size, while in 2c-2e there are some aggregated bundles of the GNPs with small size. Chithrani et al. [29] studied a relationship between particles size (14-100 nm) and cellular uptake of the GNPs in HeLa cells, in which the maximum uptake was occurred at a size of 50 nm. Jiang et al. [28] have reported that cellular uptake strongly depends on the size of the GNPs, in which the GNPs having 2-100 nm size range were coated with Herceptin and were evaluated for cell internalization against breast cancer cell lines by the ErbB2 receptor. e most efficient cellular uptake was observed with particles range of 20-50 nm. Apoptosis was also enhanced by the GNPs having 40-50 nm size [28]. From the high dark cytotoxicity at high concentration, we con�rmed that the PS-GNPs conjugate 2a and 2b showed better photodynamic activity at low concentration (3.2 g/mL) having low dark cytotoxicity (Figure 7).
In addition, 2a and 2b have higher absorbance at irradiated wavelength range, which allowed good photodynamic activity results. In 2c-2e, absorption intensity was lower than that of PS1, resulting in lower photocytotoxicity as compared to PS1. Table 3 shows the IC 50 values for PS1 and its PS-GNPs conjugates 2a-2e. At 48 h incubation time, 2a and 2b showed better IC 50 value, 4.32 and 6.38 g/mL, respectively, as compared to PS1 (8.72 g/mL). erefore, as we pointed out above, photodynamic in vitro activity of synthesized PS-GNPs conjugates (2a and 2b) is much higher than that of the free PS1. is result indicates that optimal size and welldispersed nanoparticles are important for photodynamic effect in aqueous media. Especially, bigger size (∼60 nm) of nanoparticles 2a could be useful to transport more chlorine molecules into the cancer cells by endocytosis [28,29,40].

Conclusions
In summary, a simple single-step synthesis of PS-GNPs conjugates from different molar ratios of Au(III)/water soluble ionic PS1 (purpurin-18-N-methyl-D-glucamine) has been studied without adding any particular reducing agents and surfactants. In vitro anticancer efficacy of the PS-GNPs conjugates against A549 lung cancer cell lines was evaluated. We revealed that PDT in vitro activity of synthesized PS-GNPs conjugates was higher as compared to free PS1 because of good transport of the PS into the cells by using size effect. Conjugate 2a based on molar ratio between HAuCl 4 and PS was 1 : 2 that exhibits best PDT efficiency than other conjugates having different molar ratios. is result could be useful for synthesis of new PS and PS-GNPs conjugates having different size as well as for developing good relationship between PDT activity and size effect of GNPs in aqueous media.

Con�ict of �nterests
We do not have any con�ict of interests to Sigma-Aldrich and BioSpec LED.