Porphyrin Derivative Conjugated with Gold Nanoparticles for Dual

Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Porphyrin Derivative Conjugated with Gold Nanoparticles for DualModality Photodynamic and Photothermal Therapies In Vitro Jinfeng Zeng, Wendi Yang, Dongjian Shi,* Xiaojie Li, Hongji Zhang, and Mingqing Chen Key Laboratory of Synthetic and Biological Colloids, Ministry of Education, School of Chemical and Material Engineering, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China S Supporting Information *

ABSTRACT: Gold nanoparticles (Au NPs) have been confirmed to show excellent photothermal conversion property for tumor theranostic applications. To improve the antitumor efficacy, a novel nanoplatform system composed of porphyrin derivative and Au NPs was fabricated to study the dual-modality photodynamic and photothermal therapy with laser irradiation. Modified chitosan was coated on the Au NPs surface via ligand exchange between thiol groups and Au. The chitosan-coated Au NPs (QCS-SH/Au NPs) were further conjugated with meso-tetrakis(4sulphonatophenyl)porphyrin (TPPS) via electrostatic interaction to obtain the porphyrin-conjugated Au hybrid nanoparticles (TPPS/QCSSH/Au NPs). Size, morphology, and properties of the prepared nanoparticles were confirmed by Zeta potential, nanoparticle size analyzer, transmission electron microscopy (TEM), and UV−vis spectroscopy. Moreover, both photothermal therapy (PTT) and photodynamic therapy (PDT) were investigated. Compared with alone Au NPs or TPPS, the hybrid TPPS/QCS-SH/Au NPs with lower cytotoxicity showed durable elevated temperature to around 56 °C and large amount of singlet oxygen (1O2) produced from TPPS. Thus, the hybrid nanoparticles showed a more significant synergistic therapy effect of hyperthermia from PTT as well as 1O2 from PDT, which has potential applications in the tumor therapy fields. KEYWORDS: photothermal therapy, photodynamic therapy, gold nanoparticles, porphyrin derivative

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increased to above 40 °C by the photothermal conversion effect. Correspondingly, the temperature of the tumor site where light absorbing agents were immobilized could increase up to 48−53 °C, high enough to cause irreversible cell damage by loosening of cell membranes, denaturation of proteins, and eventually leads to destruction of the diseased tissue.9 However, there is a big problem that Au NPs are easily tended to aggregate, which would reduce their photothermal conversion properties. Coating functional hydrophilic polymers on the surface of Au NPs is an efficient way to prevent from aggregation.10 The functional polymers are reported to be polyethylene glycol (PEG),11 hyaluronic acid,12 chitosan,13 and so on. These polymeric ligands have various advantages in the cancer treatment, including tuning of solubility and improving the long-term stability of Au NPs, tuning surface density of the particle shell, imparting nonimmunogenicity and reducing biotoxicity.14 Gomez11 and his co-workers reported that the poly(sodium styrenesulfonate) (PSS) and polyethylene glycol (PEG) coated Au NRs showed a long period of stability even after freeze-drying. Li et al. also prepared PEG-polyamidoamine dendrimers modified Au NPs or Au nanorods for photothermal

INTRODUCTION Photothermal therapy (PTT), which is produced by strongly absorbing visible or near-infrared (NIR) light and converting light energy into hyperthermia using photothermal agents, has been widely studied because of its unique advantages including remote controllability, low systemic toxicity, and few side effects.1,2 As is well-known, photothermal agents for PTT mainly include organic compounds, carbon nanomaterials, and noble metal nanoparticles.2 Indocyanine green, one kind of organic compound, is limited because its low photothermal conversion and severe light bleaching. Carbon nanomaterials, such as graphene, particularly has emerged as a promising candidate for PTT because of its unique electrical and optical properties.3,4 However, because there is only a single layer of carbon atoms, the absorptivity of graphene is only about 0.023 in the visible and NIR regions.5 Gold nanoparticles (Au NPs) as typical noble metal nanoparticles are well-known to have plasmon resonance property, allowing them to effectively absorb NIR light and efficiently convert into thermal energy.6 Thus, Au NPs had the strong absorption ability and efficient heat conversion property.7 As well as the low toxicity, stable photothermal conversion property, and well-defined surface chemistry of Au NPs, Au NPs are reported to be immobilized in the tumor site for the photothermal therapy (PTT) with laser irradiation or NIR light.8 The local temperature could be © XXXX American Chemical Society

Received: November 16, 2017 Accepted: February 13, 2018 Published: February 13, 2018 A

DOI: 10.1021/acsbiomaterials.7b00886 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering Scheme 1. Illustration for the Preparation of TPPS/QCS-SH/Au NPs and Its Anticancer Property by PDT/PTT

therapy.15 However, Au NPs cannot yield singlet oxygen (1O2), that causes irreversible damage to the tumor cells and even death. Since cancer cell is one kind of hypoxic tissue, the PTT therapy effect would be significantly improved, if the nanoparticles could generate singlet oxygen and heat simultaneously. Photosensitizer (PS) can absorb energy from laser irradiation and transfer photon energy to surrounding oxygen molecules, producing reactive oxygen species (ROS), such as singlet oxygen (1O2). The generated 1O2 causes irreversible damage to the tumor cells and even death,16 which realize photodynamic therapy (PDT). Unlike conventional therapy methods that might cause systemic toxicity by chemotherapy drugs and side damage to neighboring normal tissues by ionizing light during radiation therapy, PDT has clear advantages of noninvasive and low side effect.17 Porphyrin and its derivatives have been frequently used as one kind of typical PS agent, because of their high extinction coefficient in the red-light region and high singlet oxygen quantum yield.18,19 However, this small molecule porphyrin is subject to relatively low PDT, due to low stability and emission quantum yields, and poor solubility in aqueous media resulting in aggregation.20 Importantly, the selectivity of these PSs for tumor tissue over healthy tissue is poor and often results in significant off-target tissue damage. Alternatively, PS agents protected by nanoparticle-based carriers can be transported to the tumor site as much as possible for guarantee the therapy efficacy, as reported.16,21,22 Some reports prepared the porphyrin-Au complex nanoparticles, which showed high solubility of the two components and even improved their photoproperties.23 Lokesh24 and Zheng25 et al. reported that protection of Au NPs with porphyrin for the development of the artificial photosynthetic and catalytic materials. Most importantly, development of the complex that generate both 1O2 and heat upon light irradiation can enable PDT/PTT combination treatment continuously and simultaneously by combination of Au NPs and porphyrin,26 realizing a synergistic effect of PDT and PTT. However, their PDT/PTT combination treatment had scarcely any investigations.27,28 Therefore, herein, a novel nanosystem composed of porphyrin derivatives and Au NPs was fabricated for the

synergistic effect of PTT and PDT with laser irradiation to improve the antitumor efficacy. Quaternized chitosan-sulfydryl (QCS-SH) was first prepared to coat on the surface of Au NPs for keeping their stability. Then, PS agent, meso-tetrakis(4sulphonatophenyl)porphyrin (TPPS) was conjugated onto the surface of Au NPs via electrostatic interaction to obtain TPPS/ QCS-SH/Au NPs nanoparticles. To the best of our knowledge, there has been little research on combined PDT/PTT therapy using an anionic porphyrin derivative PS and positive charged Au NPs by the layer-by-layer method. Structure, size, and optical stability of the hybrid nanoparticles were characterized by Zeta potential and nanoparticle size analyzer, transmission electron microscopy (TEM), and ultravisible (UV−vis) spectroscopy. The anticancer PDT/PTT effects of TPPS/QCS-SH/ Au NPs were investigated in detail by examining the in vitro 1 O2 and hyperthermia generations under laser irradiation (see Scheme 1). Moreover, the hybrid nanoparticles were applied to study the further cell culture, including cytotoxicity, PTT, PDT, and PTT/PDT combined therapy.

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EXPERIMENTAL SECTION

Materials. CS with an average molecular weight (Mn) of 5000− 10000 Da was purchased from Haidebei Biomedical Company (Jinan, China) and used without further purification. 2,3-Epoxypropyltrimethylammonium chloride (GTMAC), thioglycolic acid, and sodium citrate were purchased from J&K Chemical. 1-Ethyl-3-(3(dimethylamino)propyl) carbodiimide hydrochloride (EDAC) was purchased from Aladdin. Chloroauric Acid (HAuCl4) was purchased from Alfa Aesar. Meso-tetrakis(4-sulphonatophenyl) porphyrin (TPPS) was purchased from TCI. Tetramethyl azo salt (MTT) was purchased from Amersco. Human hepatocellular carcinoma cell (HepG2) was obtained from Jindou Biomedical Company (Shanghai, China). All the materials were used as received. Characterization. Fourier transform infrared spectrum (FTIR) was recorded on Nicolet iS50 FTIR spectroscopy (Thermo Fisher Scientific, USA). Proton nuclear magnetic resonance (1H NMR, Bruker MSL500, Fällanden, Switzerland) was used to determine chemical structure of the synthesized polymers with D2O/CD3COOD (v/v = 98/2) as the solvent. Thermal gravimetric analysis (TGA) was measured by a TGA 1100SF under a nitrogen atmosphere at the temperature from 100 to 800 °C with a heating rate of 10 °C/min. B


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ACS Biomaterials Science & Engineering Scheme 2. Schematic Representation for the Synthesis of QCS and QCS-SH

UV−vis absorption spectrum was recorded on a spectrophotometer (UV-1100, Beijingruili, China). Fluorescence spectrum was measured on an Edinburgh instruments fluorescence spectrophotometer FS5. Morphology and structure of the obtained nanoparticles were observed by a transmission electron microscope (TEM, JEOL JEM2100, Japan). Size distribution and zeta potential of the nanoparticles were determined by Zeta potential and nanoparticle size analyzer (Zeta PALS, USA). X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, Thermo Fisher Scientific, USA) analysis was carried out to verify the conjugation of Au NPs nanoparticles to QCS-SH. Photothermal properties of the Au NPs, QCS-SH/Au NPs, TPPS, and TPPS/QCS-SH/Au NPs (2 mL) were evaluated by irradiation of a laser (635 nm, 0.85 W/cm2) for 6 min, respectively. The temperature change of the nanoparticle solutions was monitored by thermometer submerged in the solution for each minute and repeated for three times. Synthesis of Quaternized Chitosan (QCS). Quaternized chitosan (QCS) was synthesized according to the following steps. First, 2 g chitosan was dissolved in 200 mL acetic acid (2.0%, v/v). The mixture was adjusted the pH value to above 9 by NaOH (10 wt %) for precipitating chitosan completely, and further incubated at room temperature with vigorous stirring for 6 h. Second, the mixture was centrifuged at 5,000 r/min for 5 min and redispersed in deionized water at least for three times, until to neutral. The precipitant (∼1.87 g) was dispersed into 120 mL isopropanol adequately under a moderate stirring at 60 °C. Two hours later, after the system temperature raised up to 80 °C, a certain amount of 2,3epoxypropyltrimethylammonium chloride (GTMAC) (CS:GTMAC = 1:2, molar ratio) was added into the mixture at three intervals. The mixtures were further kept at 80 °C under sustained stirring for 9 h. Finally, QCS was precipitated from acetone, alternately washed by ethyl alcohol and acetone for three times, and subsequently dried in vacuum over 80 °C for 4 h. Synthesis of Sulfydryl Modified Quaternized Chitosan (QCSSH). Sulfydryl modified quaternized chitosan (QCS-SH) was synthesized by derivatization of its primary amino groups with thioglycolic acid as described previously.29,30 Briefly, 300 mg of QCS was dissolved in acetic acid (0.05%, v/v). Afterward, thioglycolic acid was activated by 100 mM 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDAC) for half an hour and subsequently added into the above chitosan solution at pH 5. The reaction mixture was kept at room temperature under permanent stirring for 3 h. To remove the excess of EDCA and thioglycolic acid, the reacted mixture was dialyzed against 5 mM HCl for 3 days in the dark, and subsequently dialyzed against the same solution but containing 1% NaCl for another 2 days to reduce the ionic

interactions. Samples were then dialyzed completely for 2 days against 1 mM HCl to adjust the pH to 4, and followed by lyophilization. Preparation of Gold Nanoparticles (Au NPs). Au NPs with approximate 25 nm diameter were synthesized according to a previously reported method.31 HAuCl4 (10 mM, 1.2 mL) was added into 50 mL of ultrapure water, and kept boiling. Then, 0.5 mL of freshly prepared citrate sodium solution (10 mg/mL) was added into the HAuCl4 solution, and the mixture was refluxed for half an hour as a color change from dark blue to red. After being cooled to room temperature, Au NPs solution was obtained and stored at 4 °C for further use, and the particle concentration was estimated to be 1.0 nM.32 Preparation of QCS-SH/Au NPs. To decorate the Au NPs with a QCS-SH shell, 10 mL Au NPs stock solution (∼1.0 nM) was added dropwise into QCS-SH solution (4 mg/mL, 10 mL) under continuously stirring for 24 h at room temperature. The obtained QCS-SH/Au NPs were centrifuged (10,000 rpm, 5 min, −10 °C) and washed with ultrapure water for three times to remove excess of QCSSH. The purified precipitant was redispersed in ultrapure water, and stored at 4 °C for further characterization. Preparation of TPPS/QCS-SH/Au NPs. TPPS (1 mL) was added into 9 mL QCS-SH/Au NPs solution and remained for 1 h to form a charged complex. Excessive TPPS was removed by centrifugation (10,000 rpm, 5 min, −10 °C) and washed with ultrapure water for three times. The final purified complex was dispersed in ultrapure water for further characterization. In order to quantitatively calculate the loading amount of TPPS on the surface of Au NPs, standard TPPS solutions with the concentrations at 1, 2, 3, 4, 5, and 6 μM were prepared and measured at 419 nm by UV−vis spectrum. The assembled amount of TPPS onto Au NPs was determined by measuring the unconjugated TPPS in the supernatants at 419 nm based on the standard curves. Generation of 1O2 with Laser Irradiation. Au NPs, TPPS solution, QCS-SH/Au NPs and TPPS/QCS-SH/Au NPs with same equivalent concentration in H2O/DMF (v/v = 1/1) were mixed with a chemical quencher of 1,3-diphenylisobenzofuran (DPBF, 2 μM) to characterize the generation of 1O2, respectively. The concentrations of TPPS in TPPS solution and in TPPS/QCS-SH/Au NPs were controlled to the same value at 2 μM. The concentrations of Au NPs were also set at the same value. These solutions were irradiated using a laser (635 nm, 0.85 W/cm2) for 6 min. Then, the fluorescence intensity decay of DPBF was followed at 455 nm by fluorescence spectroscopy. Cell Viability. Cell viability of the hybrid nanoparticles was determined by standard MTT assays using L929 cells as models. 50,000 cells per well were seeded into a 96-well plate and incubated at C


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ACS Biomaterials Science & Engineering

Figure 1. TEM images of (a) Au NPs and (b) TPPS/QCS-SH/Au NPs complexes with the QCS-SH layers throughout the Au NP surface, as indicated by arrows. (c) Zeta potential measurements of Au NPs and their hybrid nanoparticles. (d) UV−vis absorption spectra of various Au NPs and hybrid nanoparticles and TPPS compound. 37 °C in a humidified atmosphere with 5% of CO2. The nanoparticles were diluted with culture medium to achieve the predetermined concentrations. After 24 h of incubation, the growth medium was replaced with culture medium based on hybrid nanoparticles at various concentrations for another 24 h. And then 20 μL MTT solution (2.0 mg/mL) was added into the above solution, and followed by 4 h of incubation. Finally, the nutrient solution was removed thoroughly, and 150 mL dimethyl sulfoxide (DMSO) was added to dissolve the crystals. Optical density (OD) was calculated from the absorbance of each well, which was measured at 570 nm by an ELIASA (INFINITE 200 PRO, Tecan Austria Gmbh). Morphology of L929 cells cultured with different concentrations of the nanoparticles were observed and photographed under fluorescent microscopy after FDA (5 mg/mL) was used for cells staining. In vitro antitumor property of the TPPS/QCS-SH/Au NPs was determined by standard MTT assays using HepG2 cells as models. 50,000 HepG2 cells per well were seeded into a 96-well plate and incubated at 37 °C in a humidified atmosphere with 5% of CO2. The cells were treated with different reagents at different concentrations for 24 h of incubation and then exposed to a laser (635 nm, 0.85 W/cm2) with various time. After laser irradiation, cells treated with the same amount of PBS to the TPPS/QCS-SH/Au NPs were used as a control group and incubated for an additional 24 h. Finally, cell viability was evaluated using the MTT assay. To study the subcellular localization of TPPS/QCS-SH/Au NPs nanocomplex, HepG2 cells (3.0 × 104 cells/mL) were seeded on the cover glass for 24 h. After 8 h of incubation in the solutions containing free TPPS and TPPS/QCS-SH/Au NPs nanocomplex (50 μg/mL), respectively, cells were washed with PBS for three times in the dark and then stained with DAPI (Nuclear staining) for 10 min. Then, the intracellular distribution of free TPPS, TPPS/QCS-SH/Au NPs nanocomplex, and DAPI-stained nuclear were observed by confocal laser scanning microscope (TCS-SP8, Leica, Germany). Overlap between TPPS and DAPI-stained nuclear were observed by merging

the colocalization and fluorescent topographic profiles using NISElements BR 3.10 software.

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RESULTS AND DISCUSSION Synthesis and Characterization of QCS-SH. Synthesis of QCS-SH was shown as Scheme 2. QCS was first prepared by ring-opening of the epoxy group in GTMAC via the primary amine group in CS. QCS-SH was then synthesized using EDAC as catalyst via amidation reaction. Chemical structures of QCS and QCS-SH were characterized by FTIR (Figure S1) and 1H NMR spectra (Figure S2). The results showed the successful preparation of QCS and QCS-SH. Substituted degree of the quaternarization in QCS was 64.85%, which was determined by standard solution titration using AgNO3. The substituted degree of thioglycolic acid was approximately 7.89% by compared the peak areas between the methyl (1.86 ppm) in CS and the methylidyne proton (4.23 ppm) in GTMAC from the 1H NMR spectrum (Figure S2). Preparation of Au NP-Based Hybrid Nanoparticles. Au NPs were prepared with reduction of HAuCl4 in the presence of citrate sodium, according to the Frens’s method. Figure 1a shows TEM image of the Au NPs. Average size of the Au NPs was about 25 nm, which could be useful to achieve an optimal cellular uptake. Zeta potential of Au NPs was measured to be −15.25 ± 1.36 mV (Figure 1c). Then, QCS-SH compound was coated on the surface of Au NPs partially via ligand exchange between the thiol groups and Au, forming relatively strong Au− S bonds. Then, the Zeta potential value of the QCS-SH/Au NPs hybrid nanoparticles switched to the cation charge at +37.31 ± 1.90 mV (Figure 1c). TPPS was subsequently loaded on the QCS-SH/Au NPs nanoparticles via electrostatic D


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Figure 2. XPS spectra of (a) Au 4f region and (b) S 2p region of QCS-SH/Au NPs.

Figure 3. Fluorescence intensity changes of DPBF (2 μM) in (a) H2O/DMF (V/V = 1/1) as control, (b) in the presence of TPPS (2 μM), (c) QCS-SH/Au NPs, and (d) TPPS/QCS-SH/Au NPs with various irradiation time.

absorption. After coated QCS-SH on the Au NPs, the QCSSH/Au nanoparticles showed the absorbance peaks at 530 nm. For the case of TPPS/QCS-SH/Au NPs, the emergence peaks at 519, 553, 591, and 648 nm belonged to the typical TPPS absorption peaks. This result suggested that TPPS was loaded onto the positively charged QCS-SH/Au NPs surface successfully. It was worth noting that the broadening absorption peaks were observed after introducing QCS-SH and TPPS on the surface of Au NPs at around 525 nm. It was reported that Au NPs with slight aggregation would lead to the broaden absorption peak.34 Thus, the results indicated the formation of the TPPS/QCS-SH/Au hybrid nanoparticles. In addition to the physicochemical parameters, XPS of the QCS-SH/Au NPs was employed to analyze the surface characteristic bonds of the nanoparticles via the binding energies, as shown in Figure 2. The analysis of the Au 4f and S 2p spectra showed a very broad signal, similar to that previously reported adsorption of sulfur on gold.35,36 Thereinto, two main peaks were observed for the Au 4f signal (Figure 2a). The first peak located at ∼84.0 eV was corresponding to

interactions between QCS-SH and TPPS. The obtained TPPS/ QCS-SH/Au NPs morphology is shown in Figure 1b with TEM observation. The diameter of the TPPS/QCS-SH/Au NPs increased to approximate 33 nm. Moreover, an outer layer composed of TPPS and QCS-SH was observed from the TEM image, which was about 5 nm thickness by calculation. The average diameters of Au NPs and QCS-SH/Au NPs were also confirmed by a nanoparticle size analyzer (Figure S3). The sizes of the Au NPs and QCS-SH/Au NPs were about 42 and 48 nm, respectively, which correspond with the results from the TEM images. The Zeta potential value of TPPS/QCS-SH/Au NPs was changed to +13.24 ± 2.90 mV (Figure 1c), indicating that the TPPS compounds were coated on the surface of QCS-SH/ Au NPs successfully. This value would contribute to realizing the enhanced tumor uptake of nanocarriers, because of the electrostatic interaction with negatively charged cell membrane.33 Figure 1d shows UV−vis absorption spectra of TPPS and various hybrid Au NPs. The pure Au NPs showed the specific absorption of Au NPs at about 525 nm, while QCS-SH had no E


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This is the characteristic of photothermal therapy (PTT), which could be used for tumor treatment. As shown in Figure 4, following the laser irradiation at 635 nm with a power

Au 4f7/2, and a second doublet for Au 4f5/2 component was also observed at ∼87.6 eV, which indicated the existence of the Au− S bond in the surface of Au NPs. The spectrum (Figure 2b) was curve-fitted by a set of two doublets with 2p3/2 binding energies at 160.4 and 162.8 eV. The first doublet was corresponding to monatomic sulfur adsorbed on the Au NPs surface, while the second doublet was attributed to the presence of polymeric sulfur species.35 The spectrum of S 2p did not show any other components in the 163−164 eV energy range, where sulfur multilayer commonly appeared.37 This covalent interaction between thiol group in QCS-SH and Au NPs could also be determined by Raman spectroscopy (Figure S4). A Raman band occurred at 296 cm−1 for the Au−S interaction.38 Additionally, because the dissociation energy of Au−S bonds is robust (∼40 kcal/mol), higher than that of Au-COO- (∼2 kcal/mol) bonds, the Au-COO-bond could easily be displaced by QCS-SH to form the Au−S bonds.39 Therefore, the interaction of QCS-SH on Au NPs was mainly contributed to the Au−S bonds. In order to confirm the amount of the QCSSH component immobilized onto Au NPs, TGA measurement was employed to analyze each component from the thermal profile. As shown in Figure S5, for the QCS-SH/Au NPs, there were three decomposition stages at around 100, 150, and 280 °C, which belonged to water evaporation, decompositions of thioglycolic acid chains and chitosan chains, respectively. By calculating the weight remaining, the amount of QCS-SH coated on Au NPs was 75.4%. Additionally, the amount of TPPS bound on Au NPs was calculated by UV−vis calibration curve. On average, the coating efficacy of TPPS was 85.1%, whereas the molar ratio of TPPS bound on the each Au NP to TPPS/QCS-SH/Au NPs was ∼4300:1. 1 O2 Production and Temperature Elevation Induced by Laser Irradiation. 1O2 generation, that is prevailingly accounted for the cell apoptosis, is a key parameter to assess the PDT effect. Therefore, DPBF was used as a tracer agent to evaluate the 1O2 production during the TPPS/QCS-SH/Au NPs suffered the laser irradiation (635 nm, 0.85 W/cm2). As shown in Figure 3, the fluorescence intensity of DPBF exhibited no difference at the range from 420 to 600 nm under laser irradiation for 1 min in mixture solution without any nanoparticles (Figure 3a), indicating DPBF was stabilized in the mixture solution. The same phenomenon was also observed in the QCS-SH/Au NPs solution (Figure 3c). Thus, QCS-SH compound and Au NPs could not generate 1O2 under laser irradiation at 635 nm. On the contrary, in the presence of the TPPS compound, the fluorescence intensity of DPBF had a sharp decline with irradiation time (Figure 3b). In the TPPS/ QCS-SH/Au NPs solution (the equivalent concentration with TPPS, in Figure 3d), the fluorescence intensity also showed significantly decreased with time. In addition, the decline of DPBF fluorescence intensity in TPPS and TPPS/QCS-SH/Au NPs solutions was similar, indicating that the photosensitive property of TPPS had not been influenced after loading on the Au NPs. These results confirmed that 1O2 was generated from the TPPS compound and TPPS/QCS-SH/Au NPs under laser irradiation at 635 nm. Thus, TPPS/QCS-SH/Au NPs has immense clinical potential for photodynamic tumor therapy. Further investigating the UV−vis spectrum, the TPPS/QCSSH/Au hybrid nanoparticles showed the strong absorption in the wavelength range of 500−700 nm. Thus, irradiation of the nanoparticles at 500−700 nm, the local temperature of the TPPS/QCS-SH/Au NPs can be raised up with irradiation time.

Figure 4. Temperature changes of ultrapure water, Au NPs, QCS-SH/ Au NPs, TPPS, and TPPS/QCS-SH/Au NPs upon laser irradiation at 635 nm.

intensity of 0.85 W/cm2 for 6 min, the temperatures of the solutions increased to 25.3, 47.0, 45.7, 53.7, and 56.0 °C, in the presence of the ultrapure water, Au NPs, QCS-SH/Au NPs, TPPS and TPPS/QCS-SH/Au NPs with the same concentrations, respectively. As a control, the temperature of the ultrapure water showed negligible changes, when it was exposed to laser irradiation. For Au NPs and QCS-SH/Au NPs, the temperatures had increased gradually to above 45 °C, whereas the TPPS compound led the temperature to be raised up significantly. These results indicated that both Au NPs and TPPS had the PTT effect, but QCS-SH had no effect on hyperthermia. For conjugated Au NPs with TPPS, the local temperature of the TPPS/QCS-SH/Au NPs increased to 56.0 °C, higher than the temperature produced by pure Au NPs and TPPS, which is sufficient to kill cancer cells.9 Thus, the TPPS/ QCS-SH/Au NPs had highest therapeutic effect of the tumor, possibly due to the synergistic effect of Au NPs and TPPS promoting heat generation. These results clearly suggested that the TPPS/QCS-SH/Au NPs had the properties of excellent singlet oxygen generation and photothermal conversion capability, which can be used for collaborative treatment of PDT/PTT. In Vitro Cytotoxicity and PDT/PTT of TPPS/QCS-SH/Au NPs. Because the TPPS/QCS-SH/Au NPs showed the high 1 O2 generation efficacy and stable photothermal effects, their capability as a PDT/PTT dual-modal agent for light-mediated cancer therapy were further studied. Herein, the in vitro cytotoxicity of the TPPS/QCS-SH/Au NPs was investigated with L929 cells as the model cells (Figure 5). As shown in Figure 5a, the TPPS/QCS-SH/Au NPs with various concentrations showed negligible cytotoxicity on L929 cells without laser irradiation. The cell viability remained over 90% after incubation for 24 h even in the highest concentration of the TPPS/QCS-SH/Au NPs at 100 μg/mL. With laser irradiation at 635 nm, the cell viability was also remained over 95% (Figure 5b), indicating the harmless of laser to normal cells. Cell morphology was further observed with fluorescence microscopy after incubation with TPPS/QCS-SH/Au NPs for 24 h at 37 °C (Figure 6, the concentration of the nanoparticles at 2.5 and 100 μg/mL as examples). The cells exhibited good F


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Figure 5. (a) Cell viability of L929 incubated with TPPS/QCS-SH/Au NPs with the various concentrations and (b) cell viability of L929 under laser irradiation with various time.

Figure 6. Fluorescence images of L929 cells in TPPS/QCS-SH/Au NPs solutions with different concentrations, (a) as control, (b) 2.5 μg/mL, and (c) 100 μg/mL. The inset scale bar is 200 μm.

Figure 7. Representative fluorescence images of HepG2 cells incubated with TPPS/QCS-SH/Au NPs nanocomplexes (a, 50 μg/mL) and TPPS (b, 50 μg/mL) for 8 h (red fluorescence, TPPS; blue fluorescence, DIPA). The inset scale bar is 50 μm.

growth with a fibrous structure, same to the controlled sample. Thus, the TPPS/QCS-SH/Au NPs had good biocompatibility for normal cells without laser irradiation. Upon cellular uptake, the intracellular distribution of TPPS could be directly visualized under confocal laser scanning microscopy. Representative fluorescence images of HepG2 cells after incubated with TPPS and TPPS/QCS-SH/Au NPs nanocomplexes for 8 h were described in Figure 7. TPPS components as the fluorescent agent were observed inside the cells with well distribution in the cytoplasm as red spots (Figure 7a). Compared to TPPS (Figure 7b), the TPPS/QCS-SH/Au

NPs showed a stronger fluorescence intensity inside the cells, indicating that TPPS/QCS-SH/Au NPs have been effectively uptaken by cells. Moreover, QCS-SH/Au NPs as carriers could enhance the cellular uptake of TPPS, in good agreement with previous findings.40 Furthermore, the effects combined by PDT and PTT on HepG2 cells (as the model cancer cells) in vitro were also evaluated under the laser irradiation at 635 nm (0.85 W/cm2). HepG2 cells were placed in a 96-well plate and incubated with various concentrations of the QCS-SH/Au NPs, TPPS compound and TPPS/QCS-SH/Au NPs for 24 h, respectively. G


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Figure 8. Cell viability of HepG2 incubated with (a) QCS-SH/Au NPs, (b) TPPS, and (c) TPPS/QCS-SH/Au NPs at different concentrations before and after laser irradiation for 10 min at 635 nm (0.85 W/cm2). (d) Cell viability incubated with various nanoparticles at the same concentrations (50 μg/mL) of various nanoparticles by laser irradiation at 635 nm (0.85 W/cm2) for various time.

Subsequently, all samples were subjected to laser irradiation at 635 nm for 10 min. For comparison, HepG2 cells incubated with the same concentrations of QCS-SH/Au NPs, TPPS, and TPPS/QCS-SH/Au NPs without laser irradiation were also detected. As shown in Figure 8a-c, without laser irradiation, the cell viability of HepG2 showed slight decrement in the nanoparticle solutions with different concentrations. Thus, the nanoparticles had negligible cytotoxicity, which was similar to the results from Figure 5. With laser irradiation at 635 nm for 10 min, the QCS-SH/Au NPs, TPPS compound and TPPS/ QCS-SH/Au NPs showed a dose-dependent PDT/PTT effect. The HepG2 cell viability significantly decreased in the high concentration of the nanoparticles at 100 μg/mL. Moreover, the cell viability of HepG2 in the QCS-SH/Au NPs, TPPS and TPPS/QCS-SH/Au NPs with the same concentration at 50 μg/mL were also detected with various laser irradiation time. In Figure 8d, with the prolongation of the irradiation time, the cell viability incubated with the QCS-SH/Au NPs and TPPS decreased gradually to 55.1% and 41.5%, respectively. While the cell viability in TPPS/QCS-SH/Au NPs reduced drastically to 38.5% within irradiation for 2.5 min and reduced gradually to 14.3% after 2.5 to 10 min, much higher than the other two groups. Therefore, the TPPS/QCS-SH/Au NPs showed stronger phototoxicity than another two groups, possessing the highest PDT/PTT effect. This study further supported the synergistic anticancer efficiency of TPPS/QCS-SH/Au NPs by combination of PDT and PTT. Therefore, the rational design of the porphyrin conjugated on Au NPs could serve as a promising nanosystem for the efficient PTT and PDT in vitro.

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CONCLUSIONS

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ASSOCIATED CONTENT

In summary, TPPS and functional chitosan-coated gold nanoparticles (TPPS/QCS-SH/Au NPs) had been successfully prepared through the ligand exchange and electrostatic interaction. TPPS/QCS-SH/Au NPs exhibited excellent biocompatibility, stability, high 1O2 generation and high photothermal conversion efficiency. The antitumor efficiency of the TPPS/QCS-SH/Au NPs under the laser irradiation was also detailed assessed in vitro, and the results showed that combination of PTT/PDT treatment had significantly higher antitumor efficiency by using single laser irradiation. Therefore, with excellent biocompatibility, TPPS/QCS-SH/Au NPs might be a very promising dual-mode PDT/PTT therapeutic agent for future cancer therapy. This way can encourage more available design of the facile surface functionalization strategy to construct other multifunctional nanoplatforms, such as nanorods and nanoclusters.

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.7b00886. FTIR and 1H NMR spectra of (a) CS, (b) QCS, and (c) QCS-SH (Figures S1 and S2); hydrodynamic diameters, Raman spectra, and TGA curves of Au NPs and their complexes (PDF) H


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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +86-510-85917019. Fax: +81-510-85917763. ORCID

Jinfeng Zeng: 0000-0001-7500-9665 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

This study was supported by the National Natural Science Foundation of China (21571084), the Natural Science Foundation of Jiangsu Province (BK20150135), MOE & SAFEA for the 111 Project (B13025) and the Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, Jiangnan University (JDSJ2015-06).

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