Acta Biomaterialia xxx (2014) xxx–xxx

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Hydrophobic IR780 encapsulated in biodegradable human serum albumin nanoparticles for photothermal and photodynamic therapy Chenxiao Jiang a, Hao Cheng a, Ahu Yuan a, Xiaolei Tang a, Jinhui Wu a,b,⇑, Yiqiao Hu a,b,⇑ a b

State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, People’s Republic of China Jiangsu Key Laboratory for Nano Technology, Nanjing University, Nanjing 210093, China

a r t i c l e

i n f o

Article history: Received 7 August 2014 Received in revised form 29 October 2014 Accepted 18 November 2014 Available online xxxx Keywords: NIR dye Solubilization Drug delivery Antitumor therapy

a b s t r a c t It has been reported that IR780 iodide, a near-infrared dye, can be applied for cancer imaging, photodynamic therapy (PDT) and photothermal therapy (PTT). However, the hydrophobicity and toxicity of IR780 severely limit its further clinical applications. In this study, human serum albumin was used to load IR780 to form nanoparticles (HSA-IR780 NPs) by protein self-assembly. Compared to free IR-780, the solubility of HSA-IR780 NPs was greatly increased (1000-fold) while the toxicity was decreased (from 2.5 mg kg1 to 25 mg kg1). Moreover, both PTT and PDT could be observed in HSA-IR780 NPs, as determined by increased temperature and enhanced generation of singlet oxygen after laser irradiation at a wavelength of 808 nm. In vivo studies also showed a great tumor inhibition by the injection of HSA-IR780 NPs into tumor-bearing mice. Therefore, HSA-IR780 NPs may serve as a promising substitute for IR780 in further clinical PDT and PTT. Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction Near-infrared (NIR) dyes are small organic molecules that absorb radiation in the wavelength range 700–1000 nm. After absorbing NIR light with specific wavelengths, they reach an excited singlet state. Through vibronic relaxation or other non-radiative transitions ways, some energy from the excited singlet state will be converted into heat. Besides, the singlet state will drop to a lower-energy-excited triplet state via intersystem crossing, in which reactive species can be generated to induce oxidation reaction with surrounding biomacromolecules and destroy organic tissues [1]. Therefore, NIR dyes can produce heat in localized ranges and generate reactive oxygen species (ROS) upon NIR irradiation, which respectively contribute to photothermal therapy (PTT) and photodynamic therapy (PDT). IR780 iodide, as a NIR dye, is a lipophilic cation with specific absorption peak at 780 nm. In the structure of IR780, there is a rigid cyclohexenyl ring and a chlorine atom in the central, leading to its high hydrophobicity [2]. Currently, IR780 iodide has been reported to be capable of generating a singlet oxygen after laser irradiation at a wavelength of 808 nm, which can be used for photodynamic therapy [3]. Due to its affinity to organic anion transporter peptides (OATPs), IR780 showed preferential accumulation in multiple tumor cells [4,5]. Furthermore, IR780 ⇑ Corresponding authors. Tel.: +86 13913026062; fax: +86 25 83596143 (J. Wu). Tel.: +86 13601402829; fax: +86 25 83596143 (Y. Hu). E-mail addresses: [email protected] (J. Wu), [email protected] (Y. Hu).

can produce heat under laser irradiation, which may employ it to be a photothermal therapeutic agent [2,6–8]. However, due to the existing lipophilic group, it is extremely difficult to dissolve IR780 iodide in water, and this severely limits further clinical applications. Moreover, the toxicity of IR780 iodide is also a concern due to its low maximal tolerance dose of 1.5 mg kg1 in mice [6,8]. So far, much research has been devoted to overcoming these disadvantages by encapsulating IR780 into various nanomaterials (e.g. 188Re-labeled polymeric micelles [8], silica nanoparticles (NPs) [9] and heparin–folic acid NPs [6]). The radionuclide rhenium-188 labeled polymeric micelles were engineered by Peng et al. [8] to improve the water solubility of IR780 iodide. Although the water solubility of IR780 was increased, the synthesis process of polymers was relatively complicated and the tolerance dose of IR780 was scarcely increased (1.25 mg kg1). Singh et al. [9] integrated IR780 iodide into mesoporous silica matrix to solve the hydrophobicity. However, the silica NPs are non-biodegradable, which might induce severe side-effects after administration. Yue et al. [6] also synthesized heparin–folic acid NPs to increase the water solubility of IR780 iodide. But the poor tolerance of IR780 iodide in mice is still not resolved. Therefore, it is necessary to develop a simple method for delivering IR780, which not only increases the water solubility but also reduces the toxicity of IR780. In the previous work, we developed a simple method to remarkably increase the water-solubility of hydrophobic drugs by encapsulating them into human serum albumin (HSA) [10–12]. In this

http://dx.doi.org/10.1016/j.actbio.2014.11.041 1742-7061/Ó 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Jiang C et al. Hydrophobic IR780 encapsulated in biodegradable human serum albumin nanoparticles for photothermal and photodynamic therapy. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.11.041

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study, the simple method was used to encapsulate IR780 iodide into HSA NPs (HSA-IR780 NPs) for the combination therapy of PTT and PDT for cancer treatment (Scheme 1). This method not only solved the delivery of IR780 but also reduced the toxicity of IR780. HSAIR780 NPs were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), dynamic light scattering (DLS) and ultraviolet–visible (UV–vis) spectrophotometry. The PDT and PTT effects of HSA-IR780 NPs were evaluated in vitro. The antitumor efficacy of HSA-IR780 was also evaluated by injection of HSA-IR780 into tumor-bearing mice. 2. Materials and methods 2.1. Materials HSA solutions were purchased from Baxter. IR780 and 20 ,70 dichlorodihydrofluorescein diacetate (H2DCFDA) were both purchased from Sigma–Aldrich. Singlet oxygen sensor green was purchased from Invitrogen Corp. The cell counting kit-8 (CCK-8) was supplied by Dojindo Laboratories (Japan). Deionized water was used throughout the experiments. All the BALB/c mice were purchased from Yangzhou University Medical Center, and the weight of each mouse was 20–22 g.

2.3. Characterizations of HSA-IR780 NPs The morphology and particle size of HSA-IR780 NPs were analyzed by TEM (Hitachi H-7650). The NPs were dried on a copper grid coated with amorphous carbon and then observed at 200 kV. The NPs were also coated in a cathodic evaporator with a fine gold player and observed on a scanning electron microscope (Hitachi S-3400N). UV–vis spectra were measured by a UV–vis spectrophotometer (UV2450, Shimadzu Corporation). The stability of the HSA-IR780 NPs was investigated in 5% glucose and 0.9% NaCl under different temperatures (25 °C or 37 °C) by measuring their mean diameter, separately. The diameter of HSA-IR780 NPs was measured by a DLS analyzer (Brookhaven Instruments Corporation, USA). The final concentration of IR780 in the mixed solution was 15 lg ml1. The mean diameter of HSA-IR780 NPs was measured by DLS at 0, 2, 4, 8, 12 and 24 h. 2.4. Investigation of the photostability of HSA-IR780 NPs The absorption spectra of HSA-IR780 NPs dispersions (7 lg ml1 for IR780) and IR780 (5 lg ml1, dissolved in DMF) solutions were determined right after the dilution process or 1 day storage (in light or darkness) using the UV–vis spectrophotometer.

2.2. Synthesis of HSA-IR780 NPs According to our previous work, 200 mg of HSA were dissolved in 100 ml of deionized water with constant stirring. 2-Mercaptoethanol was added to expose the hydrophobic region of HSA. Then, IR780 (2.5 mg ml1, dissolved in dimethylformamide (DMF)) was slowly added into the denatured protein solution accompanied by stirring all the time until complete dissolution. The obtained green dispersions were ultrafiltrated to remove 2mercaptoethanol and unbound HSA (the molecular weight was 60 kDa) using an ultrafiltration membrane with the molecular weight cut-off of 100 kDa, then filtered through 0.22 lm filter membrane. Finally, the NP dispersions (namely HSA-IR780 NPs) were stored in 4 °C. The amount of the IR780 in HSA-IR780 NPs was determined by UV–vis absorption spectra according to standard curve. The IR780loading content and encapsulation efficiency were calculated as follows:

IR780 loading content ð%Þ ¼ weight of IR780 in NPs=weight of NPs  100%

Encapsulation efficiency ð%Þ ¼ weight of IR780 in NPs= weight of total added IR780  100%

2.5. Photothermal and photodynamic effects for HSA-IR780 NPs in aqueous solution To evaluate the photothermal effects of NPs in aqueous solution, HSA-IR780 NPs were exposed to 808 nm wavelength laser irradiation (1 W cm2) with the illumination direction from the top to the bottom of the cuvette. The final concentrations of IR780 in HSA-IR780 NPs dispersions were 20 and 60 lg ml1. The negative control was an equivalent amount of water with the same laser irradiation. The temperature of solution was determined with a thermal probe at 0, 2, 4, 6, 8 and 10 min. Each solution was measured three times. The images of temperature changes for NPs dispersions (containing 60 lg ml1 IR780) and water were taken by infrared imaging devices (ThermaCAMSC3000, Flirsystem incorporation) at 0.5 min internals for a total of 5 min. The pseudo-color images were processed by Matlab. To evaluate the photodynamic effects of NPs in aqueous solution, 200 ll HSA-IR780 NPs and 20 ll 50 lM singlet oxygen sensor green (SOSG, Invitrogen) were added into a 96-well plate, followed by 808 nm wavelength laser (1 W cm2) irradiation. The final concentrations of IR780 in HSA-IR780 NPs dispersions were 2, 1 and 0.5 lg ml1. Singlet oxygen generated in the procedure interacted with the anthracene component of SOSG and produce an endoperoxide emitting green fluorescence [13,14]. Thereby, the generation of ROS could be detected based on SOSG, which is a fluorescence

Scheme 1. The principle of HSA-IR780 NPs with NIR irradiation for antitumor therapy.

Please cite this article in press as: Jiang C et al. Hydrophobic IR780 encapsulated in biodegradable human serum albumin nanoparticles for photothermal and photodynamic therapy. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.11.041

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indicator for detecting ROS in aqueous solution. The fluorescence intensity was measured at 0, 10, 20 and 30 s and at an excitation of 504 nm and an emission of 525 nm using a multifunctional microplate reader (Safire, TECAN). 2.6. Detection of ROS generation of HSA-IR780 NPs in cancer cells H2DCFDA acts as a fluorescent probe for ROS in cells, which is non-fluorescent before penetrating into cells [15,16]. Once entering the cells, the diacetate group of H2DCFDA would be removed by intracellular esterases. The remaining part would be oxidized rapidly and emit green fluorescence in the presence of ROS. Based on this principle, pre-seeded MCF-7 cells in a confocal 12-well plate were co-incubated with HSA-IR780 NPs dispersions or phosphate buffered saline (PBS) and H2DCFDA (40 lM) for 4 h at an atmosphere of 37 °C, 5% CO2. The final concentration of IR780 in HSA-IR780 NPs dispersions was 20 lg ml1. Subsequently, the cells were washed with PBS and the center region in the 12-well plate was exposed to 808 nm wavelength laser irradiation (1 W cm2) for 5 min. The spot size of the laser used for irradiation is 0.6 cm  0.5 cm. Fluorescence images in the irradiation region were immediately captured on a confocal fluorescence microscope (Olympus FV1000) using an excitation of 504 nm and an emission of 510–560 nm. 2.7. Cytotoxicity experiments in vitro Cytotoxicity of HSA-IR780 NPs can be assessed by propidium iodide (PI) staining and cell counting kit (CCK-8) assays. MCF-7 cells were seeded into a confocal 12-well plate at a density of 2  105 cells per well overnight. The cells were treated with HSA-IR780 NPs dispersions or PBS for 4 h. The final concentration of IR780 in HSA-IR780 NPs dispersions was 20 lg ml1. Then, the center region in the 12-well plate was exposed to 808 nm wavelength laser irradiation (1 W cm2) for 5 min. The spot size of the laser used for irradiation is 0.6 cm  0.5 cm. After that, the cells were washed twice with PBS and stained with PI for 15 min. Fluorescence images in the irradiation region of cells were acquired from the same confocal microscope at an excitation of 535 nm and an emission of 600–680 nm. MCF-7 cells were seeded into a 96-well plate at a density of 4  104 cells per well and cultured with HSA-IR780 NPs at different concentrations for 6 h (included in the 24 h), followed by 808 nm laser irradiation (1 W cm2) for 5 min each well. Another group was cultured with HSA-IR780 NPs at the same concentrations without NIR irradiation for 24 h. The cells without any drugs and NIR irradiation were taken as the negative control. After the cultured medium was removed, the plates were washed once with PBS. Then, the mixed solution consisting of CCK-8 (10 ll) and fresh medium (100 ll) was added into each well, and the cells were incubated for an additional 2 h at 37 °C, 5% CO2. Finally, the absorbance was measured at 450 nm using a microplate reader. Data were analyzed using Prism 4 (GraphPad Software, San Diego, CA) and expressed as mean ± standard deviation. Multiple groups were compared using one-way analysis of variance (ANOVA) followed by the Tukey–Kramer test for post hoc comparisons. Statistical significance was set at P < 0.05. 2.8. NIR fluorescence imaging and photothermal effect of HSA-IR780 NPs in vivo

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imaging system (IVIS Lumina XR, Caliper Life Sciences). After 48 h, the mice were killed. To evaluate the photothermal effect of HSA-IR780 NPs in vivo, the tumor-bearing mice were intravenously injected with HSAIR780 NPs at different concentrations (5, 10 and 20 mg kg1 for IR780, n = 3) when the tumor volumes reached 100 mm3. Mice treated with the equivalent amount of saline were the control group (n = 3). After 24 h, all the mice were exposed to 808 nm wavelength laser irradiation (1 W cm2). The temperature changes were recorded using a visual IR thermometer (Fluke Corporation) at different time points for a total of 10 min. The IR images were captured by IR imaging devices (ThermaCAMSC3000, Flirsystem incorporation) at 0, 0.5, 1, 3 and 5 min. The pseudo-color images were processed by Matlab. 2.9. Photo-therapeutic efficacy of HSA-IR780 NPs in vivo The CT26 colon adenocarcinoma cells were subcutaneously inoculated to the male BALB/c mice. When the tumor volumes reached 100–200 mm3, the mice were intravenously injected with HSA-IR780 NPs (20 mg kg1 for IR780, n = 4–9) as the test groups. Mice treated with the same volume of saline (n = 4–9) were taken as the control groups. After 24 h post-injection, the mice were illuminated with 808 nm wavelength laser irradiation (1 W cm2, 5 min). After 3 days, the test groups were intratumorally injected with 0.05 ml HSA-IR780 NPs (2.5 mg kg1 for IR780) and the control groups were intratumorally injected with 0.05 ml saline, both followed by 808 nm illumination (2 W cm2, 2 min). Tumor diameters were measured using an vernier caliper, and the weights of mice were recorded each day. The volume (V) of the tumor was estimated according to this formula: V = D  d2/2 (where D is the longest diameter of tumor and d is the shortest diameter of tumor). Relative tumor volumes were calculated as V/V0 (V0 is the initial tumor volume when the treatment began). Data were analyzed using Prism 4 (GraphPad Software, San Diego, CA) and expressed as mean ± standard error. Multiple groups were compared using ANOVA followed by the Tukey–Kramer test for post hoc comparisons. Statistical significance was set at P < 0.01. 2.10. Toxicity tests of HSA-IR780 NPs in vivo The male BALB/c mice were intravenously injected with IR780 iodide (2.5 mg kg1, n = 3) and HSA-IR780 NPs at different concentrations (25 and 2.5 mg kg1 for IR780, n = 3), respectively. The weights of mice were noted within 16 days. At the 16th day all the mice were killed. Male BALB/c mice (n = 5) were injected with HSA-IR780 NPs (25 mg kg1 for IR780, 0.5 ml per mice) in the tail vein. BALB/c mice (n = 5) injected with saline were the control group. After 14 days, blood samples from the retro-orbital venous plexus were collected in heparinized microhematocrit tubes and centrifuged at a speed of 3000 rpm for 10 min. Then, the mice were killed. The upper sera were sent to be analyzed at the Nanjing Drum Tower Hospital (the affiliated hospital of Nanjing University Medical School). Besides, the tissues including heart, liver, spleen, kidney and brain were harvested, fixed in formalin and sent for analysis to the Jiangsu Institute for Food and Drug Control. All animal procedures were approved by the institutional animal care and use committee at Nanjing University. 3. Results and discussion

The CT26 colon adenocarcinoma cells were subcutaneously inoculated into the male BALB/c mice. When the tumor volumes reached 100 mm3, HSA-IR780 NPs (0.2 mg kg1 for IR780) were intravenously injected into BALB/c mice. The NIR images were captured at 0.5, 1, 2, 4, 6, 8, 11, 24, 32 and 48 h using an animal optical

3.1. Synthesis and characterization of HSA-IR780 NPs HSA-IR780 NPs were synthesized according to our previous methods.[10–12] We first destroyed the disulfide bond of HSA by

Please cite this article in press as: Jiang C et al. Hydrophobic IR780 encapsulated in biodegradable human serum albumin nanoparticles for photothermal and photodynamic therapy. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.11.041

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2-mercaptoenthanol. Then the hydrophobic domains in HSA were exposed. IR780, a strong hydrophobic molecule, tended to integrate with the exposed hydrophobic parts of HSA to form the NPs. Hence, IR780 and hydrophobic groups of HSA were enclosed in the center of the NPs, while the hydrophilic groups of HSA were exposed to water. The obtained NPs dispersions were ultrafiltrated to remove 2-mercaptoethanol, and high performance liquid chromatography was used to ensure a low residue of 2-mercaptoethanol in HSA-IR780 NPs. The morphology and particle size of the NPs were characterized by TEM and SEM, as shown in Fig. 1a. The SEM image shows that NPs formed by self-assembly are spherical and have a diameter of 250 nm with a smooth surface. From the TEM image, the NPs are uniformly dispersed in aqueous solution and have a diameter of 100–250 nm with a smooth surface. According to the pharmacokinetic study of AbraxaneÒ, this size range of NP might be mainly excreted via bile and faeces [17,18]. We then investigated the stability of HSA-IR780 NPs in 0.9% NaCl and 5% glucose, at 25 °C and 37 °C, respectively (Fig. 1b). In 24 h, the size of NPs remains consistent (220 nm) with the pH of 6.8, indicating that HSA-IR780 NPs can encapsulate the photosensitizer drug with little leakage. In addition, we measured the solubility of both IR780 and HSA-IR780 NPs. The water solubility of IR780 was lower than 0.4 lg ml1, while the water solubility of IR780 after encapsulating into HSA was higher than 400 lg ml1. Hence, the solubility of IR780 in water can be improved by over, 1000-fold via encapsulation into HSA carriers.

DMF and diluted with water) has a strong absorption peak at 780 nm. After loading IR780, HSA-IR780 NPs dispersions (7 lg ml1 for IR780) present the same absorption peak at 278 nm as HSA. But the absorption peak at 780 nm has a 9 nm right-shift to 789 nm, which might be due to the hydrophobic interaction and the changes in solvent polarity. IR780 iodide was first dissolved in DMF. In the presence of 2-mercaptoethanol, IR780 was non-covalently entrapped in the hydrophobic region of HSA to form spherical NPs through self-assembly, which were dissolved in water. It is the changes in solvent polarity and hydrophobic interaction that contribute to the bathochromic shift in the NIR absorption spectral band. The spectrum of HSA-IR780 NPs possesses the characteristic peaks of both HSA and IR780, demonstrating that IR780 has been successfully encapsulated into HSA NPs. To investigate the photostability of IR780 and HSA-IR780 NPs, we studied the spectra of free IR780 and HSA-IR780 NPs right after the dilution process or 1 day of storage (in light or darkness). After 1 day of storage at room temperature, the maximum absorbance at 780 nm of IR780 (Fig. 1d) declined, demonstrating that IR780 is unstable in water. In light storage, the maximum absorbance of IR780 decreased greater than those of IR780 stored in darkness, indicating that IR780 is more sensitive to light. However, the maximum absorbance of HSA-IR780 NPs remains almost the same either stored in darkness or light (Fig. 1e), revealing that HSA NPs can perfectly protect IR780 from aggregation and hydrolysis in water, and hence be eligible for further cells and animal experiments.

3.2. Optical characteristics of HSA-IR780 NPs

3.3. Photothermal and photodynamic effects of HSA-IR780 NPs in aqueous solution

The IR780 loading efficiency in HSA-IR780 NPs was 1.6%, with the encapsulation efficiency over 73%. From UV–vis spectra shown in Fig. 1c, HSA solution has an absorption peak at 278 nm and no absorption in the NIR region, while IR780 (7 lg ml1, dissolved in

The phototherapeutic effects of HSA-IR780 NPs were confirmed by a series of experiments. To verify the photothermal effects of HSA-IR780 NPs, different concentrations of HSA-IR780 NPs disper-

Fig. 1. Characterizations and photostability of HSA-IR780 NPs. (a) TEM images of HSA-IR780 NPs. The scale bar is 200 nm. Inset: SEM images of HSA-IR780 NPs. The scale bar is 200 nm. (b) Stability of HSA-IR780 NPs in different conditions (black square: 25 °C, 5% glucose; red circle: 25 °C, 0.9% NaCl; blue triangle: 37 °C, 5% glucose; green inverted triangle: 37 °C, 0.9% NaCl). (c) UV–vis spectra of 7 lg ml1 IR780, 7 lg ml1 HSA-IR780 NPs (calculated as IR780) and 120 lg ml1 HSA (black: IR780; red: HSA-IR780 NPs; blue: HSA). UV–vis spectra of IR780 (d) and HSA-IR780 NPs (e) determined right after the dilution process or 1 day of storage (black: fresh solution; red: 1 day of storage in darkness; blue: 1 day of storage in light).

Please cite this article in press as: Jiang C et al. Hydrophobic IR780 encapsulated in biodegradable human serum albumin nanoparticles for photothermal and photodynamic therapy. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.11.041

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sions and water were exposed to 808 nm wavelength laser irradiation at a power density of 1 W cm2. From Fig. 2a, only a slight temperature raise of 8 °C for water can be seen. But the temperature for 20 lg ml1 HSA-IR780 NPs increased in the first 3 min before decreasing during the residual time, indicating that IR780 in the NPs is degraded due to the exposure to NIR irradiation, which can be acquired from Fig. S.2. On the other hand, the temperature of 60 lg ml1 HSA-IR780 NPs increases from 22 °C to 45 °C within 10 min, which is enough for tumor photothermal treatment. The higher concentration of IR780 in NPs leads to much higher temperatures. These help HSA-IR780 NPs to be a powerful photothermal agent. In addition, to observe visually the temperature changes, Fig. 2c shows the visible photos and IR thermal images of 60 lg ml1 HSAIR780 NPs and water exposed to 808 nm wavelength laser irradiation for 5 min recorded every 0.5 min. For HSA-IR780 NPs group, the pseudo-color signals are observed gradually deepening with the irradiation time increase, showing directly that the temperature rises from room temperature to nearly 46 °C. On the other hand, the pseudo-color signals for water slightly change. This result is in accordance with Fig. 2a, further proof of the photothermal effects of HSA-IR780 NPs. To verify the photodynamic effects of NPs, the ROS generation of HSA-IR780 NPs is manifested by the fluorescence intensity of SOSG. As shown in Fig. 2b, after being exposed to 808 nm wavelength laser irradiation for 30 s, the fluorescence intensity of HSA-IR780 NPs gradually increases with the concentration increase of NPs. The fluorescence intensity of 2 lg ml1 NPs increases from 473.75 to 2386, while the fluorescence intensity of water remained almost the same. Besides, in vitro release studies were carried out in PBS at 37 °C. As shown in Fig. S.1, less than 5% of IR780 was released with or without laser irradiation, since IR780 was closely bound with HSA by strong hydrophobic interactions in the core of NPs, and IR780 could not escape from HSA into water due to its extremely low water solubility. This result demonstrates that ROS are generated within NPs upon laser irradiation and then diffused into the surroundings.

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These results indicate that HSA-IR780 NPs can simultaneously generate heat and ROS after laser irradiation at a wavelength of 808 nm, which is important for the combination therapy of PTT and PDT. 3.4. ROS generation of HSA-IR780 NPs in cancer cells Upon exposure to 808 nm wavelength laser irradiation, ROS generation of HSA-IR780 NPs can oxidize H2DCF, which is a nonfluorescent part of H2DCFDA, and exhibit green fluorescence in human breast cancer cell lines (MCF-7). As shown in Fig. 3, no fluorescence can be seen in cells treated by the control groups of PBS, PBS with laser and HSA-IR780 NPs. However, cells treated by HSAIR780 NPs plus NIR irradiation exhibit obvious green fluorescence, demonstrating an amount of ROS generation in cells and further predicting the application of NPs for PDT therapy. 3.5. Cytotoxicity assays of HSA-IR780 NPs in vitro HSA-IR780 NPs are expected to be cytotoxic due to PTT and PDT effects when exposed to laser irradiation of 808 nm wavelength. To evaluate its cytotoxicity, dead cellular staining by PI and standard cell viability assays was carried out. MCF-7 cells were treated with 60 lg ml1 HSA-IR780 NPs or PBS for 4 h and then exposed to 808 nm wavelength laser irradiation (1 W cm2) for 5 min. Dead cells were stained by PI as shown in red fluorescence. As shown in Fig. 4, red fluorescence can be observed only in the cells treated with HSA-IR780 NPs plus NIR irradiation, indicating dead cancer cells. In contrast, a little red fluorescence could be observed in HSA-IR780 NPs, PBS and PBS plus NIR groups. All these indicate that HSA-IR780 NPs with NIR illumination show prospective abilities to kill cancer cells, while without NIR illumination they are relatively harmless and do not cause cell death. To verify the cytotoxicity of HSA-IR780 NPs, standard cell viability assays were also carried out. The cells without any drug and laser were the control group, and the cells with only NIR laser

Fig. 2. Photothermal and photodynamic effects of HSA-IR780 NPs in aqueous solution. (a) Heating curves of water and HSA-IR780 NPs solutions (20 and 60 lg ml1 for IR780, 1 ml) exposed to 808 nm laser irradiation (1 W cm2). Data are expressed as mean ± SD (n = 3). (b) Fluorescence intensity of SOSG combined with HSA-IR780 NPs solutions at different concentrations (0, 0.5, 1 and 2 lg ml1 for IR780, 200 ll) exposed to 808 nm laser irradiation (1 W cm2). Data are expressed as mean ± SD (n = 3). (c) Visible photos and IR thermal images of water and HSA-IR780 NPs (60 lg ml1 for IR780, 500 ll) solutions exposed to 808 nm laser irradiation at a power density of 1 W cm2 recorded at different time intervals. Black arrow represent the irradiation direction of the 808 nm laser.

Please cite this article in press as: Jiang C et al. Hydrophobic IR780 encapsulated in biodegradable human serum albumin nanoparticles for photothermal and photodynamic therapy. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.11.041

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Fig. 5. Cell viability values (%) of MCF-7 cells. MCF-7 cells incubated with HSAIR780 NPs (red, middle) and HSA-IR780 NPs followed by NIR (808 nm, 1 W cm2, 5 min) irradiation (blue, right) at different concentrations. The control represents the cells without any drug and laser. The laser represents the cells with only NIR laser (808 nm, 1 W cm2, 5 min). Data are expressed as mean ± SD (n = 4–6). * indicates P < 0.05. NS indicates no significance. Fig. 3. Confocal fluorescence images of ROS generation in MCF-7 cells. From top to bottom: PBS, PBS plus NIR laser (808 nm, 1 W cm2, 5 min), HSA-IR780 NPs and HSA-IR780 NPs plus NIR laser. The final concentration of HSA-IR780 NPs (calculated as IR780) was 20 lg ml1. The green fluorescence color represents oxidatively stressed cells affected with ROS. Fluorescence images were collected at 510– 560 nm, under an excitation at 504 nm. The scale bar is 50 lm.

viabilities of the control and the laser group are 100% and 101%, respectively. This negligible difference of cell viabilities between the control and the laser group indicates that laser alone has almost no toxicity to tumor cells. With the increase of the concentration of IR780, the cell viabilities of both the NPs group and NPs with the laser group gradually decline. However, the cell viabilities of NPs are higher than that of NPs with laser at the final concentration of IR780 between 1.56 and 12.5 lg ml1. The above results reveal that HSA-IR780 NPs can display more toxicity to cancer cells when exposed to NIR irradiation compared to those without NIR irradiation. Due to the effects of PTT and PDT combination therapy, which have been proven in aqueous solution and cell levels, the HSA-IR780 NPs can greatly suppress the growth of tumor cells.

3.6. In vivo NIR fluorescence imaging and photothermal effect of HSA-IR780 NPs

Fig. 4. Confocal fluorescence images of MCF-7 cells staining with PI. From top to bottom: PBS, PBS plus NIR (808 nm, 1 W cm2, 5 min), HSA-IR780 NPs and HSAIR780 NPs plus NIR. The final concentration of IR780 in HSA-IR780 NPs solutions was 20 lg ml1. The cells were stained with PI as shown in red. Fluorescence images were collected at 600–680 nm, under an excitation at 535 nm. The scale bar is 50 lm.

(808 nm, 1 W cm2, 5 min) were the laser group (Fig. 5). The other two groups were the cells incubated with HSA-IR780 NPs and HSAIR780 NPs combining with NIR laser (808 nm, 1 W cm2, 5 min) respectively. As shown in Fig. 5, it can be seen that the cell

To evaluate the feasibility of HSA-IR780 NPs as an ideal photosensitizer, in vivo NIR fluorescence imaging and photothermal tests were implemented on CT26 tumor-bearing mice. NIR fluorescence imaging was applied on tumor-bearing mice which were injected with HSA-IR780 NPs via the tail vein. As shown in Fig. 6a, the fluorescence signals in the tumor region strengthen with time during 24 h post-injection, revealing that HSA-IR780 NPs can preferentially accumulate in tumors. The preferential accumulation in tumors might be due to the enhanced permeability and retention effects [19,20]. After 24 h post-injection, the fluorescence signals gradually decline, indicating that 24 h post-injection is the best time for laser irradiation. To evaluated the photothermal effects in vivo, tumor-bearing mice were intravenously administered HSA-IR 780 NPs and exposed to 808 nm wavelength laser irradiation (1 W cm2) for 10 min at 24 h post-injection. The temperature changes of the tumor were recorded by a visual IR thermometer. As shown in Fig. 6b, after laser irradiation, the temperature in the tumor increases greatly. The longer the irradiation time, the higher the temperature observed in the tumor. In the 20 mg kg1 HSA-IR780 group, the temperature in the tumor region rises rapidly and reaches near 46 °C after laser irradiation for 3 min. It remains

Please cite this article in press as: Jiang C et al. Hydrophobic IR780 encapsulated in biodegradable human serum albumin nanoparticles for photothermal and photodynamic therapy. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.11.041

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Fig. 6. NIR fluorescence imaging and photothermal effects of HSA-IR780 NPs in vivo. (a) NIR images of tumor-bearing mice following intravenous injection of HSA-IR780 NPs during 48 h. The tumors are circled with a dotted line. (b) Heating curves of tumor-bearing mice with different concentrations of HSA-IR780 NPs and saline. Data are expressed as mean ± SD (n = 3). (c) IR thermal images of tumor-bearing mice. Mice were intravenously administered HSA-IR780 NPs and saline and exposed to 808 nm laser (1 W cm2) at 24 h post-injection.

almost constant during the residual time, which is sufficient to irreversibly damage the tumor. On the other hand, an increase of only 1.8 °C in the saline group is observed under the same irradiation conditions. Besides, the IR thermal images of the tumor-bearing mice treated with 20 mg kg1 HSA-IR780 NPs or saline were also monitored. Fig. 6c displays visually the temperature increase in the tumor region, which is consistent with Fig. 6b. These results indicate that HSA-IR780 NPs can accumulate in the tumor region after intravenous injection. Under the laser irradiation, HSAIR780 NPs produce heat effects which help it to be an ideal photothermal agent.

3.7. Photo-therapeutic efficacy of HSA-IR780 NPs in vivo Antitumor studies were carried out to estimate the phototherapeutic efficacy of HSA-IR780 NPs in vivo. When the tumor volume reached 100–200 mm3, mice bearing CT26 tumor were intravenously injected with HSA-IR780 NPs or saline. At 24 h post-injection, mice were illuminated with NIR laser irradiation. As shown in Fig. 7a, after laser irradiation, the tumor of mice treated with HSA-IR780 NPs declines significantly and it almost disappears on day 14 (Fig. 7b). And the scar tissues gradually heal during the 14 days (Fig. S.3). However, in other three groups (saline, saline plus

Fig. 7. Photo-therapeutic efficacy of HSA-IR780 NPs in vivo. (a) Tumor growth of mice bearing CT26 tumor after various treatments as indicated. The tumor volumes were normalized to their initial values. (Red and blue arrows represent the day of injection.) Data are expressed as mean ± standard error (n = 3–9). ** indicates P < 0.01, compared to the NPs with laser group. (b) Photos of mice bearing CT26 tumor on day 14 after various treatments as indicated.

Please cite this article in press as: Jiang C et al. Hydrophobic IR780 encapsulated in biodegradable human serum albumin nanoparticles for photothermal and photodynamic therapy. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.11.041

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Fig. 8. Toxicity tests of HSA-IR780 NPs in vivo. (a) Toxicity study of mice injected with different concentrations of HSA-IR780 NPs. (b) Table of serum biochemical indicators acquired from mice on the 14th day post-injection with saline or HSA-IR780 NPs (25 mg kg1 for IR780, n = 5). Data are expressed as mean ± SD. (c) Histological analysis of the organs acquired from mice on the 14th day post-injection with saline or HSA-IR780 NPs (25 mg kg1 for IR780).

NIR and NPs), the volumes of tumors increase greatly by more than 12-fold on day 14 compared with those on day 0 (day 0 was the initial day when the mice bearing tumors were intravenously injected with HSA-IR780 NPs or saline). These results demonstrate that HSAIR780 NPs have prominent phototherapeutic efficacy to suppress tumor growth. 3.8. Histological assessments and hemanalysis results of mice injected with HSA-IR780 NPs To compare the toxicity between IR780 iodide and HSA-IR780 NPs, IR780 iodide (dissolved in DMF and diluted in water) and HSA-IR780 NPs were injected to mice via the tail vein. For the IR780 group, all the mice were dead right after injection with IR780 at the dose of 2.5 mg kg1. Thus, IR780 was too toxic to be applied in the efficacy experiments in vivo. However, for HSAIR780 NPs group (25 and 2.5 mg kg1), no death was observed and all the mice survived over 16 days. The results demonstrate that the HSA encapsulation greatly reduces the toxicity of IR780 iodide and enhances the tolerance of IR780 iodide (Fig. 8a). To further assess the safety of HSA-IR780 NPs in vivo, blood samples and organs of healthy BABL/c mice injected with HSA-IR780 NPs (25 mg kg1) via the tail vein were harvested and analyzed on the 14th day post-injection. Comparing the saline group with the NPs group, no apparent lesion (no necrocytosis, edema, inflammatory infiltration and hyperplasia) can be observed in the sections of the six tissues (heart, liver, spleen, lung, kidney and brain) (Fig. 8c). In addition, two indicators, ALT (alanine aminotransferase)

and AST (aspartate aminotransferase) for hepatic function, and another two, UREA (urea nitrogen) and CREA (creatinine) for renal function, were evaluated via blood samples from mice. From Fig. 8b, the levels of enzymes are in normal ranges, and no visible differences of the four indicators can be observed. Histological sections and serum biochemistry results prove that HSA-IR780 NPs at the tested dose have no cytotoxic effects on healthy mice. 4. Conclusions In this study, HSA-IR780 NPs were successfully produced for in vitro and in vivo photothermal and photodynamic combination therapy for cancer. Encapsulation with HSA not only improves the water solubility and optical stability of IR780 iodide in water, but also significantly reduces the toxicity of IR780 iodide to mice. Moreover, HSA-IR780 NPs have the ability to target tumors. Furthermore, upon 808 nm wavelength laser irradiation at a density of 1 W cm2, HSA-IR780 NPs can simultaneously produce hyperthermia and generate amounts of ROS to severely destroy tumors both in vitro and in vivo. In summary, the novel self-assembly HSA-IR780 NPs could be an ideal photosensitizer for cancer phototherapy since they can remain easily dissolved and stable in aqueous solution, increase the tolerance of IR780 iodide and show photothermal and photodynamic effects both in vitro and in vivo. Disclosure The authors declare no conflict of interest.

Please cite this article in press as: Jiang C et al. Hydrophobic IR780 encapsulated in biodegradable human serum albumin nanoparticles for photothermal and photodynamic therapy. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.11.041

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Acknowledgements This paper was supported by the Natural Science Foundation of Jiangsu BK2011572 and BK2011539, the National Natural Science Foundation (No. 81202474, 30973651, 81171786), the Changzhou Special Project of Biotechnology and Biopharmacy (No. CE20105006) and the Science Bridges China-Changzhou Biotechnology and Pharmaceutical Technology Special Project (Grant Number: CE20105006). Appendix A. Figures with essential colour discrimination Certain figures in this article, particularly Figs. 1–8 and Scheme 1, are difficult to interpret in black and white. The full colour images can be found in the on-line version, at http://dx.doi.org/ 10.1016/j.actbio.2014.11.041. Appendix B. Supplementary data Total release rate of IR780 in HSA-IR780 NPs with or without laser during 24 h is shown in Fig. S.1. Photos of mice bearing CT26 tumor received various treatments as indicated are shown in Fig. S3. This material is available free of charge via the Internet. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actbio.2014.11.041. References [1] Yuan A, Wu J, Tang X, Zhao L, Xu F, Hu Y. Application of near-infrared dyes for tumor imaging, photothermal, and photodynamic therapies. J Pharm Sci 2013;102:6–28. [2] Zhang C, Wang S, Xiao J, Tan X, Zhu Y, Su Y, et al. Sentinel lymph node mapping by a near-infrared fluorescent heptamethine dye. Biomaterials 2010;31:1911–7. [3] Wilk KA, Zielinska K, Pietkiewicz J, Skolucka N, Choromanska A, Rossowska J, et al. Photo-oxidative action in MCF-7 cancer cells induced by hydrophobic cyanines loaded in biodegradable microemulsion-templated nanocapsules. Int J Oncol 2012;41:105–16.

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Please cite this article in press as: Jiang C et al. Hydrophobic IR780 encapsulated in biodegradable human serum albumin nanoparticles for photothermal and photodynamic therapy. Acta Biomater (2014), http://dx.doi.org/10.1016/j.actbio.2014.11.041

Hydrophobic IR780 encapsulated in biodegradable human serum albumin nanoparticles for photothermal and photodynamic therapy.

It has been reported that IR780 iodide, a near-infrared dye, can be applied for cancer imaging, photodynamic therapy (PDT) and photothermal therapy (P...
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