Journal of Photochemistry and Photobiology B: Biology 142 (2015) 212–219

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Photosensitizer-encapsulated amphiphilic chitosan derivative micelles: Photoactivity and enhancement of phototoxicity against human pancreatic cancer cells Huajie Li a,1, Zhong Yu b,1, Shuangping Wang a, Xi Long a, Li-Ming Zhang a, Zhaohua Zhu b, Liqun Yang a,⇑ a Institute of Polymer Science, School of Chemistry and Chemical Engineering, Key Laboratory of Designed Synthesis and Application of Polymer Material, Key Laboratory for Polymeric Composite and Functional Materials of Ministry of Education, Sun Yat-Sen University, Guangzhou 510275, China b Department of Gastroenterology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, China

a r t i c l e

i n f o

Article history: Received 1 May 2014 Received in revised form 10 September 2014 Accepted 26 October 2014 Available online 13 December 2014

a b s t r a c t Photosensitizer-encapsulated amphiphilic chitosan derivative (Photosan-DA-Chit) micelles with controlled photoactivity have been prepared using a simple self-assembly method in phosphate-buffered saline (pH 6.2). The fluorescence quantum yield and fluorescence lifetime of the Photosan-DA-Chit micelles were lower than those of free Photosan, which indicated that the micelles were suppressing the photoactivity of Photosan. However, upon incubation with human pancreatic cancer cells (i.e., Panc-1 cells), the Photosan-DA-Chit micelles showed higher fluorescence activity than free Photosan and generated higher levels of reactive oxygen species under laser illumination. The Photosan-DA-Chit micelles therefore exhibited strong phototoxicity, which led to significant levels of apoptosis in the Panc-1 cells. In the absence of light, however, the Photosan-DA-Chit micelles showed no cytotoxicity. These results indicated that they could be used as a potential photodynamic therapy in pancreatic cancer. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Pancreatic cancer is one of the most common forms of cancer, and the survival rate for this disease is very low. Most pancreatic cancer patients will die within the first year of diagnosis, with only 5% of patients surviving beyond 5 years [1]. Current therapeutic options for the treatment of pancreatic cancer are limited, and there is therefore an urgent need for the development of new therapeutic strategies for the treatment of this disease. Photodynamic therapy (PDT) was introduced as a clinical strategy for the treatment of cancer because of its fundamental specificity, selectivity, low lesion and administration of a non-toxic drug known as a photosensitizer [2], when it was approved for use in several North American, European and Asian countries in 1990s [3]. PDT was recently reported as a viable treatment option for pancreatic cancer [4–7]. During the PDT process, a photosensitizer is intravenously administered to the patient. Subsequent light activation of the targeted tissues leads to the generation of reactive oxygen species (ROS), including singlet oxygen (1O2), superoxide (O 2) and peroxide anions (O2 2 ), which oxidize subcellular organelles and several other biomolecules, leading to light-induced cell death ⇑ Corresponding author. Tel.: +86 20 84110934; fax: +86 20 84112245. 1

E-mail address: [email protected] (L. Yang). Both authors contributed equally to this work.

http://dx.doi.org/10.1016/j.jphotobiol.2014.10.020 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.

[8]. An ideal photosensitizer system should possess the following properties: (1) the ability to avoid damaging of normal tissues during systemic circulation (i.e., suppressible phototoxicity) [9]; (2) the ability to exhibit phototoxic activity when it reaches the tumor site [9,10]; and (3) the ability to specifically target tumor cells to allow for increasing concentrations of the photosensitizer to reach the tumor site with lower concentrations in normal tissue [11]. There are, however, several limitations associated with the clinical use of photosensitizers. For example, patients being treated with photosensitizers must carry sun glasses and avoid exposure to bright light after being injected photosensitizers and prior to being irradiate with laser to avoid phototoxicity to their eyes and skin. Furthermore, the quantum yield of the ROS formed in tumor tissues during PDT is generally low. Those are related to the photosensitizer’s lack of a tumor tissue targeting property [11,12]. Amphiphilic polysaccharide derivatives are potential drug carriers because of their good cellular compatibility and biodegradability properties [13,14]. Furthermore, amphiphilic polysaccharide derivatives can form nanoscale micelles, which can lead to an increase in tumor selectivity through enhanced permeability and retention effects, as well as being able to avoid rapid renal clearance and unwanted uptake by the reticuloendothelial system [9,12]. Encapsulated drugs could therefore be efficiently delivered to tumor tissues where they could be released in a sustained manner [11,12].

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Chitosan is a non-toxic, cationic polysaccharide that is being used in a growing number of biomedical applications because of its excellent biocompatibility and biodegradability properties, as well as its low immunogenecity and biological activity [15]. We previously reported an amphiphilic chitosan derivative conjugated with deoxycholic acid groups (DA-Chit, Fig. 1A), which exhibited good cytocompatibility towards human retinal pigment epithelial cells [16]. This DA-Chit derivative could be used to form nanoscale drug-loaded micelles using a self-assembly method, which would allow for the controlled release of ocular drugs. In this study, we have developed photosensitizer-encapsulated DA-Chit micelles with controlled photoactivity as a PDT for the treatment of pancreatic cancer. Photosan, which is also known as Photofrin, is a porphyrin oligomer bearing sodium carboxylate groups that has been approved for clinic use as an anionic watersoluble photosensitizer [3,17,18]. Photosan-encapsulated DA-Chit derivative (Photosan-DA-Chit) micelles were prepared using a simple self-assembly method in a phosphate-buffered solution (PBS, pH 6.2) (Fig. 1B). The mechanism for the formation of the micelles was investigated using UV–vis and fluorescence spectroscopy. The fluorescence-quenching properties of the Photosan in the Photosan-DA-Chit micelles were studied using steady-state fluorescence analysis including evaluation of the fluorescence quantum yield and fluorescence lifetime. The fluorescence activity, ROS generation and phototoxicity of the Photosan-DA-Chit micelles, as well as their ability to induce apoptosis, were assessed in human pancreatic cancer (Panc-1) cells. 2. Experimental 2.1. Materials Chitosan with a weight average molecular weight of 4.5  105 g/mol and the deacetylation degree of 90.0% was bought from Shanghai Bo’ao Biological Technology Co., Ltd (Shanghai, China). Following the methods used in our previous work [16], the DA-Chit derivatives were synthesized (Supplementary Data). Photosan (Porfimer sodium) was purchased as a freeze-dried injectable powder from SeeLab Inc. (Wesselburen, Germany). Rhodamine B was purchased from Acros Organics (Janssens Pharmaceutica, Belgium). RPMI 1640 culture medium and penicillin– streptomycin were purchased from Gibco Co. (Carlsbad, CA, USA), and fresh fetal calf serum was purchased from Hangzhou Jinuo Biomedical Co (Hangzhou, China). A 20 ,70 -dichlorofluorescein-diacetate (DCFH-DA) kit was purchased from the Beyotime Institute of Biotechnology (Shanghai, China). 2.2. Preparation of the Photosan-DA-Chit micelles The DA-Chit derivative (30.0 mg) was added to 15 mL of PBS (pH 6.2), and the resulting mixture was stirred at room temperature

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overnight to give a solution. Fifteen microliters of PBS (pH 6.2) containing Photosan (150 lg/mL) was then added to the DA-Chit derivative, and the resulting mixture was stirred at room temperature in the absence of light for 24 h. The mixture was then subjected to dialysis in PBS (pH 6.2) in the absence of light for 1 day to remove any un-encapsulated Photosan. A working curve for the Photosan concentration in PBS (pH 6.2) and a wavelength of 362 nm was used to determine the amount of Photosan in the solution outside of the dialysis bag (R2 = 0.998), which was analyzed by UV–vis spectrophotometry (UV-3150, Shimadzu, Japan). The amount of Photosan encapsulated in the micelles was calculated based on the reduction in the Photosan concentration. The Photosan encapsulating capacity was then calculated using Eq. (1).

Encapsulating capacity ð%Þ ¼ ½ðA  BÞ=C  100

ð1Þ

where A is the total weight of Photosan used, B is the weight of unencapsulated Photosan, and C is the weight of the DA-Chit derivative. Consequently, the Photosan encapsulating capacities of the Photosan-DA-Chit micelles were determined to be 5.4%. The UV– vis spectra of Photosan before and after encapsulation in the micelles were obtained in PBS (pH 6.2) at a Photosan concentration of 2 lg/mL. 2.3. Physicochemical properties of the Photosan-DA-Chit micelles The morphology of the Photosan-DA-Chit micelles was investigated using transmission electron microscopy (TEM). The hydrodynamic diameter distribution of the micelles was detected by dynamic light scattering analysis in PBS (pH 6.2). Detailed information pertaining to the analytical methods used in this study is provided in the Supplementary Data. The zeta potential of the micelles was measured in the distilled water using ZetaPALS (Brookhaven Instruments Corporation, Holtsville, NY, USA) at 25 °C. Each measurement was performed in triplicate. 2.4. Steady-state fluorescence measurements Steady-state fluorescence measurements were carried out on a Combined Fluorescence Lifetime and Steady State Spectrometer (FLSP920, Edinburgh Instruments Ltd., Edinburgh, UK). Fluorescence emission spectra were obtained with a maximum excitation wavelength of 395 nm over a scanning wavelength range of 550– 750 nm, using excitation and emission slits of 4 and 5 nm, respectively. 2.4.1. Measurement of fluorescence lifetime Fluorescence lifetime experiments were carried out based on the time-correlated single-photon counting technique [19]. Fluorescence was collected through a 615-nm emission filter with the excitation length of 405 nm and the slit width of 13 nm. A measurement time of 3–5 min was used to acquire high-quality data

Fig. 1. (A) Chemical structure of the DA-Chit derivative, and (B) preparation of the Photosan-DA-Chit micelles by self-assembly.

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with 5  104 counts. The fluorescence lifetime (s) was determined by an exponential model function illustrated in Eq. (2) [19].

RðtÞ ¼

n X Bi et=si

ð2Þ

i¼1

where R(t) is the fluorescence time response at t time, i is the fluorescence constituent, Bi and si are the fluorescence proportion and lifetime of the i fluorescence constituent. 2.4.2. Measurement of fluorescence quantum yield According to a previously published procedure [20], the fluorescence quantum yields of Photosan and the Photosan-DA-Chit micelles were measured using Eqs. (3) and (4).

I AS  IS A

ð3Þ

If ;k dk

ð4Þ

U ¼ US 

I¼

Z

k2 k1

where U, I and A present fluorescence quantum yield, integral fluorescence intensity and absorbance of samples, and US, IS and AS are fluorescence quantum yield, integral fluorescence intensity and absorbance of the reference. Rhodamine B was used as the reference, of which US is 0.31 [21]. Fluorescence emission spectra were obtained using the same experimental parameters as mentioned previously, and then I and IS values were integrated. The UV–vis absorption spectra were recorded from 300 to 500 nm, and the A and AS values were measured at k = 395 nm. 2.5. Cell culture The Panc-1 cell line was purchased from the American Type Culture Collection (Manassas, VA, USA) and cultured in an RPMI1640 complete medium containing penicillin 100 U/mL, streptomycin 100 mg/mL, and 10% fresh fetal calf serum. The cells were then transferred to an incubator with a 5% CO2 atmosphere and incubated at 37 °C. The cell density was (12)  106/mL. One passage was made every 3–4 days, and the logarithmic growth phase was used for the experiments. 2.6. Fluorescence activity of the Photosan-DA-Chit micelles in Panc-1 cells Panc-1 cells (2  105 per dish) were seeded onto cell culture dishes of 3.5 cm in diameter and cultured at 37 °C for 24 h. The cells were then washed three times with PBS (pH 7.4) and the medium was replaced with the fresh medium. Solutions of the Photosan-DA-Chit micelles and free Photosan with different Photosan concentrations were then added to the cells, which were incubated for 8 h. The Photosan concentrations were 0.5, 1.0, 2.0, 3.0, 4.0 ,6.0 and 8.0 lg/mL and matched between the groups. The cells were then washed three times with PBS (pH 7.4). Untreated cells were used as a control. The fluorescence intensity of Photosan was detected on an FACS Calibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) using excitation and emission filters of 488 and 670 nm. The resulting data were analyzed using Cell Quest software (Becton Dickinson, Franklin Lakes, NJ, USA). The level of available intracellular Photosan was determined based on the relative fluorescence intensity of the cells. All of the experiments were conducted in triplicate. Confocal microscope imaging studies were then performed on a Carl Zeiss LSM710 laser scanning confocal microscope (Carl Zeiss Far East Co., Ltd., Jena, Germany) equipped with argon (488 nm) and He–Ne (543 nm) lasers. Intracellular Photosan was then observed using confocal microscopy.

2.7. Detection of the ROS produced in Panc-1 cells ROS produced by the light treatment of the Panc-1 cells were detected using the dichlorofluorescein probe method [22]. Panc-1 cells (1  104 per well) were seeded onto 96-well plates and cultured for 24 h. The cells were then washed three times with PBS (pH 7.4) and the medium was replaced with fresh medium. Solutions of the Photosan-DA-Chit micelles and free Photosan at a Photosan concentration of 2 lg/mL were then added to the cells, which were incubation was carried out for 8 h. The light treatment experiments were performed on a Biolitec PDT 630 semiconductor laser therapeutic apparatus (CeramOptec GmbH Co, Siemensstrasse, Germany). Panc-1 cells were illuminated with the laser (k = 630 nm, power = 300 mW, light dose density = 5 J/cm2) for 8 min. The cells were then cultured for 1 h in the absence of light, incubated with DCFH-DA (10 lmol/L) for 30 min and then were washed with serum-free culture medium. The cells were observed using an Olympus IX71 fluorescence microscope (Tokyo, Japan) with excitation and emission filters at 488 and 525 nm, respectively. The fluorescence intensity of the 2’,7’-dichlorofluorescein in the cells was evaluated using an FACS Calibur flow cytometer with excitation and emission filters at 488 and 525 nm, respectively. The results of these experiments have been given as a percent relative to the oxidative stress of the control cells, which was set as 100%. All of the experiments were performed in triplicate. 2.8. Evaluation of phototoxicity in Panc-1 cells Panc-1 cells (1  104 per well) were seeded onto 96-well plates and were cultured for 24 h. The cells were then washed three times with PBS (pH 7.4) and the medium was replaced with the fresh medium. Solutions of the Photosan-DA-Chit micelles and free Photosan with different Photosan concentrations were then added to the cells and the resulting mixture were incubated for 8 h. Light treatment experiments were conducted under conditions as mentioned previously. After being cultured for 24 h in the absence of light, the relative survival rates of the cells were evaluated using a Cell Counting Kit-8 (CCK-8) assay [23]. The culture medium was replaced, and 10 lL of the CCK-8 reagent (Dojindo, Kumamoto, Japan) was added to each well. The cells were then incubated for 4 h and the absorbance values of the samples were measured at 450 nm using a SpectraMax M5 multi-function microplate reader (Molecular Devices, Silicon Valley, CA, USA). Cell viability (%) was calculated using Eq. (5). Dark toxicity was assessed using Panc-1 cells that were incubated with the Photosan-DA-Chit micelles and free Photosan but without laser light exposure.

Cell viability ð%Þ ¼ ðA450s =A450c Þ  100

ð5Þ

where A450-s and A450-c were obtained for the light treated and untreated cells, respectively. All experiments were conducted in triplicate. The Panc-1 cells undergoing the light treatment were washed with PBS (pH 7.4) before being fixed in 4% paraformaldehyde for 1 h. The cells were then washed three times with PBS (pH 7.4) and dried at room temperature under vacuum for 2 weeks. Images of Panc-1 cells were obtained using an ultra-depth three-dimensional microscope (VHX-1000C, Osaka, Japan). 2.9. Apoptosis assay Apoptosis of the Panc-1 cells was evaluated using flow cytometry. The apoptotic cell death mechanisms were investigated by evaluating caspase-3 protein activation using a western blot assay. Further details of these experiments have been provided in the Supplementary Data.

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2.10. Statistical analysis All statistical analyses were performed using the SPSS17.0 software. All of the data have been expressed as the mean ± standard deviation ð x  sÞ. The means of multiple samples were compared using one-way analysis of variance. Samples with two factors and multiple levels were analyzed using an analysis of the variance of factorial design, followed by Fisher’s LSD test or Student-Newman–Keuls post hoc tests. P < 0.05 was considered as statistically significant. 3. Results and discussion 3.1. Preparation and physicochemical properties of the Photosan-DAChit micelles Photosan contains a large number of sodium carboxylate groups and therefore exhibits good water-solubility. This compound could form micelles with the DA-Chit derivative through intermolecular interactions in PBS (pH 6.2). From the TEM image as shown in Fig. 2, it was observed that the Photosan-DA-Chit micelles had a spherical morphology with diameters in the range of 200– 300 nm. Dynamic light scattering analysis showed that the hydrodynamic diameter of the Photosan-DA-Chit micelles was similar to that of the DA-Chit micelles (Table 1). However, the zeta potential of the Photosan-DA-Chit micelles was smaller than that of the DAChit micelles (Table 1), which was attributed to an increase in the surface density of the negatively charged carboxylic groups (ACOO) on the micelles according to the literature [18]. 3.2. Formation mechanism of the Photosan-DA-Chit micelles The mechanism for the formation of the Photosan-DA-Chit micelles was investigated using UV–vis and fluorescence spectroscopy, and the results are summarized in Table 1. The UV–vis spectrum of free Photosan (Fig. 3A–a) contained absorption peaks at 364 nm as well as additional peaks in the range of 450–650 nm, which were characteristic of porphyrin rings of Photosan, including the Soret band and the Q-bands [18]. Interestingly, the Soret band of Photosan was found to be red shifted to 403 nm (Fig. 3A–b) after it had been encapsulated in the DA-Chit micelles. Fig. 3B shows the fluorescence emission spectra of free Photosan and the PhotosanDA-Chit micelles. The maximum emission wavelength of Photosan was also red shifted from 615 nm to 628 nm following its encapsulation in the micelles, which suggested that the DA-Chit micelles

Fig. 2. Hydrodynamics diameter distribution and TEM image of the Photosan- DAChit micelles.

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were having a significant impact on the spectroscopic properties of Photosan. Based on information from the literature [24–26], the red shifts observed in the UV–vis and fluorescence spectra of Photosan were attributed to the formation of hydrogen bonding interactions between the DA-Chit derivatives and Photosan in the micelles in PBS (pH 6.2). 3.3. Fluorescence quenching of Photosan in the Photosan-DA-Chit micelles Fig. 3C shows simulated fluorescence time responses with photon counts for free Photosan and the Photosan-DA-Chit micelles. The values shown in the figure were simulated by Reconvolution Fit of the assay model functions (Eq. (2)). This process was conducted using an experimental instrument response function (IRF) following the addition of Poissonian noise [19]. The results of this exercise implied that the fluorescence lifetime of Photosan became shorter following its encapsulation in the DA-Chit micelles (Table 1). The appearance of two fluorescence lifetime values was attributed to the Photosan being composed of various fluorescence oligo-porphyrin constituents. Furthermore, the fluorescence quantum yield (U) of Photosan was determined using Rhodamine B as a reference according to Eqs. (3) and (4) (Fig. S1). These results indicated that the U value for Photosan was reduced from 2.0  102 to 8.2  103 following its encapsulation in the DA-Chit micelles (Table 1). This shortening of the fluorescence lifetime and the reduction in the fluorescence quantum yield of Photosan implied that its fluorescence activity was being suppressed in the Photosan-DA-Chit micelles. The fluorescence-quenching properties of the DA-Chit micelles could lead to a reduction in the phototoxicity of Photosan towards normal tissues during its systemic circulation. It is noteworthy that the fluorescence-quenching properties of the Photosan-DA-Chit micelles were in good agreement with those of the polysaccharide/photosensitizer conjugate-based micelles [9,27]. 3.4. Fluorescence activity of Photosan in Panc-1 cells Although the encapsulation of Photosan in the DA-Chit micelles would suppress the fluorescence activity and cytotoxicity of Photosan towards normal tissues, this phenomenon could also lead to a decrease in the phototoxicity of Photosan at the tumor site. To make Photosan-encapsulated micelles that exhibit sufficiently high levels of phototoxicity towards tumor cells, it would therefore be necessary to enhance the fluorescence activity of Photosan. The fluorescence intensities of free Photosan and the Photosan-DA-Chit micelles were quantitatively estimated in the Panc-1 cells using a flow cytometry assay. As shown in Fig. 4A, the fluorescence intensity observed in cells incubated with the Photosan-DA-Chit micelles was significantly stronger than that observed in the cells incubated with free Photosan, by comparison with the control cells. Furthermore, concentration-dependent changes in the fluorescence intensities of the cells were quantitatively estimated (Fig. 4B), and it was found that the fluorescence intensity increased with increasing Photosan concentration. The fluorescence intensities of the cells in the Photosan-DA-Chit micelle group were generally two-fold higher than those in the free Photosan group. This result suggested that the Photosan-DA-Chit micelles could be used to enhance the fluorescence activity of Photosan in Panc-1 cells. This increase in the fluorescence activity of Photosan could be attributed to two different factors. First, the Photosan-DA-Chit micelles would strongly adhere to the Panc-1 cell membrane because of excellent cellular adhesion and cytocompatibility properties of the DA-Chit derivative [16], which would result in the increased cellular uptake of Photosan. This would suggest that the recovery of the fluorescence signal is related to the cellular

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Table 1 Hydrodynamic diameters, zeta potentials and photophysical parameters of the Photosan-DA-Chit micelles and free Photosan at a Photosan concentration of 2 lg/mL in PBS (pH 6.2). Samples

Hydrodynamic diameter (nm)

Zeta potential (mv)

Soret band (nm)

Emission wavelength (nm)

Lifetime (ns)

s1 Photosan Photosan-DA-Chit micelle DA-Chit micelle a

— 272 275

— 4.0 ± 0.7 11.9 ± 1.3

364 403 —

615, 676 628, 672 —

13.2 (80.3%) 11.4 (78.2%) —

U

s2 a

a

7.0 (19.7%) 1.3 (21.8%) —

2.0  102 a

8.2  103 a

—

The relative percentage of the fluorescence constituent.

Fig. 3. (A) UV–vis absorption spectra, (B) fluorescence emission spectra and (C) simulation of fluorescence lifetime assay of (a) free Photosan and (b) the Photosan-DA-Chit micelles in PBS (pH 6.2) at a Photosan concentration of 2 lg/mL for all samples, (c) IRF. (D) Residual functions for all evaluated time responses shown in panel C (the values of v2 for (a) free Photosan and (b) the DA-Chit-Photosan micelles were 1.0 and 1.2, respectively).

Fig. 4. (A) Histograms of flow cytometry of the Panc-1 cells incubated with free Photosan and the Photosan-DA-Chit micelles at a Photosan concentration of 0.5 lg/mL compared with the control. (B) Quantitative determination of fluorescence intensity of Photosan by flow cytometry with different Photosan concentrations in the Panc-1 cells (n = 5, P < 0.05, significant statistical difference between the different groups of free Photosan and the Photosan-DA-Chit micelles, and within the same group for different Photosan concentrations). (C) Laser confocal microscope images of Panc-1 cells following uptake of (a) free Photosan and (b) the Photosan-DA-Chit micelles (scale bar: 20 lm, I: fluorescence images, II: optical images; All Photosan concentrations were maintained at 8 lg/mL).

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Fig. 5. (A) Detection of ROS produced in Panc-1 cells by fluorescence microscopy with the fluorescent DCFH-DA probe following 8 h of incubation with (a) free Photosan, (b) the Photosan-DA-Chit micelles (40). (B) Quantitative determination of ROS produced in Panc-1 cells by flow cytometry (n = 3, ⁄P < 0.05, compared with the free Photosan group). All Photosan concentrations were maintained at 2 lg/mL. Panc-1 cells treated with the different Photosan samples were incubated with DCFH-DA (10 lmol/L) for 30 min following light treatment (k = 630 nm, power = 300 mW, light dose density = 5 J/cm2, 8 min).

concentration effect of Photosan. Second, the observed recovery in the fluorescence activity could be attributed to the release of Photosan from the micelles, as shown in our previous work [16]. Confocal laser scanning microscopy was used to examine the fluorescence intensity of Photosan in Panc-1 cells. This analysis revealed that the cells treated with the Photosan-DA-Chit micelles (Fig. 4C-b-I) contained larger and more intense regions of red fluorescence than those treated with free Photosan (Fig. 4C–a-I). This result was in agreement with the results of the flow cytometry analysis, and further confirmed that the Photosan-DA-Chit micelles could enhance the fluorescence activity of Photosan in Pan-1 cells.

same order as the cells incubated with different Photosan samples (Fig. 5A). Quantitative flow cytometric analysis confirmed that the fluorescence intensity was significantly stronger in the cells treated with the Photosan-DA-Chit micelles than those treated with free Photosan, compared with the control group (Fig. 5B).The fluorescence intensity observed in the cells treated with the Photosan-DA-Chit micelles was about three-fold higher than that of the cells treated with free Photosan. These results strongly suggested that the Photosan-DA-Chit micelles could deliver Photosan to the Panc-1 cells and consequently produce higher levels of ROS compared with free Photosan.

3.5. Efficiency of ROS generation in Panc-1 cells

3.6. Enhancement of phototoxicity in Panc-1 cells

The efficiency of light-induced cell death in PDT is dependent on the formation of ROS to achieve a high level of photochemical oxidation [8,28]. With this in mind, fluorescence microscopy and flow cytometry were used to measure the formation of ROS in Panc-1 cells. Following 8 h of incubation with the Photosan-DAChit micelles or free Photosan, the Panc-1 cells were subjected to the light treatment process. The cells were incubated with a fluorescent DCFH-DA probe for 30 min to evaluate the formation of ROS. DCFH-DA is transported across the cell membrane and deacetylated by esterases to form 20 ,70 -dichlorofluorescein in the presence of ROS, which emits green fluorescence upon illumination with a laser [22]. It was assumed that the amount of ROS produced in the cells would be dependent on the fluorescence activity of the Photosan in the cells. Based on the aforementioned result, the fluorescence intensity of free Photosan was lower than that of the PhotosanDA-Chit micelles in the cells. As expected, the green fluorescence intensity of the DCFH-DA-detected ROS was observed to have the

The phototoxicity of the Photosan-DA-Chit micelles was evaluated in Panc-1 cells both with and without light treatment, and the results were compared with those achieved with free Photosan. After 8 h of incubation with the Photosan-DA-Chit micelles or free Photosan, the Panc-1 cells were irradiated with laser light. The cells were then incubated for 24 h in the absence of light, and the cell viability was evaluated using a CCK-8 assay (Fig. 6A). No significant differences were observed in the dark cytotoxicity when the Panc-1 cells were treated with Photosan-DA-Chit micelles or free Photosan in the absence of laser light illumination at Photosan concentrations less than 8 lg/mL. However, the cell viability decreased dramatically with increasing in the Photosan concentration when the cells were irradiated with laser light, which suggested that the Photosan was strongly phototoxic against Panc-1 cells. Importantly, the phototoxicity of the Photosan-DA-Chit micelles was significantly higher than that of free Photosan over all the concentrations tested. Since ROS is believed to be the main cytotoxic agent produced by laser illumination [8,18,28], the

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Fig. 6. (A) Phototoxicity (solid lines) and dark-toxicity (broken lines) of the Photosan-DA-Chit micelles and free Photosan with different Photosan concentrations (n = 3, P < 0.05). (B) Ultra-depth three-dimensional images of Panc-1 cells incubated with different samples following light treatment: (a) the control group, (b) the free Photosan group, (c) the Photosan-DA-Chit micelle group (scale bar: 10 lm). Light treatment conditions were k = 630 nm, power = 300 mW, light dose density = 5 J/cm2, irradiation time = 8 min.

Fig. 7. Western blot analysis of the caspase-3 activation in Panc-1 cells: (a) the control group, (b) the free Photosan group, (c) the Photosan-DA-Chit micelle group. All Photosan concentrations were maintained at 2 lg/mL. Light treatment conditions were k = 630 nm, power = 300 mW, light dose density = 5 J/cm2, irradiation time = 8 min (n = 3, ⁄P < 0.05, compared with the free Photosan group).

enhanced phototoxicity of the Photosan-DA-Chit micelles relative to free Photosan is most likely related to the larger amount of ROS being generated by the Photosan-DA-Chit micelles in the Panc-1 cells (Fig. 5). Ultra-depth three-dimensional microscopy was used to monitor changes in the morphological features of the Panc-1 cells following the light treatment process (Fig. 6B). The results of this analysis revealed differences in the morphologies of the cells belonging to the free Photosan- and Photosan-DA-Chit micelle-treated groups, compared with the control group (Fig. 6B–a). The free Photosantreated cells underwent a gradual shrinkage whilst maintaining their pseudopodial structures (Fig. 6B–b). In contrast, the cells treated with the Photosan-DA-Chit micelles shrank significantly and experienced membrane damage, which resulted in the disappearance of the initial shape of the cells (Fig. 6B–c). Taken together, the results of the CCK-8 assay and the morphological analysis implied that the Photosan-DA-Chit micelles exhibited stronger phototoxicity towards Panc-1 cells than free Photosan, and that they could therefore be used as a potential PDT for the treatment of pancreatic cancer.

3.7. Enhancement of apoptosis of Panc-1 cells induced by the phototoxicity The death mechanism of the Panc-1 cells induced by the phototoxicity was evaluated using flow cytometry (Fig. S2A). The result indicated that the number of cells undergoing apoptosis was much higher than the number of cells undergoing necrosis, which suggested that apoptosis plays a leading role in the cell death process induced by Photosan phototoxicity. This was in agreement with the result of previous work [29]. Furthermore, the level of apoptosis in the Panc-1 cells treated with Photosan-DA-Chit micelles was higher than that of in the Panc-1 cells treated with free Photosan because of the higher phototoxicity of the Photosan-DA-Chit micelles. This result indicated that the level of apoptosis in Panc1 cells was enhanced by the phototoxicity of the Photosan-DA-Chit micelles. Caspases-3 is strongly associated with apoptosis and is commonly activated by numerous death signals, where it cleaves a variety of important cellular proteins [29–31]. For this reason, caspase-3 activation was evaluated in the Panc-1 cells using western

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blot analysis, as shown in Fig. 7. The cells that underwent the light treatment were able to express the caspase-3 protein, and the level of caspase-3 activation was higher in the Panc-1 cells treated with the Photosan-DA-Chit micelles than those treated with free Photosan. This result provided further confirmation that the Photosan-DA-Chit micelles were enhancing the apoptosis of the Panc-1 cells through induced phototoxicity. 4. Conclusions Photosan-DA-Chit micelles were prepared by the self-assembly of Photosan and a DA-Chit derivative in PBS (pH 6.2). The photoactivity of Photosan was suppressed when it was encapsulated in the DA-Chit micelles. However, the Photosan-DA-Chit micelles showed high levels of fluorescence activity and ROS generation than free Photosan in Panc-1 cells following laser light illumination, which resulted in strong phototoxicity. The phototoxicity of the Photosan-DA-Chit micelles led to a high level of apoptosis in the Panc-1 cells. In contrast, no dark cytotoxicity was observed from the Photosan-DA-Chit micelles or free Photosan. These results demonstrate that the Photosan DA-Chit micelles developed in the current study could potentially be use as photodynamic therapy for the treatment of pancreatic cancer. Acknowledgements This work was supported by the National Natural Science Foundation of China (21244005, 20974130), and the Science and Technology Planning Project of Guangdong Province, China (2012B091100457, 2012B091100356). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jphotobiol.2014. 10.020. References [1] W. Hartwig, J. Werner, D. Jager, J. Debus, M.W. Buchler, Improvement of surgical results for pancreatic cancer, Lancet Oncol. 14 (2013) e476–e485. [2] C.A. Robertson, D.H. Evans, H. Abrahamse, Photodynamic therapy (PDT): A short review on cellular mechanisms and cancer research applications for PDT, J. Photochem. Photobiol. B-Biol. 96 (2009) 1–8. [3] J.P. Celi, B.Q. Spring, I. Rizvi, C.L. Evans, K.S. Samkoe, S. Verma, B.W. Pogue, T. Hasan, Imaging and photodynamic therapy: mechanisms, monitoring, and optimization, Chem. Rev. 110 (2010) 2795–2838. [4] K.S. Samkoe, A. Chen, I. Rizvi, J.A. O’Hara, P.J. Hoopes, S.P. Pereira, T. Hasan, B.W. Pogue, Imaging tumor variation in response to photodynamic therapy in pancreatic cancer xenograft models, Int. J. Radiat. Oncol. Biol. Phys. 76 (2010) 251–259. [5] S.G. Bown, A.Z. Rogowska, D.E. Whitelaw, W.R. Lees, L.B. Lovat, P. Ripley, L. Jones, P. Wyld, A. Gillams, A.W.R. Hatfield, Photodynamic therapy for cancer of the pancreas, Gut 50 (2002) 549–557. [6] D. Mitton, R. Ackroyd, A brief overview of photodynamic therapy in Europe, Photodiagn. Photody. Ther. 5 (2008) 103–111. [7] L. Ayaru, S.G. Bown, S.P. Pereira, Photodynamic therapy for pancreatic carcinoma: experimental and clinical studies, Photodiagn. Photody. Ther. 1 (2004) 145–155. [8] N. Nishiyama, Y. Morimoto, W.D. Jang, K. Kataoka, Design and development of dendrimer photosensitizer-incorporated polymericmicelles for enhanced photodynamic therapy, Adv. Drug Deliver. Rev. 61 (2009) 327–338.

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