Journal of Photochemistry and Photobiology B: Biology 140 (2014) 49–56
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Anticancer efficacy of photodynamic therapy with hematoporphyrin-modified, doxorubicin-loaded nanoparticles in liver cancer Ji-Eun Chang a, In-Soo Yoon b, Ping-Li Sun a,c, Eunjue Yi a, Sanghoon Jheon a,d, Chang-Koo Shim e,⇑ a
Department of Thoracic and Cardiovascular Surgery, Seoul National University Bundang Hospital, Seongnam-Si, Gyeonggi-do, Republic of Korea College of Pharmacy and Natural Medicine Research Institute, Mokpo National University, Jeonnam, Republic of Korea Department of Pathology, Seoul National University Bundang Hospital, Seongnam-Si, Gyeonggi-do, Republic of Korea d Department of Thoracic and Cardiovascular Surgery, Seoul National University College of Medicine, Seoul, Republic of Korea e Department of Pharmaceutics, College of Pharmacy, Seoul National University, Seoul, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 11 April 2014 Received in revised form 23 June 2014 Accepted 10 July 2014 Available online 24 July 2014 Keywords: Photodynamic therapy Hematoporphyrin Doxorubicin Nanoparticles Liver cancer
a b s t r a c t Photodynamic therapy (PDT) in combination with chemotherapy has great potential for cancer treatment. However, there have been very few attempts to developing cancer-targeted co-delivered systems of photosensitizers and anticancer drugs. We developed hematoporphyrin (HP)-modified doxorubicin (DOX)-loaded nanoparticles (HP-NPs) to improve the therapeutic effect of PDT in treating liver cancer. HP is not only a ligand for low density lipoprotein (LDL) receptors on the hepatoma cells but also a well-known photosensitizer for PDT. In vitro phototoxicity in HepG2 (human hepatocellular carcinoma) cells and in vivo anticancer efficacy in HepG2 tumor-bearing mice of free HP and HP-NPs were evaluated. The in vitro phototoxicity in HepG2 cells determined by MTT assay, annexin V-FITC staining and FACS analysis was enhanced in HP-NPs compared with free HP. Furthermore, compared with free HP-based PDT, in vivo anticancer efficacy in HepG2 tumor-bearing mice was markedly improved by HP-NPs-based PDT. Moreover, in both cases, the therapeutic effect was increased according to the irradiation time and number of PDT sessions. In conclusion, the HP-NPs prepared in this study represent a potentially effective co-delivery system of photosensitizer (HP) and anticancer drug (DOX) which improved the effects of PDT in liver cancer. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Photodynamic therapy (PDT) using photosensitizers has emerged as an effective therapeutic option for various cancers [1]. Upon irradiation with the proper wavelength of light, photosensitizers transfer energy to molecular oxygen, leading to the production of highly reactive singlet oxygen, which can damage adjacent tissues [2,3]. Moreover, photosensitizers remain effective even if they are conjugated to other molecules [4]. Among the various photosensitizers, hematoporphyrin (HP) is known as the first photosensitizer used in PDT [5]. However, the clinical application of PDT has been often hindered by the limited aqueous solubility and tumor specificity of photosensitizers [6]. Therefore, to over⇑ Corresponding author. Address: College of Pharmacy, Seoul National University, 599 Gwanangno, Gwanak gu, Seoul, 151–742, Republic of Korea. Tel.: +82 2 880 7873; fax: +82 2 888 5969. E-mail address: [email protected] (C.-K. Shim). http://dx.doi.org/10.1016/j.jphotobiol.2014.07.005 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.
come these limitations, various drug delivery systems such as lipid- or polymer-based nanoparticles and polymer conjugates have been extensively explored for the cancer-specific delivery of photosensitizers [7–9]. PDT in combination with chemotherapy has great potential for cancer treatment, because it may permit low doses of photosensitizer and anticancer drug, thereby diminishing undesirable sideeffects [10]. Several studies have reported that the combination of PDT and chemotherapy could produce an additive or synergistic anticancer efficacy in vitro and in vivo [10–13]. To date, vast number of investigations have focused on developing cancer-targeted drug delivery systems for either photosensitizers or anticancer drugs alone. However, there have been very few studies on the co-delivery of photosensitizers and anticancer drugs in cancer. Among various cancer-targeted drug delivery systems, we previously developed HP-modified bovine serum albumin nanoparticles (HP-NPs) for the cancer targeting of doxorubicin (DOX) [14]. DOX is one of the most widely used anticancer drugs for the
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treatment of lymphoma, sarcomas, carcinomas, and melanoma [15]. However, its therapeutic effects have been limited due to serious side effects such as dose-dependent irreversible cardiomyopathy and congestive heart failure [15]. HP is a ligand binding to low-density lipoprotein (LDL) receptors on tumor cell membrane [16], and albumin nanoparticles could accumulate more in the cancer without targeting [17]. The HP-NPs enhanced the delivery of DOX to liver tumors in rats and reduced the distribution of DOX to the heart, suggesting their usefulness as a cancer-specific delivery system for DOX [14]. Since HP is also a widely used photosensitizer, we hypothesized that the HP-NPs could combine HP-based PDT and DOX-based chemotherapy with enhanced cancer specificity, potentially leading to an improved anticancer efficacy and reduced toxicity. There are very few reports on photosensitizermodified cancer-specific nanoparticulate systems for PDT in combination with chemotherapy. Herein, we report on PDT using HP-NPs for cancer treatment. The HP-NPs were prepared as previously reported, and the in vitro phototoxicity of PDT using HP-NPs or free HP with varying light irradiation conditions in HepG2 (human hepatocellular carcinoma) cells was evaluated by MTT assay, annexin V-FITC staining and FACS analysis. Subsequently, the in vivo anticancer efficacy of PDT using HP-NPs or free HP with varying light irradiation conditions in HepG2 tumor-bearing mice was further evaluated in terms of tumor growth and immunohistology. 2. Materials and methods 2.1. Materials Doxorubicin hydrochloride (DOX), bovine serum albumin (BSA, purity P98%), glutaraldehyde 8% solution, N-hydroxysuccinimide (NHS, 98%), fetal bovine serum (FBS), phosphate buffered saline (PBS), penicillin–streptomycin, thiazolyl blue tetrazolium bromide (MTT) and Dulbecco’s Modified Eagle medium (DMEM) were obtained from Sigma–Aldrich Co. (St. Louis, MO, USA). Hematoporphyrin dihydrochloride (HP) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). N,N-dicyclohexylcarbodiimide (DCC, purity >99%) was purchased from Fluka (Tokyo, Japan). Ethanol, dimethyl sulfoxide (DMSO), acetonitrile, and all other reagents were obtained from Fisher Scientific Korea Ltd. (Seoul, Korea). 2.2. Preparation and characterization of HP-NPs HP-NPs were prepared in the same way as previously reported [14]. Briefly, first, DOX-loaded nanoparticles (NPs) were prepared by a desolvation technique. 10 mg of DOX and 200 mg of BSA were dissolved in 10 mL of double-distilled water (DDW). The solution was then stirred and ethanol was added under constant stirring at room temperature. For the desolvation process, 8% glutaraldehyde solution was added as a crosslinking agent. The resulting nanoparticles were purified by centrifugation (Centrifuge 5415R, Eppendorf AG Hamburg, Germany) at 16,000g for 12 min; then redispersion in DDW was repeated 3 times to eliminate the ethanol and glutaraldehyde. Secondly, HP was conjugated to prepare NPs. HP, NHS and DCC were added to DMSO, and the mixture was filtered. HP-NHS complex was isolated by mixing the filtrate with excess diethyl ether and washed with methanol. This step was repeated once. The sample was lyophilized and stored at 20 °C. HP-NHS was dissolved in an aqueous suspension of NPs and stirred overnight. Centrifugation and redispersion in DDW were repeated 3 times. The mean particle size and zeta potential of prepared HP-NPs were measured using an electrophoretic light scattering spectrophotometer (ELS-8000, Otsuka Electronics Co., Ltd. Osaka, Japan).
The morphology of HP-NPs was then observed by field-emission scanning electron microscopy (FE-SEM) (SUPRA 55VP, Carl Zeiss, Oberkochen, Germany) and transmission electron microscopy (TEM) (JEM 1010, JEOL, Tokyo, Japan). 2.3. In vitro phototoxicity HepG2 (human hepatocellular carcinoma, ATCC, Manassas, VA, USA) cells were seeded in 12-well cell culture plates at a density of 1 105 cells/well in DMEM medium containing 10% (v/v) FBS and 1% (w/v) penicillin–streptomycin and incubated for 1 day at 37 °C in a humidified 5% CO2 and 95% air atmosphere. After cell attachment, the medium was washed off and replaced with serum-free media containing free HP or HP-NPs (2 lM as HP). After 4-h incubation, the cells were washed twice with cold PBS to eliminate the remaining drugs, and then fresh culture medium was added. For MTT assay, the cells were illuminated once or twice with a PDT laser (Diomed Inc, Andover, MA, USA) (630 nm, 400 mW/cm2) for 0, 5, 10, 25, 35, and 50 s (0, 2, 4, 10, 14, and 20 J/cm2, respectively.). For the double irradiation, the interval time was 2 h and the conditions of first and second irradiation session were the same. The irradiated cells were incubated for 1 day, and cell viability was performed by MTT assay [18]. The cells were incubated for 4 h with 50 lL/well of medium containing 1 mg/mL of MTT agent, and 500 lL/well of DMSO was added. The plates were shaken gently, and absorbance was read at a wavelength of 540 nm using an absorbance microplate reader (Spectramax plus384, Molecular Devices Corporation, Sunnyvale, CA, USA). Cytotoxicity was represented as the percentage of the control. All the experiments were performed in triplicate. For microscopic analysis, the cells were illuminated with a PDT laser (630 nm, 400 mW/cm2) for 50 s (20 J/cm2). The irradiated cells were incubated for 1 day and washed with cold PBS twice. Apoptotic cells were identified by using an annexin V-FITC fluorescence microscope kits (BD bioscience, San Jose, CA, USA) [19]. The cells were stained with both annexin V-FITC and DAPI, and the apoptotic cells were examined by light microscope (Axioskop 40, Carl Zeiss, Gottingen, Germany) with a X-Cite 120Q excitation light source (Lumen Dynamics Group Inc., Mississauga, Ontario, Canada). For flow cytometry analysis, the cells were illuminated single or double with a PDT laser (630 nm, 400 mW/cm2) for 25 or 50 s (10 or 20 J/cm2). For the double irradiation, the interval time was 2 h and the conditions of first and second irradiation were the same. The irradiated cells were incubated for 1 day and washed with cold PBS twice. Then, the cells were stained with both annexin V-FITC and PI, and the apoptotic cells were measured by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA, USA) using CellQuest software (BD Immunocytometry Systems, Mountain View, CA, USA). 2.4. In vivo anticancer efficacy in tumor bearing mice All animal study protocols were approved by the Institutional Animal Care and Use Committee of Seoul National University Bundang Hospital. BALB/C male nude mice (6–7 weeks, 20–22 g) were purchased from Orientbio (Kyungki-Do, Korea). 1 106 HepG2 cells in 0.1 mL DMEM medium were injected subcutaneously into the left flanks of the mice. When the tumors grew to approximately 200 mm3 in volume, the mice were randomly divided into 12 groups (n = 5 each) and injected with PBS, free HP or HP-NPs once or twice (on day 0 and 7). Each injection was made into a tail vein at an HP dose of 2 mg/kg in PBS. At 24 h post-injection, tumor sites were irradiated with a PDT laser (630 nm, 400 mW/cm2) once or
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twice for 250 or 500 s (100 or 200 J/cm2). The conditions for each drug were as follows; single injection without PDT, single injection with single PDT (100 J/cm2), single injection with single PDT (200 J/cm2), and double injection with double PDT (200 J/cm2). The tumor volume of each mouse was monitored for 4 weeks to evaluate the therapeutic efficacy. Tumors were measured with a caliper, and the tumor volume (mm3) was calculated as (length width2)/2. Two weeks after PDT treatment, the tumor tissue of each group was isolated, fixed in 10% formalin, embedded in paraffin, and stained with Hematoxylin and Eosin (H&E) to evaluate the induction of necrosis or apoptosis. 2.5. Statistical analysis All data were presented as the mean ± standard deviation. The statistical significance between the groups was determined by using the Student’s t-test for 2 groups or one-way ANOVA with Tukey’s post hoc test for more than 3 groups. It was considered significant when p values were less than 0.05. 3. Results 3.1. Characterization of HP-NPs Fig. 1 shows the size distribution, TEM and FE-SEM images of HP-NPs. The prepared nanoparticle formulations were submicron polydisperse systems having moderate size distribution and their average particle sizes were 350 ± 15 nm (Fig. 1a). The zeta potential was 21.1 ± 1.1 mV. In TEM and FE-SEM images, smooth and spherical particles were observed (Fig. 1b). 3.2. In vitro phototoxicity In vitro viability of HepG2 cells after 4-h incubation with PBS, free HP and HP-NPs followed by single or double light irradiation for 0, 10, 15, 25, 35, or 50 s is shown in Fig. 2. No significant change in cell viability was observed in the control (PBS) group with or
Fig. 2. In vitro viability of HepG2 cells after 4-h incubation with PBS, free HP and HP-NPs followed by single (PBS, d; free HP, .; HP-NPs, j) or double (PBS 2, s; free HP 2, 4; HP-NPs 2, h) light irradiation for 0, 10, 15, 25, 35, or 50 s (n = 3).
without light irradiation and free HP group without light irradiation, while significant reduction in cell viability was observed in the free HP group with light irradiation. This result is consistent with those of a previous study which reported the utility of HP as a photosensitizer [5]. In the free HP group, the cell viability after double light irradiation was significantly lower than after single light irradiation, and it tended to be reduced as the irradiation time increased. This result suggests that the in vitro phototoxicity of HP may depend on the extent of exposure to light irradiation. Moreover, under the same irradiation conditions, HP-NPs showed higher cytotoxicity compared with free HP. Empty nanoparticles showed no cytotoxicity at all (data not shown). In vitro annexin V-FITC staining of apoptotic HepG2 cells after 4-h incubation with PBS, free HP and HP-NPs followed by single light irradiation for 0 or 20 J/cm2 is shown in Fig. 3. In the control (PBS) group with or without light irradiation and free HP group without light irradiation, negligible green fluorescence (indicating apoptotic cells) intensity was observed, while significant green
Fig. 1. Size distribution (a) and TEM (left) and FE-SEM (right) (b) images of HP-NPs.
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Fig. 3. In vitro annexin V-FITC staining of apoptotic HepG2 cells after 4-h incubation with PBS, free HP and HP-NPs followed by single light irradiation for 0 or 20 J/cm2. DAPI stain (blue, first column) and annexin V-FITC stain (green, second column) indicate nucleus and apoptotic cells, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
fluorescence was observed in the free HP group with light irradiation. Moreover, the green fluorescence intensity of HP-NPs group was higher than that of free HP groups in the same light irradiation conditions. These results were in agreement with the cell viability data (Fig. 2). FACS analysis of HepG2 cells after 4-h incubation with PBS, free HP and HP-NPs followed by single or double light irradiation for 25 or 50 s is shown in Fig. 4. The combination staining of annexin V and PI depicts apoptotic stages of the cells [20]. In the control (PBS) group, negligible annexin V and PI fluorescence was observed, while significant fluorescence was observed in both free HP and HP-NPs groups. Under the same irradiation conditions, the fluorescence intensity of HP-NPs group was higher than that of free HP groups. These results were consistent with the cell viability and microscope analysis data (Figs. 2 and 3). Furthermore, under the same irradiation conditions, the portion of late apoptosis was increased in the HP-NPs group. Taken together, compared with free HP, the cellular phototoxicity determined by MTT assay, microscopy and FACS analysis was enhanced in HP-NPs (Figs. 2–4). These results may be attributed
to the enhanced cellular uptake of HP-NPs and/or the cytotoxicity of DOX itself [14]. It was reported that HP-loaded polymeric micelles or solid lipid nanoparticles enhanced the cellular uptake of HP in A549 or KB cells, respectively [21,22]. 3.3. In vivo anticancer efficacy in tumor bearing mice In vivo tumor growth in HepG2 tumor-bearing mice during 4 weeks after single or double (on day 0 and 7) intravenous injection of PBS, free HP and HP-NPs with or without 100 or 200 J/cm2 light irradiation 1 day after each injection is shown in Fig. 5. The tumor growth of the free HP group with light irradiation was significantly lower than that of the control (PBS) group with or without light irradiation and free HP group without light irradiation, indicating the anticancer efficacy of HP-based PDT. Moreover, in the same irradiation conditions, the tumor growth of HP-NPs was significantly lower than that of the free HP group, which was consistent with the in vitro phototoxicity studies (Figs. 2–4). Moreover, within each free HP or HP-NPs group, the antitumor efficacy was increased according to irradiation time and the
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Fig. 4. FACS analysis of HepG2 cells after 4-h incubation with PBS, free HP and HP-NPs followed by single or double light irradiation for 25 or 50 s. Both annexin V and PI negative cells are considered undamaged; annexin V positive and PI negative cells are early apoptotic; both annexin V and PI positive cells are late apoptotic; and annexin V negative and PI positive cells are either late apoptotic or necrotic.
Fig. 5. In vivo tumor growth in HepG2 tumor-bearing mice during 4 weeks after single (+) or double (++) (on day 0 and 7) intravenous injection of PBS, free HP and HP-NPs with or without 100 or 200 J/cm2 light irradiation 1 day after each injection (n = 4).
number of PDT session. It was consistent with the in vitro cell viability study (Fig. 4) showing that the in vitro phototoxicity of free HP and HP-NPs may depend on the extent of exposure to light irradiation. Images of tumor tissues in HepG2 tumor-bearing mice before PDT and on day 1, 3, 8, and 15 after double (on day 0 and 7) intravenous injection of PBS, free HP and HP-NPs with 200 J/cm2 light irradiation 1 day after each injection (on day 1 and 8) are shown in Fig. 6. The day after PDT, HP-NPs-treated mice showed
a hemorrhage compartment where the laser had been delivered and severe necrosis was noted. In the free HP treated group, tumor necrosis developed more slowly than in the HP-NPs treated groups. Moreover, H&E staining of tumor tissues in HepG2 tumor-bearing mice on day 7 and 14 after double (on day 0 and 7) intravenous injection of PBS, free HP and HP-NPs with 200 J/cm2 light irradiation on 1 day after each injection (on day 1 and 8) is shown in Fig. 7. Most tumor cells were severely damaged in mice treated with HP-NPs, while tumor cell death was incomplete in mice treated with PBS or free HP. Based on the morphologies of tumor tissues, the tumor growth of the three groups was in the following order: control (PBS) > free HP > HP-NPs, which was consistent with the in vivo tumor volume data (Fig. 5). Taken together, compared with free HP-based PDT, in vivo anticancer efficacy was markedly improved by HP-NPs-based PDT (Figs. 5–7). These results may be attributed to the in vivo tumor specificity of HP-NPs and/or the cytotoxicity of DOX itself [14]. 4. Discussion In this study, we developed photosensitizer (HP)-modified, anticancer agent (DOX)-loaded nanoparticles to improve the therapeutic efficacy of PDT in treating liver cancer. PDT is an ideal local cancer treatment using tumor tissue-penetrating lasers after the administration of tumor selective photosensitizers [23]. To increase the targeting ability of photosensitizers for the tumor site, we selected nanoparticles as a targeting carrier. Nanoparticles are ideal cancer targeting carriers because they have both passive and active targeting ability [24]. For passive targeting, nano-sized particles are easily uptaken by the tumor vessels which are very leaky
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Fig. 6. Images of tumor tissues in HepG2 tumor-bearing mice before treatment and 1, 3, 8, and 15 days after double (on day 0 and 7) intravenous injection of PBS, free HP and HP-NPs with 200 J/cm2 light irradiation 1 day after each injection (on day 1 and 8).
Fig. 7. H&E staining of tumor tissues in HepG2 tumor-bearing mice on day 7 and 14, after double (on day 0 and 7) intravenous injection of PBS, free HP and HP-NPs with 200 J/cm2 light irradiation 1 day after each injection (on day 1 and 8).
and this is called EPR (enhanced permeability and retention) effect [25]. Furthermore, for active targeting, it is easy to conjugate cancer targeting moieties such as photosensitizers on the surface of the nanoparticles. Hematoporphyrin is not only a targeting ligand for LDL receptors which is overexpressed on hepatoma cells but is also a well-known photosensitizer for PDT [14]. Doxorubicin which is a widely used antineoplastic agent used for liver cancer [26] was selected for the combination therapy with chemotherapy and PDT. In a previous study, we reported that HP-modified, DOX-loaded nanoparticles (HP-NPs) significantly enhanced the targeting ability for liver cancer sites, with fewer adverse reactions in normal tissues [14]. The HP-NPs were successfully prepared by the desolvation method and characterized in terms of particle size distribution and morphology (Fig. 1). To investigate the PDT efficacy of prepared HP-NPs, we performed both in vitro and in vivo experiments with various conditions of laser irradiation. The wavelength was fixed at 630 nm which was suitable for hematoporphyrin [1] and the output power was set at 400 mW/cm2 in order not to damage any tissues not pretreated with photosensitizers. When the output
power exceeded 400 mW/cm2, the cells and the skin of the animals were damaged by the irradiation itself. Furthermore, the distance from the PDT fiber to the cells or the tumor sites of the animals was an extremely important factor which influenced the effects of the PDT. We tested several pilot studies to find out the most effective distance, and 1 cm was set as the optimal distance. For in vitro studies, HP concentration was determined to be 2 lM at which there is no cytotoxicity without light irradiation. In both in vitro and in vivo studies, HP-NPs were more efficient than free HP under the same laser irradiation conditions. The reasons are considered in the following ways; first, nano-sized formulation stimulated the accumulation of the drugs into the tumor site by passive diffusion, secondly, hematoporphyrin played a pivotal role as a cancer targeting ligand, finally, the dual effects of PDT and chemotherapy with doxorubicin significantly enhanced the anticancer efficacy. Zhou et al. [27] and Hwang et al. [28] also studied about combination of chemotherapy and photodynamic therapy in cancer. The phototoxicity increased according to the irradiation time and the number of PDT sessions in both free HP and HP-NPs. In
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our preliminary study, the free HP treated group showed no antitumor efficacy at lower irradiation doses (less than 50 J/cm2), while HP-NPs treated group showed therapeutic effects under the same conditions (unpublished data). Thus, it appears that the anti-tumor efficacy of HP may depend more on its formulation than irradiation conditions. However, the exact relative contribution of each factor, i.e. formulation vs. irradiation condition, to the anti-tumor efficacy of HP cannot be clarified based only on the present data, which require further investigation. Many researchers have consistently reported that the nano sized formulation of photosensitizers enhanced the PDT efficacy in various tumors such as liver cancer by Ismail et al. [29], lung cancer by Lkhagvadulam et al. [30], cervical cancer by Benito et al. [31], ovarian cancer by Zeisser-Labouèbe et al. [32], melanoma by Mohammadi et al. [33], Camerin et al. [34], and Lima et al. [35], laryngeal carcinoma by Lima et al. [35], epidermoid carcinoma by Master et al. [36], colon cancer by Oh et al. [37], tongue carcinoma by Li et al. [38], fibrosarcoma tumor by Khaing Oo et al. [39], and breast cancer by Vargas et al. [40] and Navarro et al. [41]. This study has some limitations. First, the study only investigated the animal models, no studies of human subjects. Before starting the human studies, the biodistribution study of HP-NPs in the animal models should be performed first. The tissue distribution data are important reference to predict potential adverse reactions. The pharmacokinetic data from the plasma samples represent an ideal index for beginning the clinical study. After completing the biodistribution study in the animal models, the clinical study should be planned. Secondly, the study was performed with a single photosensitizer: hematoporphyrin. The results of this study encourage the study of other photosensitizers for evaluation of co-delivery of therapeutic agents. We are planning to study other photosensitizers such as Photofrin, which is widely used in clinical PDT. All of the results may be attributed to the in vitro/in vivo cancer specificity of HP-NPs and/or the cytotoxicity of DOX itself. Therefore, the HP-NPs prepared in this study represent a potentially effective co-delivery system of photosensitizer (HP) and anticancer drug (DOX). 5. Abbreviations
PDT HP DOX HPNPs LDL BSA PBS
photodynamic therapy hematoporphyrin doxorubicin hematoporphyrin-modified, doxorubicin-loaded bovine serum albumin nanoparticles low density lipoprotein bovine serum albumin phosphate-buffered saline
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[34] M. Camerin et al., The in vivo efficacy of phthalocyanine–nanoparticle conjugates for the photodynamic therapy of amelanotic melanoma, Eur. J. Cancer 46 (10) (2010) 1910–1918. [35] A.M. Lima et al., Hypericin encapsulated in solid lipid nanoparticles: phototoxicity and photodynamic efficiency, J. Photochem. Photobiol. B: Biol. 125 (2013) 146–154. [36] A.M. Master et al., Optimization of a nanomedicine-based silicon phthalocyanine 4 photodynamic therapy (Pc 4-PDT) strategy for targeted treatment of EGFR-overexpressing cancers, Mol. Pharm. 9 (8) (2012) 2331– 2338. [37] I.-H. Oh et al., Cancer cell-specific photoactivity of pheophorbide a glycol chitosan nanoparticles for photodynamic therapy in tumor-bearing mice, Biomaterials 34 (27) (2013) 6454–6463.
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Journal of Photochemistry and Photobiology B: Biology journal homepage: www.elsevier.com/locate/jphotobiol
Anticancer efficacy of photodynamic therapy with hematoporphyrin-modified, doxorubicin-loaded nanoparticles in liver cancer Ji-Eun Chang a, In-Soo Yoon b, Ping-Li Sun a,c, Eunjue Yi a, Sanghoon Jheon a,d, Chang-Koo Shim e,⇑ a
Department of Thoracic and Cardiovascular Surgery, Seoul National University Bundang Hospital, Seongnam-Si, Gyeonggi-do, Republic of Korea College of Pharmacy and Natural Medicine Research Institute, Mokpo National University, Jeonnam, Republic of Korea Department of Pathology, Seoul National University Bundang Hospital, Seongnam-Si, Gyeonggi-do, Republic of Korea d Department of Thoracic and Cardiovascular Surgery, Seoul National University College of Medicine, Seoul, Republic of Korea e Department of Pharmaceutics, College of Pharmacy, Seoul National University, Seoul, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 11 April 2014 Received in revised form 23 June 2014 Accepted 10 July 2014 Available online 24 July 2014 Keywords: Photodynamic therapy Hematoporphyrin Doxorubicin Nanoparticles Liver cancer
a b s t r a c t Photodynamic therapy (PDT) in combination with chemotherapy has great potential for cancer treatment. However, there have been very few attempts to developing cancer-targeted co-delivered systems of photosensitizers and anticancer drugs. We developed hematoporphyrin (HP)-modified doxorubicin (DOX)-loaded nanoparticles (HP-NPs) to improve the therapeutic effect of PDT in treating liver cancer. HP is not only a ligand for low density lipoprotein (LDL) receptors on the hepatoma cells but also a well-known photosensitizer for PDT. In vitro phototoxicity in HepG2 (human hepatocellular carcinoma) cells and in vivo anticancer efficacy in HepG2 tumor-bearing mice of free HP and HP-NPs were evaluated. The in vitro phototoxicity in HepG2 cells determined by MTT assay, annexin V-FITC staining and FACS analysis was enhanced in HP-NPs compared with free HP. Furthermore, compared with free HP-based PDT, in vivo anticancer efficacy in HepG2 tumor-bearing mice was markedly improved by HP-NPs-based PDT. Moreover, in both cases, the therapeutic effect was increased according to the irradiation time and number of PDT sessions. In conclusion, the HP-NPs prepared in this study represent a potentially effective co-delivery system of photosensitizer (HP) and anticancer drug (DOX) which improved the effects of PDT in liver cancer. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Photodynamic therapy (PDT) using photosensitizers has emerged as an effective therapeutic option for various cancers [1]. Upon irradiation with the proper wavelength of light, photosensitizers transfer energy to molecular oxygen, leading to the production of highly reactive singlet oxygen, which can damage adjacent tissues [2,3]. Moreover, photosensitizers remain effective even if they are conjugated to other molecules [4]. Among the various photosensitizers, hematoporphyrin (HP) is known as the first photosensitizer used in PDT [5]. However, the clinical application of PDT has been often hindered by the limited aqueous solubility and tumor specificity of photosensitizers [6]. Therefore, to over⇑ Corresponding author. Address: College of Pharmacy, Seoul National University, 599 Gwanangno, Gwanak gu, Seoul, 151–742, Republic of Korea. Tel.: +82 2 880 7873; fax: +82 2 888 5969. E-mail address: [email protected] (C.-K. Shim). http://dx.doi.org/10.1016/j.jphotobiol.2014.07.005 1011-1344/Ó 2014 Elsevier B.V. All rights reserved.
come these limitations, various drug delivery systems such as lipid- or polymer-based nanoparticles and polymer conjugates have been extensively explored for the cancer-specific delivery of photosensitizers [7–9]. PDT in combination with chemotherapy has great potential for cancer treatment, because it may permit low doses of photosensitizer and anticancer drug, thereby diminishing undesirable sideeffects [10]. Several studies have reported that the combination of PDT and chemotherapy could produce an additive or synergistic anticancer efficacy in vitro and in vivo [10–13]. To date, vast number of investigations have focused on developing cancer-targeted drug delivery systems for either photosensitizers or anticancer drugs alone. However, there have been very few studies on the co-delivery of photosensitizers and anticancer drugs in cancer. Among various cancer-targeted drug delivery systems, we previously developed HP-modified bovine serum albumin nanoparticles (HP-NPs) for the cancer targeting of doxorubicin (DOX) [14]. DOX is one of the most widely used anticancer drugs for the
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treatment of lymphoma, sarcomas, carcinomas, and melanoma [15]. However, its therapeutic effects have been limited due to serious side effects such as dose-dependent irreversible cardiomyopathy and congestive heart failure [15]. HP is a ligand binding to low-density lipoprotein (LDL) receptors on tumor cell membrane [16], and albumin nanoparticles could accumulate more in the cancer without targeting [17]. The HP-NPs enhanced the delivery of DOX to liver tumors in rats and reduced the distribution of DOX to the heart, suggesting their usefulness as a cancer-specific delivery system for DOX [14]. Since HP is also a widely used photosensitizer, we hypothesized that the HP-NPs could combine HP-based PDT and DOX-based chemotherapy with enhanced cancer specificity, potentially leading to an improved anticancer efficacy and reduced toxicity. There are very few reports on photosensitizermodified cancer-specific nanoparticulate systems for PDT in combination with chemotherapy. Herein, we report on PDT using HP-NPs for cancer treatment. The HP-NPs were prepared as previously reported, and the in vitro phototoxicity of PDT using HP-NPs or free HP with varying light irradiation conditions in HepG2 (human hepatocellular carcinoma) cells was evaluated by MTT assay, annexin V-FITC staining and FACS analysis. Subsequently, the in vivo anticancer efficacy of PDT using HP-NPs or free HP with varying light irradiation conditions in HepG2 tumor-bearing mice was further evaluated in terms of tumor growth and immunohistology. 2. Materials and methods 2.1. Materials Doxorubicin hydrochloride (DOX), bovine serum albumin (BSA, purity P98%), glutaraldehyde 8% solution, N-hydroxysuccinimide (NHS, 98%), fetal bovine serum (FBS), phosphate buffered saline (PBS), penicillin–streptomycin, thiazolyl blue tetrazolium bromide (MTT) and Dulbecco’s Modified Eagle medium (DMEM) were obtained from Sigma–Aldrich Co. (St. Louis, MO, USA). Hematoporphyrin dihydrochloride (HP) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). N,N-dicyclohexylcarbodiimide (DCC, purity >99%) was purchased from Fluka (Tokyo, Japan). Ethanol, dimethyl sulfoxide (DMSO), acetonitrile, and all other reagents were obtained from Fisher Scientific Korea Ltd. (Seoul, Korea). 2.2. Preparation and characterization of HP-NPs HP-NPs were prepared in the same way as previously reported [14]. Briefly, first, DOX-loaded nanoparticles (NPs) were prepared by a desolvation technique. 10 mg of DOX and 200 mg of BSA were dissolved in 10 mL of double-distilled water (DDW). The solution was then stirred and ethanol was added under constant stirring at room temperature. For the desolvation process, 8% glutaraldehyde solution was added as a crosslinking agent. The resulting nanoparticles were purified by centrifugation (Centrifuge 5415R, Eppendorf AG Hamburg, Germany) at 16,000g for 12 min; then redispersion in DDW was repeated 3 times to eliminate the ethanol and glutaraldehyde. Secondly, HP was conjugated to prepare NPs. HP, NHS and DCC were added to DMSO, and the mixture was filtered. HP-NHS complex was isolated by mixing the filtrate with excess diethyl ether and washed with methanol. This step was repeated once. The sample was lyophilized and stored at 20 °C. HP-NHS was dissolved in an aqueous suspension of NPs and stirred overnight. Centrifugation and redispersion in DDW were repeated 3 times. The mean particle size and zeta potential of prepared HP-NPs were measured using an electrophoretic light scattering spectrophotometer (ELS-8000, Otsuka Electronics Co., Ltd. Osaka, Japan).
The morphology of HP-NPs was then observed by field-emission scanning electron microscopy (FE-SEM) (SUPRA 55VP, Carl Zeiss, Oberkochen, Germany) and transmission electron microscopy (TEM) (JEM 1010, JEOL, Tokyo, Japan). 2.3. In vitro phototoxicity HepG2 (human hepatocellular carcinoma, ATCC, Manassas, VA, USA) cells were seeded in 12-well cell culture plates at a density of 1 105 cells/well in DMEM medium containing 10% (v/v) FBS and 1% (w/v) penicillin–streptomycin and incubated for 1 day at 37 °C in a humidified 5% CO2 and 95% air atmosphere. After cell attachment, the medium was washed off and replaced with serum-free media containing free HP or HP-NPs (2 lM as HP). After 4-h incubation, the cells were washed twice with cold PBS to eliminate the remaining drugs, and then fresh culture medium was added. For MTT assay, the cells were illuminated once or twice with a PDT laser (Diomed Inc, Andover, MA, USA) (630 nm, 400 mW/cm2) for 0, 5, 10, 25, 35, and 50 s (0, 2, 4, 10, 14, and 20 J/cm2, respectively.). For the double irradiation, the interval time was 2 h and the conditions of first and second irradiation session were the same. The irradiated cells were incubated for 1 day, and cell viability was performed by MTT assay [18]. The cells were incubated for 4 h with 50 lL/well of medium containing 1 mg/mL of MTT agent, and 500 lL/well of DMSO was added. The plates were shaken gently, and absorbance was read at a wavelength of 540 nm using an absorbance microplate reader (Spectramax plus384, Molecular Devices Corporation, Sunnyvale, CA, USA). Cytotoxicity was represented as the percentage of the control. All the experiments were performed in triplicate. For microscopic analysis, the cells were illuminated with a PDT laser (630 nm, 400 mW/cm2) for 50 s (20 J/cm2). The irradiated cells were incubated for 1 day and washed with cold PBS twice. Apoptotic cells were identified by using an annexin V-FITC fluorescence microscope kits (BD bioscience, San Jose, CA, USA) [19]. The cells were stained with both annexin V-FITC and DAPI, and the apoptotic cells were examined by light microscope (Axioskop 40, Carl Zeiss, Gottingen, Germany) with a X-Cite 120Q excitation light source (Lumen Dynamics Group Inc., Mississauga, Ontario, Canada). For flow cytometry analysis, the cells were illuminated single or double with a PDT laser (630 nm, 400 mW/cm2) for 25 or 50 s (10 or 20 J/cm2). For the double irradiation, the interval time was 2 h and the conditions of first and second irradiation were the same. The irradiated cells were incubated for 1 day and washed with cold PBS twice. Then, the cells were stained with both annexin V-FITC and PI, and the apoptotic cells were measured by flow cytometry (FACSCalibur, BD Biosciences, San Jose, CA, USA) using CellQuest software (BD Immunocytometry Systems, Mountain View, CA, USA). 2.4. In vivo anticancer efficacy in tumor bearing mice All animal study protocols were approved by the Institutional Animal Care and Use Committee of Seoul National University Bundang Hospital. BALB/C male nude mice (6–7 weeks, 20–22 g) were purchased from Orientbio (Kyungki-Do, Korea). 1 106 HepG2 cells in 0.1 mL DMEM medium were injected subcutaneously into the left flanks of the mice. When the tumors grew to approximately 200 mm3 in volume, the mice were randomly divided into 12 groups (n = 5 each) and injected with PBS, free HP or HP-NPs once or twice (on day 0 and 7). Each injection was made into a tail vein at an HP dose of 2 mg/kg in PBS. At 24 h post-injection, tumor sites were irradiated with a PDT laser (630 nm, 400 mW/cm2) once or
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twice for 250 or 500 s (100 or 200 J/cm2). The conditions for each drug were as follows; single injection without PDT, single injection with single PDT (100 J/cm2), single injection with single PDT (200 J/cm2), and double injection with double PDT (200 J/cm2). The tumor volume of each mouse was monitored for 4 weeks to evaluate the therapeutic efficacy. Tumors were measured with a caliper, and the tumor volume (mm3) was calculated as (length width2)/2. Two weeks after PDT treatment, the tumor tissue of each group was isolated, fixed in 10% formalin, embedded in paraffin, and stained with Hematoxylin and Eosin (H&E) to evaluate the induction of necrosis or apoptosis. 2.5. Statistical analysis All data were presented as the mean ± standard deviation. The statistical significance between the groups was determined by using the Student’s t-test for 2 groups or one-way ANOVA with Tukey’s post hoc test for more than 3 groups. It was considered significant when p values were less than 0.05. 3. Results 3.1. Characterization of HP-NPs Fig. 1 shows the size distribution, TEM and FE-SEM images of HP-NPs. The prepared nanoparticle formulations were submicron polydisperse systems having moderate size distribution and their average particle sizes were 350 ± 15 nm (Fig. 1a). The zeta potential was 21.1 ± 1.1 mV. In TEM and FE-SEM images, smooth and spherical particles were observed (Fig. 1b). 3.2. In vitro phototoxicity In vitro viability of HepG2 cells after 4-h incubation with PBS, free HP and HP-NPs followed by single or double light irradiation for 0, 10, 15, 25, 35, or 50 s is shown in Fig. 2. No significant change in cell viability was observed in the control (PBS) group with or
Fig. 2. In vitro viability of HepG2 cells after 4-h incubation with PBS, free HP and HP-NPs followed by single (PBS, d; free HP, .; HP-NPs, j) or double (PBS 2, s; free HP 2, 4; HP-NPs 2, h) light irradiation for 0, 10, 15, 25, 35, or 50 s (n = 3).
without light irradiation and free HP group without light irradiation, while significant reduction in cell viability was observed in the free HP group with light irradiation. This result is consistent with those of a previous study which reported the utility of HP as a photosensitizer [5]. In the free HP group, the cell viability after double light irradiation was significantly lower than after single light irradiation, and it tended to be reduced as the irradiation time increased. This result suggests that the in vitro phototoxicity of HP may depend on the extent of exposure to light irradiation. Moreover, under the same irradiation conditions, HP-NPs showed higher cytotoxicity compared with free HP. Empty nanoparticles showed no cytotoxicity at all (data not shown). In vitro annexin V-FITC staining of apoptotic HepG2 cells after 4-h incubation with PBS, free HP and HP-NPs followed by single light irradiation for 0 or 20 J/cm2 is shown in Fig. 3. In the control (PBS) group with or without light irradiation and free HP group without light irradiation, negligible green fluorescence (indicating apoptotic cells) intensity was observed, while significant green
Fig. 1. Size distribution (a) and TEM (left) and FE-SEM (right) (b) images of HP-NPs.
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Fig. 3. In vitro annexin V-FITC staining of apoptotic HepG2 cells after 4-h incubation with PBS, free HP and HP-NPs followed by single light irradiation for 0 or 20 J/cm2. DAPI stain (blue, first column) and annexin V-FITC stain (green, second column) indicate nucleus and apoptotic cells, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
fluorescence was observed in the free HP group with light irradiation. Moreover, the green fluorescence intensity of HP-NPs group was higher than that of free HP groups in the same light irradiation conditions. These results were in agreement with the cell viability data (Fig. 2). FACS analysis of HepG2 cells after 4-h incubation with PBS, free HP and HP-NPs followed by single or double light irradiation for 25 or 50 s is shown in Fig. 4. The combination staining of annexin V and PI depicts apoptotic stages of the cells [20]. In the control (PBS) group, negligible annexin V and PI fluorescence was observed, while significant fluorescence was observed in both free HP and HP-NPs groups. Under the same irradiation conditions, the fluorescence intensity of HP-NPs group was higher than that of free HP groups. These results were consistent with the cell viability and microscope analysis data (Figs. 2 and 3). Furthermore, under the same irradiation conditions, the portion of late apoptosis was increased in the HP-NPs group. Taken together, compared with free HP, the cellular phototoxicity determined by MTT assay, microscopy and FACS analysis was enhanced in HP-NPs (Figs. 2–4). These results may be attributed
to the enhanced cellular uptake of HP-NPs and/or the cytotoxicity of DOX itself [14]. It was reported that HP-loaded polymeric micelles or solid lipid nanoparticles enhanced the cellular uptake of HP in A549 or KB cells, respectively [21,22]. 3.3. In vivo anticancer efficacy in tumor bearing mice In vivo tumor growth in HepG2 tumor-bearing mice during 4 weeks after single or double (on day 0 and 7) intravenous injection of PBS, free HP and HP-NPs with or without 100 or 200 J/cm2 light irradiation 1 day after each injection is shown in Fig. 5. The tumor growth of the free HP group with light irradiation was significantly lower than that of the control (PBS) group with or without light irradiation and free HP group without light irradiation, indicating the anticancer efficacy of HP-based PDT. Moreover, in the same irradiation conditions, the tumor growth of HP-NPs was significantly lower than that of the free HP group, which was consistent with the in vitro phototoxicity studies (Figs. 2–4). Moreover, within each free HP or HP-NPs group, the antitumor efficacy was increased according to irradiation time and the
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Fig. 4. FACS analysis of HepG2 cells after 4-h incubation with PBS, free HP and HP-NPs followed by single or double light irradiation for 25 or 50 s. Both annexin V and PI negative cells are considered undamaged; annexin V positive and PI negative cells are early apoptotic; both annexin V and PI positive cells are late apoptotic; and annexin V negative and PI positive cells are either late apoptotic or necrotic.
Fig. 5. In vivo tumor growth in HepG2 tumor-bearing mice during 4 weeks after single (+) or double (++) (on day 0 and 7) intravenous injection of PBS, free HP and HP-NPs with or without 100 or 200 J/cm2 light irradiation 1 day after each injection (n = 4).
number of PDT session. It was consistent with the in vitro cell viability study (Fig. 4) showing that the in vitro phototoxicity of free HP and HP-NPs may depend on the extent of exposure to light irradiation. Images of tumor tissues in HepG2 tumor-bearing mice before PDT and on day 1, 3, 8, and 15 after double (on day 0 and 7) intravenous injection of PBS, free HP and HP-NPs with 200 J/cm2 light irradiation 1 day after each injection (on day 1 and 8) are shown in Fig. 6. The day after PDT, HP-NPs-treated mice showed
a hemorrhage compartment where the laser had been delivered and severe necrosis was noted. In the free HP treated group, tumor necrosis developed more slowly than in the HP-NPs treated groups. Moreover, H&E staining of tumor tissues in HepG2 tumor-bearing mice on day 7 and 14 after double (on day 0 and 7) intravenous injection of PBS, free HP and HP-NPs with 200 J/cm2 light irradiation on 1 day after each injection (on day 1 and 8) is shown in Fig. 7. Most tumor cells were severely damaged in mice treated with HP-NPs, while tumor cell death was incomplete in mice treated with PBS or free HP. Based on the morphologies of tumor tissues, the tumor growth of the three groups was in the following order: control (PBS) > free HP > HP-NPs, which was consistent with the in vivo tumor volume data (Fig. 5). Taken together, compared with free HP-based PDT, in vivo anticancer efficacy was markedly improved by HP-NPs-based PDT (Figs. 5–7). These results may be attributed to the in vivo tumor specificity of HP-NPs and/or the cytotoxicity of DOX itself [14]. 4. Discussion In this study, we developed photosensitizer (HP)-modified, anticancer agent (DOX)-loaded nanoparticles to improve the therapeutic efficacy of PDT in treating liver cancer. PDT is an ideal local cancer treatment using tumor tissue-penetrating lasers after the administration of tumor selective photosensitizers [23]. To increase the targeting ability of photosensitizers for the tumor site, we selected nanoparticles as a targeting carrier. Nanoparticles are ideal cancer targeting carriers because they have both passive and active targeting ability [24]. For passive targeting, nano-sized particles are easily uptaken by the tumor vessels which are very leaky
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Fig. 6. Images of tumor tissues in HepG2 tumor-bearing mice before treatment and 1, 3, 8, and 15 days after double (on day 0 and 7) intravenous injection of PBS, free HP and HP-NPs with 200 J/cm2 light irradiation 1 day after each injection (on day 1 and 8).
Fig. 7. H&E staining of tumor tissues in HepG2 tumor-bearing mice on day 7 and 14, after double (on day 0 and 7) intravenous injection of PBS, free HP and HP-NPs with 200 J/cm2 light irradiation 1 day after each injection (on day 1 and 8).
and this is called EPR (enhanced permeability and retention) effect [25]. Furthermore, for active targeting, it is easy to conjugate cancer targeting moieties such as photosensitizers on the surface of the nanoparticles. Hematoporphyrin is not only a targeting ligand for LDL receptors which is overexpressed on hepatoma cells but is also a well-known photosensitizer for PDT [14]. Doxorubicin which is a widely used antineoplastic agent used for liver cancer [26] was selected for the combination therapy with chemotherapy and PDT. In a previous study, we reported that HP-modified, DOX-loaded nanoparticles (HP-NPs) significantly enhanced the targeting ability for liver cancer sites, with fewer adverse reactions in normal tissues [14]. The HP-NPs were successfully prepared by the desolvation method and characterized in terms of particle size distribution and morphology (Fig. 1). To investigate the PDT efficacy of prepared HP-NPs, we performed both in vitro and in vivo experiments with various conditions of laser irradiation. The wavelength was fixed at 630 nm which was suitable for hematoporphyrin [1] and the output power was set at 400 mW/cm2 in order not to damage any tissues not pretreated with photosensitizers. When the output
power exceeded 400 mW/cm2, the cells and the skin of the animals were damaged by the irradiation itself. Furthermore, the distance from the PDT fiber to the cells or the tumor sites of the animals was an extremely important factor which influenced the effects of the PDT. We tested several pilot studies to find out the most effective distance, and 1 cm was set as the optimal distance. For in vitro studies, HP concentration was determined to be 2 lM at which there is no cytotoxicity without light irradiation. In both in vitro and in vivo studies, HP-NPs were more efficient than free HP under the same laser irradiation conditions. The reasons are considered in the following ways; first, nano-sized formulation stimulated the accumulation of the drugs into the tumor site by passive diffusion, secondly, hematoporphyrin played a pivotal role as a cancer targeting ligand, finally, the dual effects of PDT and chemotherapy with doxorubicin significantly enhanced the anticancer efficacy. Zhou et al. [27] and Hwang et al. [28] also studied about combination of chemotherapy and photodynamic therapy in cancer. The phototoxicity increased according to the irradiation time and the number of PDT sessions in both free HP and HP-NPs. In
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our preliminary study, the free HP treated group showed no antitumor efficacy at lower irradiation doses (less than 50 J/cm2), while HP-NPs treated group showed therapeutic effects under the same conditions (unpublished data). Thus, it appears that the anti-tumor efficacy of HP may depend more on its formulation than irradiation conditions. However, the exact relative contribution of each factor, i.e. formulation vs. irradiation condition, to the anti-tumor efficacy of HP cannot be clarified based only on the present data, which require further investigation. Many researchers have consistently reported that the nano sized formulation of photosensitizers enhanced the PDT efficacy in various tumors such as liver cancer by Ismail et al. [29], lung cancer by Lkhagvadulam et al. [30], cervical cancer by Benito et al. [31], ovarian cancer by Zeisser-Labouèbe et al. [32], melanoma by Mohammadi et al. [33], Camerin et al. [34], and Lima et al. [35], laryngeal carcinoma by Lima et al. [35], epidermoid carcinoma by Master et al. [36], colon cancer by Oh et al. [37], tongue carcinoma by Li et al. [38], fibrosarcoma tumor by Khaing Oo et al. [39], and breast cancer by Vargas et al. [40] and Navarro et al. [41]. This study has some limitations. First, the study only investigated the animal models, no studies of human subjects. Before starting the human studies, the biodistribution study of HP-NPs in the animal models should be performed first. The tissue distribution data are important reference to predict potential adverse reactions. The pharmacokinetic data from the plasma samples represent an ideal index for beginning the clinical study. After completing the biodistribution study in the animal models, the clinical study should be planned. Secondly, the study was performed with a single photosensitizer: hematoporphyrin. The results of this study encourage the study of other photosensitizers for evaluation of co-delivery of therapeutic agents. We are planning to study other photosensitizers such as Photofrin, which is widely used in clinical PDT. All of the results may be attributed to the in vitro/in vivo cancer specificity of HP-NPs and/or the cytotoxicity of DOX itself. Therefore, the HP-NPs prepared in this study represent a potentially effective co-delivery system of photosensitizer (HP) and anticancer drug (DOX). 5. Abbreviations
PDT HP DOX HPNPs LDL BSA PBS
photodynamic therapy hematoporphyrin doxorubicin hematoporphyrin-modified, doxorubicin-loaded bovine serum albumin nanoparticles low density lipoprotein bovine serum albumin phosphate-buffered saline
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