Photodynamic therapy (PDT) is an alternative choice for lung cancer treatment. To increase the therapeutic effect of PDT, lung cancer-targeted nanoparticles combined with chemotherapy were developed. Both in vitro and in vivo anticancer efficacies of PDT with prepared nanoparticles were evaluated.
Photodynamic therapy (PDT) is a non-invasive and non-surgical method representing an attractive alternative choice for lung cancer treatment. Photosensitizers selectively accumulate in tumor tissue and lead to tumor cell death in the presence of oxygen and the proper wavelength of light.
To increase the therapeutic effect of PDT, we developed both photosensitizer- and anticancer agent-loaded lung cancer-targeted nanoparticles. Both enhanced permeability and retention (EPR) effect-based passive targeting and hyaluronic-acid-CD44 interaction-based active targeting were applied. CD44 is a well-known hyaluronic acid receptor that is often introduced as a biomarker of non-small cell lung cancer.
In addition, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may increase anticancer therapeutic effects and reduce adverse reactions.
We chose hypocrellin B (HB) as a novel photosensitizer in this study. It has been reported that HB causes higher anticancer efficacy of PDT compared to hematoporphyrin derivatives1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for lung cancer2.
The antitumor efficacies of photosensitizer (HB) solution, photosensitizer encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT were compared both in vitro and in vivo. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.
The HB-P-NP treatment group showed the most effective anticancer effect after PDT. In conclusion, the HB-P-NPs prepared in the present study represent a potential and novel photosensitizer delivery system in treating lung cancer with PDT.
Photodynamic therapy (PDT) is composed of three major factors: photosensitizers, light, and oxygen. PDT is reported as a promising treatment for various cancers3. When the photosensitizers are administered into the cancer patient, they selectively accumulate in the tumor tissues. When the proper wavelength of light is applied, the highly reactive singlet oxygen and other free radicals lead to tumor cell damage4.
Lung cancer was introduced as one of the first applications for PDT in the early 1980s5. PDT provides several advantages in treating lung cancer. Since PDT is a non-invasive and non-surgical treatment, it is an attractive alternative choice for the patients in whom surgical resection is inappropriate.
There have been many challenges to enhance the cancer-targeting efficacy of the photosensitizers. Increasing photosensitizer accumulation in cancer sites and decreasing accumulation in normal tissues are the identical goals for the cancer-targeting studies. A variety of targeted drug delivery systems, such as polymers, liposomes, and nanoparticles are adopted as photosensitizer carriers6-8. In our previous studies, nanoparticles effectively increased the cancer-targeting abilities of the photosensitizers9,10. Nanoparticles are ideal cancer-targeting carriers since they possess both passive and active targeting abilities. The leaky tumor vessels provide opportunity for nano-sized carriers to accumulate easily in tumors, which is well-known as the enhanced permeability and retention (EPR) effect11,12. The interaction between the nanoparticles and the specific receptors on cancer cells enables active cancer targeting. In this study, we prepared hyaluronic acid-based nanoparticles to interact with CD44, the major hyaluronic acid receptor that is overexpressed on lung cancer cells13.
To maximize the anticancer efficacy, a combination of PDT and chemotherapy is adopted in the present study. This combination concept may permit an increased therapeutic effect. Furthermore, decreased doses of both the photosensitizer and the anticancer drug can diminish adverse effects. We selected hypocrellin B (HB) as a novel photosensitizer in the present study. HB is isolated from Chinese medicinal fungus Hypocrella bambuase. Shang et al. reported that HB-based PDT possesses a higher anticancer efficacy when compared to hematoporphyrin derivative-based PDT1. Paclitaxel was selected as the anticancer drug since it has proven to be a potential treatment for various cancers, including lung cancer2.
Herein, we compared the anticancer efficacies of photosensitizer (hypocrellin B, HB) solution, photosensitizer-encapsulated hyaluronic acid-ceramide nanoparticles (HB-NPs), and both photosensitizer- and anticancer agent (paclitaxel)-encapsulated hyaluronic acid-ceramide nanoparticles (HB-P-NPs) after PDT. The in vitro phototoxicity in A549 (human lung adenocarcinoma) cells and the in vivo antitumor efficacy in A549 tumor-bearing mice were evaluated.
NOTE: All animal study protocols were approved by the Institutional Animal Care and Use Committee of Seoul National University Bundang Hospital (BA1308-134/072-01).
1. Synthesis of Hyaluronic Acid-Ceramide (HACE)
2. Preparation of the Nanoparticles
3. In Vitro Phototoxicity
4. In vivo anticancer efficacy in tumor-bearing mice
We prepared both HB-NPs and HB-P-NPs with the techniques mentioned above. The mean diameters of HB-NPs and HB-P-NPs were 220.9 ± 3.2 nm and 211.9 ± 1.6 nm, respectively.
The cell viability of A549 cells after 4 hr of incubation with PBS, empty NPs, HB-NPs, and HB-P-NPs followed by light irradiation (0 to 16 J/cm2) is shown in Figure 1. Without light, the HB-NP treatment group showed no cytotoxicity at all, while HB-P-NP-treated cells showed 81.28 ± 0.14% cell viability. This may be due to the anticancer effect of paclitaxel, which was released from the nanoparticles. Under PDT, the cell viability was decreased according to the light exposure time in both HB-NP- and HB-P-NP-treated cells. The HB-P-NP-treated cells showed increased phototoxicity than the HB-NPs group under the same irradiation conditions. When 16 J/cm2 of irradiation was given, only 7.52 ± 0.38% of the cells survived in the HB-P-NP treatment group.
The consistent results were visualized with annexin V-FITC staining (Figure 2). The PDT-induced apoptotic cells were stained with annexin V-FITC, expressing green fluorescence. The strongest green fluorescence signal was detected in the HB-P-NP-treated cells under the same irradiation conditions.
The stages of the PDT-induced apoptotic cells were classified by flow cytometry. By double-staining with annexin V-FITC and PI, early apoptotic cells (only annexin V-FITC positive), late apoptotic cells (both annexin-V and PI positive) and necrotic cells (only PI positive) were distinguished. Under the same PDT conditions, the portion of late apoptotic cells was increased in the HB-P-NP-treated group (Figure 3).
In vivo tumor growth in lung tumor-bearing mice after double intravenous injection of PBS, free HB, HB-NPs, and HB-P-NPs followed by light irradiation is shown in Figure 4. The surfaces of the tumor sites were also examined (Figure 5). The HB-P-NP-treated mice showed the fastest reactions, including hemorrhage and necrosis, and the slowest tumor growth. Moreover, histological analysis confirmed the most severely damaged tumor cells were in the HB-P-NP treatment group (Figure 6).
Figure 1: In vitro Phototoxicity of A549 Cells. A549 cells were treated with PBS, empty NPs, HB-NPs, or HB-P-NPs, and light irradiation was given for 0, 5, 10, 20, 30, or 40 sec (0, 2, 4, 8, 12, or 16 J/cm2). Under the same irradiation time, the HB-P-NP treatment group showed higher phototoxicity than the HB-NP treatment group. Values are expressed as means ± standard deviations (SD; n = 3). *** p < 0.001, compared to the PBS-treated group. ## p < 0.01, ### p < 0.001, compared to the HB-NP treatment group. Adapted from Chang et al.10 Please click here to view a larger version of this figure.
Figure 2: Microscopic Analysis of Apoptotic A549 Cells. A549 cells were treated with PBS, HB-NPs or HB-P-NPs, and light irradiation was given for 0, 2, or 4 J/cm2. The double-stain of DAPI (blue, first column) and annexin V-FITC (green, second column) indicate nuclei and apoptotic cells, respectively. The HB-P-NP-treated cells showed stronger green fluorescence signals than the HB-NP-treated cells under the same irradiation conditions, indicating a much higher phototoxicity. Scale bar = 200 µm, magnification = 100X. Adapted from Chang et al.10 Please click here to view a larger version of this figure.
Figure 3: FACS Analysis of Apoptotic A549 Cells. A549 cells were treated with PBS, HB-NPs, or HB-P-NPs, and light irradiation was given for 0, 2, or 4 J/cm2. The cells were categorized into four groups. i) Both annexin V-FITC- and PI-negative cells are considered undamaged, ii) annexin V-FITC-positive and PI-negative cells are early apoptotic, iii) both annexin V-FITC- and PI-positive cells are late apoptotic, and iv) annexin V-FITC-negative and PI-positive cells are either late apoptotic or necrotic. In accordance with the microscopic analysis, under the same irradiation conditions, the HB-P-NP-treated cells showed higher phototoxicity than the HB-NP-treated cells, and late apoptotic cell levels were increased. Adapted from Chang et al.10 Please click here to view a larger version of this figure.
Figure 4: Tumor Volume Monitoring of A549 Tumor-bearing Mice. On days 0 and 7, the double injections of PBS, free HB, HB-NPs, and HB-P-NPs were performed. The PDT (200 J/cm2) was performed 1 day after each injection, on days 1 and 8. The treatments showed different levels of therapeutic effect, with HB < HB-NPs < HB-P-NPs. On day 45, the HB-P-NPs treatment group showed a significantly decreased tumor volume compared to the other groups. Values are presented as means ± standard deviations (SD; n = 4). The tumor volume (mm3) was defined as (length × width2) / 2. Adapted from Chang et al.10 Please click here to view a larger version of this figure.
Figure 5: The Surface Alterations on the Tumor Site of A549 Tumor-bearing Mice. The tumor sites were monitored after double intravenous injections (on days 0 and 7) of PBS, free HB, HB-NPs, and HB-P-NPs with 200 J/cm2 light irradiation 1 day after each injection (on days 1 and 8). Both the HB-NPs and the HB-P-NPs treatment groups started to show reactions the day after the first PDT. The HB-P-NPs treatment group showed severe hemorrhaging and the fastest development of necrosis. Adapted from Chang et al.10 Please click here to view a larger version of this figure.
Figure 6: Histological Analysis of Tumor Tissues in A549 Tumor-bearing Mice. On day 16, the histological confirmation was performed by an H&E stain of excised tumor tissues. The HB-P-NPs treatment group demonstrated the most complete tumor cell death. Scale bar = 500 µm, magnification = 40X. Adapted from Chang et al.10 Please click here to view a larger version of this figure.
The most critical step in this study is selecting the proper laser conditions: wavelength, power, and irradiation time. The proper wavelength of light suitable for the specific photosensitizer is necessary for the PDT. We used a 630-nm laser that was appropriate for hypocrellin B. The output power was another important factor, which was set at 400 mW/cm2 based on many pilot studies. Output powers exceeding 400 mW/cm2 damaged the cells or the skin surface of the animals due to the irradiation itself, while output powers below 400 mW/cm2 were too weak to show any therapeutic effect. The appropriate irradiation times for the in vitro and in vivo studies were 40 sec (16 J/cm2) and 500 sec (200 J/cm2), respectively. The distance from the PDT fiber to the cells or to the tumor tissues of the animal models was also a critical factor in this study. To cover the entire cell or tumor surface with the light, 1 cm was found to be the optimal distance.
If a different photosensitizer is applied, use of the proper wavelength of light can maximize the PDT efficacy. The output power and irradiation time should be fixed through trial and error. When the skin surface starts to burn during the light irradiation, the output power should be set lower. When the tumor surface shows no change at all the day after PDT, it may indicate the necessity to increase the output power.
This method has some limitations. First, though the distance from the PDT fiber to the target is an important factor in this study, it is hard to ensure uniformity. The distance may vary slightly because it is controlled by humans. Secondly, for the animal model with tumors in organs such as the lung, this protocol is difficult to apply since endoscopy is necessary and respiratory movement unavoidable.
Though surgical resection is the first option for treating lung cancer, PDT has several advantages as a non-invasive and non-surgical alternative treatment. When the operation is not appropriate, such as in multiple lesion cases, PDT can be a valid option. Also, preoperative PDT may reduce the tumor size and lessen the degree of surgery17. PDT has fewer side effects when compared to surgery, radiation therapy, or endobronchial brachytherapy. In addition, PDT is irrelevant to mutations that provide resistance to radiation therapy or chemotherapy18.
In this study, we suggested both photosensitizer- and anticancer drug-encapsulated lung cancer-targeted nanoparticles as a novel photosensitizer delivery system for PDT. This concept may be applied to other photosensitizers and anticancer drugs.
The authors have nothing to disclose.
This study was supported by grant no. 14-2014-017 from the SNUBH Research Fund.
The authors are indebted to J. Patrick Barron, Professor Emeritus, Tokyo Medical University and Adjunct Professor, Seoul National University Bundang Hospital for his pro bono editing of this manuscript.
oligo hyaluronic acid | Bioland Co., Ltd. | _ | |
DS-Y30 (ceramide 3B; mainly N-oleoyl-phytosphingosine) | Doosan Biotech Co., Ltd. | _ | |
adipic acid dihydrazide | Sigma Aldrich | A0638 | |
N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide | Sigma Aldrich | 39391 | |
4-(chloromethyl)benzoyl chloride | Sigma Aldrich | 270784 | |
Tween 80 | Tokyo Chemical Industry Co., Ltd. | T0546 | |
syringe filter | Sartorius Stedim Biotech GmbH | 17762 | 15 mm, RC, PP, 0.45 µm |
triethylamine | Sigma Aldrich | T0886 | |
Mini-GeBAflex tubes | Gene Bio-Application Ltd. | D070-12-100 | |
Paclitaxel | Taihua Corporations | _ | |
RPMI-1640 | Gibco Life Technologies, Inc. | 11875 | |
Penicillin–streptomycin | Gibco Life Technologies, Inc. | 15070 | |
Fetal bovine serum | Gibco Life Technologies, Inc. | 16140071 | |
Celite (Filter agent) | Sigma Aldrich | 6858 | See step 1.4 |