Name: anticancer efficacy of photodynamic therapy with lung cancer-targeted nanoparticles
Uploaded: 2016-12-01T13:00:00.000+00:00
Duration: 8 min 3 s
Description: Watch this Scientific Journal Video about Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles at JoVE.com

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.

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)
<ol>
	<li>Solubilize 12.21 mmol of hyaluronic acid (HA) oligomer and 9.77 mmol of tetra-n-butylammonium hydroxide (TBA) in 60 ml of double-distilled water (DDW). Stir for 30 min.</li>
	<li>To synthesize the DS-Y30 linker, dissolve 8.59 mmol of DS-Y30 ceramide and 9.45 mmol of triethylamine in 25 ml of tetrahydrofuran (THF). Mix with 8.59 mmol of 4-chloromethylbenzoyl chloride in THF. Stir for 6 hr at 60 &#176;C.</li>
	<li>Dissolve the synthesized 8.10 mmol of HA-TBA and 0.41 mmol of DS-Y30 linker in a mixture of THF and acetonitrile (4:1, v/v). Stir for 5 hr at 40 &#176;C.</li>
	<li>Remove impurities by filtering with a filter agent, and eliminate the organic solvent by vacuum evaporation. Purify the product using a dialysis membrane (molecular weight cut-off: 3.5 kDa) and lyophilize.</li>
</ol>
2. Preparation of the Nanoparticles
<ol>
	<li>Dissolve 1 mg of HB and 1 mg of paclitaxel in 0.5 ml of dimethyl sulfoxide (DMSO) and blend with 0.5 ml of DDW by vortex-mixing for 5 min. Then, solubilize HACE in that mixture by vortex-mixing for a further 5 min.</li>
	<li>To eliminate the solvent, heat at 70 &#176;C for 4 hr under a gentle stream of nitrogen gas.</li>
	<li>Resuspend the film composed of HACE, HB, and paclitaxel with 1 ml of DDW. Filter with a syringe filter (0.45 &#181;m pore size) to remove unencapsulated drugs.</li>
</ol>
3. In Vitro Phototoxicity
<ol>
	<li>Uptake of nanoparticles in lung cancer cell lines
	<ol>
		<li>Prepare RPMI-1640 medium-containing 10% (v/v) fetal bovine serum and 1% (w/v) penicillin-streptomycin.</li>
		<li>Seed A549 cells in 24-well cell culture plates at a density of 1 &#215; 105 cells/well (triplicates for each group). Incubate for 24 hr at 37 &#176;C in a humidified 5% CO2 and 95% air atmosphere.</li>
		<li>After cell attachment, remove the medium and wash the cells by adding 1 ml of phosphate-buffered saline (PBS).</li>
		<li>Dissolve the nanoparticles in PBS to a final concentration of 2 &#181;M/ml HB. Then, incubate the cells with 1 ml of PBS, empty NPs, HB-NPs, or HB-P-NPs in each well for 4 hr in the dark.</li>
		<li>Remove all of the solution and wash the cells by adding 1 ml of cold PBS. Repeat the washing step once more. Add fresh culture medium.</li>
	</ol>
	</li>
	<li>Cell viability assay
	<ol>
		<li>Place the cell culture plate under the PDT fiber (with 1 cm of distance from the PDT fiber to the well). Wear laser safety glasses and illuminate the cells with a PDT laser (630 nm, 400 mW/cm2) in the dark for various periods of time: 0, 5, 10, 20, 30, and 40 sec (0, 2, 4, 8, 12, and 16 J/cm2). Then, incubate the cells for 24 hr in the dark.</li>
		<li>Aspirate the medium and wash the cells by adding 1 ml of cold PBS. Repeat the washing step once more.</li>
		<li>Add 10 &#181;l of cytotoxicity measuring solution to each well. Incubate in the dark for 2 hr.</li>
		<li>Measure the absorbance at 450 nm using a microplate reader.</li>
	</ol>
	</li>
	<li>Microscopic analysis
	<ol>
		<li>Place the cell culture plate under the PDT fiber (with 1 cm of distance from the PDT fiber to the well). Wear laser safety glasses and illuminate the cells with a PDT laser (630 nm, 400 mW/cm2) in the dark for 0, 20, or 40 sec (0, 8, or 16 J/cm2). Then, incubate the cells for 24 hr in the dark.</li>
		<li>Aspirate the medium and wash the cells by adding 1 ml of cold PBS. Repeat the washing step once more.</li>
		<li>Add 50 &#181;l of annexin V-FITC and 50 &#181;l of 4&#39;,6-diamidino-2-phenylindole (DAPI, 1.5 &#181;g/ml). Gently shake the plate and incubate it for 15 min at room temperature in the dark. 
		NOTE: Use annexin V-FITC from the fluorescence microscope kit.</li>
		<li>Wash the cells by adding 1 ml of cold PBS. Repeat the washing step once more. Keep the cells in fresh PBS. Identify the apoptotic cells using light microscopy at 100X magnification.</li>
	</ol>
	</li>
	<li>Fluorescence activated cell sorting (FACS) analysis
	<ol>
		<li>Place the cell culture plate under the PDT fiber (with 1 cm of distance from the PDT fiber to the well). Wear laser safety glasses and illuminate the cells with a PDT laser (630 nm, 400 mW/cm2) for 0, 20, or 40 sec (0, 8, or 16 J/cm2). Then, incubate the cells for 24 hr in the dark.</li>
		<li>Aspirate the medium and wash the cells by adding 1 ml of cold PBS. Repeat the washing step once more.</li>
		<li>Resuspend the cells in 1 ml of 1&#215; binding buffer (dilute 1 part of the 10x binding buffer; 0.1 M Hepes/NaOH (pH 7.4), 1.4 M NaCl, and 25 mM CaCl2 to 9 parts distilled water) and transfer 100 &#181;l of the sample solution to a 5-ml culture tube.</li>
		<li>Add 5 &#181;l of annexin V-FITC and 5 &#181;l of propidium iodide (PI). Gently vortex the tube and incubate for 15 min at room temperature(RT) in the dark. Add 400 &#181;l of 1&#215; binding buffer. 
		NOTE: Use annexin V-FITC and PI from the fluorescence microscope kit.</li>
		<li>Identify the apoptotic cells by using FACS14. The excitation laser lines of PI and annexin V-FITC are 488 nm and 635 nm, respectively. Measure the fluorescence emission of PI and annexin V-FITC at 610 &#177; 20 nm and 660 &#177; 20 nm, respectively. Collect the acquired cells on the flow cytometer per 10,000 events.</li>
	</ol>
	</li>
</ol>
4. In vivo anticancer efficacy in tumor-bearing mice
<ol>
	<li>Lung cancer-induced mouse model
	<ol>
		<li>Prepare 1 &#215; 106 A549 cells in 0.1 ml RPMI-1640 medium; keep it in ice.</li>
		<li>Anesthetize the mice with an i.p. injection of a xylazine and a mixture of tiletamine and zolazepam (1:2, 1 ml/kg). Confirm proper anesthetization by gently pinching a small fold of mouse skin. Use vet ointment on the eyes to prevent dryness while under anesthesia. 
		NOTE: If no movement is observed, the animal is sufficiently anesthetized to start the experiments.</li>
		<li>Inject the cells subcutaneously into the left flanks of BALB/C male nude mice (6 - 7 weeks old, 20 - 22 g).</li>
		<li>Keep observing the mice until they start to move around the cage. 
		NOTE: Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return an animal that has undergone surgery to the company of other animals until it has fully recovered. Keep the mice under specific pathogen-free (SPF) conditions.</li>
		<li>Measure the tumor size with calipers every day. Calculate the tumor volume (mm3) as (length &#215; width2) / 2. When the tumor size reaches approximately 200 mm3 in volume, start the experiment.</li>
	</ol>
	</li>
	<li>Anticancer efficacy study
	<ol>
		<li>Randomly divide the mice into 4 groups (n = 10 for each group).</li>
		<li>Anesthetize the mice with an i.p. injection of a xylazine and a mixture of tiletamine and zolazepam (1:2, 1 ml/kg). Confirm proper anesthetization by gently pinching a small fold of mouse skin. Use vet ointment on the eyes to prevent dryness while under anesthesia. 
		NOTE: If no movement is observed, the animal is sufficiently anesthetized to start the experiments.</li>
		<li>Dissolve the nanoparticles in PBS to a final concentration of 2 mg/ml HB. Inject PBS, free HB, HB-NPs, or HB-P-NPs via the tail vein (2 mg/kg as HB) twice on days 0 and 7.</li>
		<li>Keep observing the mice until they start to move around the cage. 
		NOTE: Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return an animal that has undergone surgery to the company of other animals until fully recovered. Keep the cages dark and under specific SPF conditions.</li>
		<li>24 hr after each injection, anesthetize the mice with an i.p. injection of a xylazine and a mixture of tiletamine and zolazepam (1:2, 1 ml/kg). Confirm proper anesthetization by gently pinching a small fold of mouse skin. Use vet ointment on the eyes to prevent dryness while under anesthesia. 
		NOTE: If no movement is observed, the animal is sufficiently anesthetized to start the experiments.</li>
		<li>Place the tumor site under the PDT fiber (with 1 cm of distance from the PDT fiber to the tumor). Wear laser safety glasses, turn off the switch, and illuminate the tumor with a PDT laser (630 nm, 400 mW/cm2) for 500 sec (200 J/cm2) twice on days 1 and 8.</li>
		<li>Keep observing the mice until they start to move around the cage. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency. Do not return an animal that has undergone surgery to the company of other animals until fully recovered.</li>
		<li>Keep the cages under SPF conditions. Maintain the cages in the dark for 24 hr after laser treatment.</li>
		<li>Visually monitor the tumor volume and the changes at the tumor site every day. Measure the tumor size with calipers, and calculate the volume as (length &#215; width2) / 2 (mm3). Take pictures of the tumor sites every day to check for tumor surface alterations after PDT.</li>
		<li>On day 16, sacrifice five mice per group by terminal anesthesia using isoflurane.</li>
		<li>With forceps and scissors, cut the outer skin and expose the tumors15. Carefully harvest them. Fix them in 10% formalin, embed them in paraffin, and stain them with hematoxylin and eosin (H&#38;E) for the histological analysis16.</li>
		<li>After 45 days of monitoring, sacrifice the remaining mice by terminal anesthesia using isoflurane.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

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.

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.

<table><tbody><tr><td>oligo hyaluronic acid</td><td>Bioland Co., Ltd.</td><td>_</td><td></td></tr><tr><td>DS-Y30 (ceramide 3B; mainly &lt;em&gt;N&lt;/em&gt;-oleoyl-phytosphingosine)</td><td>Doosan Biotech Co., Ltd.</td><td>_</td><td></td></tr><tr><td>adipic acid dihydrazide</td><td>Sigma Aldrich</td><td>A0638</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-(3-dimethylaminopropyl)-&lt;em&gt;N&amp;prime;&lt;/em&gt;-ethylcarbodiimide</td><td>Sigma Aldrich</td><td>39391</td><td></td></tr><tr><td>4-(chloromethyl)benzoyl chloride</td><td>Sigma Aldrich</td><td>270784</td><td></td></tr><tr><td>Tween 80</td><td>Tokyo Chemical Industry Co., Ltd.</td><td>T0546</td><td></td></tr><tr><td>syringe filter</td><td>Sartorius Stedim Biotech GmbH</td><td>17762</td><td>15 mm, RC, PP, 0.45 &amp;micro;m</td></tr><tr><td>triethylamine</td><td>Sigma Aldrich</td><td>T0886</td><td></td></tr><tr><td>Mini-GeBAflex tubes</td><td>Gene Bio-Application Ltd.</td><td>D070-12-100</td><td></td></tr><tr><td>Paclitaxel</td><td>Taihua Corporations</td><td>_</td><td></td></tr><tr><td>RPMI-1640</td><td>Gibco Life Technologies, Inc.</td><td>11875</td><td></td></tr><tr><td>Penicillin&amp;ndash;streptomycin</td><td>Gibco Life Technologies, Inc.</td><td>15070</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco Life Technologies, Inc.</td><td>16140071</td><td></td></tr><tr><td>Celite (Filter agent)</td><td>Sigma Aldrich</td><td>6858</td><td>See step 1.4</td></tr></tbody></table>

anticancer efficacy of photodynamic therapy with lung cancer-targeted nanoparticles

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.

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 &#177; 3.2 nm and 211.9 &#177; 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 &#177; 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 &#177; 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).
<img alt="Figure 1" src="/files/ftp_upload/54865/54865fig1.jpg" /> 
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 &#177; standard deviations (SD; n = 3). *** p &#60; 0.001, compared to the PBS-treated group. ## p &#60; 0.01, ### p &#60; 0.001, compared to the HB-NP treatment group. Adapted from Chang et al.10 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54865/54865fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54865/54865fig2.jpg" /> 
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 &#181;m, magnification = 100X. Adapted from Chang et al.10 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54865/54865fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54865/54865fig3.jpg" /> 
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 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54865/54865fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54865/54865fig4.jpg" /> 
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 &#60; HB-NPs &#60; 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 &#177; standard deviations (SD; n = 4). The tumor volume (mm3) was defined as (length &#215; width2) / 2. Adapted from Chang et al.10 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54865/54865fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54865/54865fig5.jpg" /> 
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 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54865/54865fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54865/54865fig6.jpg" /> 
Figure 6: Histological Analysis of Tumor Tissues in A549 Tumor-bearing Mice. On day 16, the histological confirmation was performed by an H&#38;E stain of excised tumor tissues. The HB-P-NPs treatment group demonstrated the most complete tumor cell death. Scale bar = 500 &#181;m, magnification = 40X. Adapted from Chang et al.10 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54865/54865fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles at JoVE.com

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

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. ...

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Nanyang Technological University

Research

JoVE Journal

Cancer Research

9.0K Views.  Seoul National University Bundang Hospital. 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.

Video: Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

Photodynamic Therapy with Blended Conducting Polymer/Fullerene Nanoparticle Photosensitizers

In this article a method for the fabrication and reproducible in-vitro evaluation of conducting polymer nanoparticles blended with fullerene as the next generation photosensitizers for Photodynamic Therapy (PDT) is reported. The nanoparticles are formed by hydrophobic interaction of the semiconducting polymer MEH-PPV (poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene]) with the fullerene PCBM (phenyl-C61-butyric acid methyl ester) in the presence of a non-compatible solvent. MEH-PPV has a high extinction coefficient that leads to high rates of triplet formation, and efficient charge and energy transfer to the fullerene PCBM. The latter processes enhance the efficiency of the PDT system through fullerene assisted triplet and radical formation, and ultrafast deactivation of MEH-PPV excited stated. The results reported here show that this nanoparticle PDT sensitizing system is highly effective and shows unexpected specificity to cancer cell lines.

This protocol describes a method for the fabrication of conducting polymer nanoparticles blended with fullerene. These nanoparticles were investigated for their potential use as a next generation photosensitizers for Photodynamic Therapy (PDT).

In this article a method for the fabrication and reproducible in-vitro evaluation of conducting polymer nanoparticles blended with fullerene as the next ...

Chemistry

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor cells, CAFs regulate tumor progression and provide protection from antitumor immunity. Emerging anticancer strategies aim to remodel the tumor microenvironment through the ablation of pro-tumorigenic CAFs or reprogramming of CAFs functions and their activation status. A promising approach is the development of nanosized delivery agents able to target CAFs, thus allowing the specific delivery of drugs and active molecules. In this context, a cellular model of CAFs may provide a useful tool for in vitro screening and preliminary investigation of such nanoformulations.
This study describes the isolation and culture of primary CAFs from the syngeneic 4T1 murine model of triple-negative breast cancer. Magnetic beads were used in a 2-step separation process to extract CAFs from dissociated tumors. Immunophenotyping control was performed using flow cytometry after each passage to verify the process yield. Isolated CAFs can be employed to study the targeting capability of different nanoformulations designed to tackle the tumor microenvironment. Fluorescently labeled H-ferritin nanocages were used as candidate nanoparticles to set up the method. Nanoparticles, either bare or conjugated with a targeting ligand, were analyzed for their binding to CAFs. The results suggest that ex vivo extraction of breast CAFs may be a useful system to test and validate nanoparticles for the specific targeting of tumorigenic CAFs.

This paper aims to provide a protocol for the isolation and culture of primary cancer-associated fibroblasts from a syngeneic murine model of triple-negative breast cancer and their application for the preclinical study of novel nanoparticles designed to target the tumor microenvironment.&#160;

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor ...

5-Aminolevulinic Acid-mediated Photodynamic Therapy on Primary Bone Tumor and Bone Metastases Cell Lines

Bone metastases are associated with poor prognosis and low quality of life for the affected patients. Photodynamic therapy (PDT) emerges as a noninvasive therapy that can target local metastatic bone lesions. This paper presents an in vitro method to study the PDT effect in adherent cell lines. To this end, we demonstrate a step-by-step approach to subject both primary (giant cell bone tumor) and human bone metastatic cancer cell lines (derived from a primary invasive ductal breast carcinoma and renal carcinoma) to 5-aminolevulinic acid (5-ALA)-mediated PDT. 
After 24 h post 5-ALA-PDT irradiation (blue light-wavelength 436 nm), the therapeutic effect was assessed in terms of cell migration potential, viability, apoptotic features, and cellular growth arrest (senescence). Post 5-ALA-PDT irradiation, musculoskeletal-derived cell lines respond differently to the same doses and exposure of PDT. Depending on the extent of cellular damage triggered by PDT exposure, two different cell fates-apoptosis and senescence were noted. Variable sensitivity to PDT therapy among different bone cancer cell lines provides useful information for selecting more appropriate PDT settings in clinical settings. This protocol is designed to exemplify the use of PDT in the context of musculoskeletal neoplastic cell lines. It may be adjusted to investigate the therapeutic effect of PDT on various cancer cell lines and various photosensitizers and light sources.

This protocol presents a step-by-step methodological approach of exposing bone metastatic cells lines and primary bone tumors to 5-aminolevulinic acid-mediated photodynamic therapy (PDT). The effects on cell migration potential/invasiveness, viability, apoptosis, and senescence potential are also analyzed following PDT exposure.

Bone metastases are associated with poor prognosis and low quality of life for the affected patients. Photodynamic therapy (PDT) emerges as a noninvasive ...

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.
Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response1. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)2.
The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.
In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

The protocol reported here allows the evaluation of the efficacy of photodynamic therapy (PDT) in cell lines and the optimization of the PDT settings before applying the therapy in animal models.

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic ...

Medicine

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...

An In-House-Built and Light-Emitting-Diode-Based Photodynamic Therapy Device for Enhancing Verteporfin Cytotoxicity in a 2D Cell Culture Model

This paper describes a novel, simple, and low-cost device to perform in vitro photodynamic therapy (PDT) assays, named the PhotoACT. The device was built using a set of conventional programmable light-emitting diodes (LEDs), a liquid crystal display (LCD) module, and a light sensor connected to a commercial microcontroller board. The box-based structure of the prototype was made with medium-density fiberboards (MDFs). The internal compartment can simultaneously allocate four cell culture multiwell microplates.
As a proof of concept, we studied the cytotoxic effect of the photosensitizer (PS) verteporfin against the HeLa cell line in two-dimensional (2D) culture. HeLa cells were treated with increasing concentrations of verteporfin for 24 h. The drug-containing supernatant medium was discarded, the adherent cells were washed with phosphate-buffered saline (PBS), and drug-free medium was added. In this study, the effect of verteporfin on cells was examined either without light exposure or after exposure for 1 h to light using red-green-blue (RGB) values of 255, 255, and 255 (average fluence of 49.1 &#177; 0.6 J/cm2). After 24 h, the cell viability was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Experimental results showed that exposure of cells treated with verteporfin to the light from the device enhances the drug&#39;s cytotoxic effect via a mechanism mediated by reactive oxygen species (ROS). In addition, the use of the prototype described in this work was validated by comparing the results with a commercial PDT device. Thus, this LED-based photodynamic therapy prototype represents a good alternative for in vitro studies of PDT.

Here, we describe a novel, simple, and low-cost device to successfully perform in vitro photodynamic therapy (PDT) assays using two-dimensional HeLa cell culture and verteporfin as a photosensitizer.

This paper describes a novel, simple, and low-cost device to perform in vitro photodynamic therapy (PDT) assays, named the PhotoACT. The device was built ...

Biochemistry

Near Infrared Photoimmunotherapy for Mouse Models of Pleural Dissemination

The efficacy of photoimmunotherapy can be evaluated more accurately with an orthotopic mouse model than with a subcutaneous one. A pleural dissemination model can be used for the evaluation of treatment methods for intrathoracic diseases such as lung cancer or malignant pleural mesothelioma.
Near-infrared photoimmunotherapy (NIR-PIT) is a recently developed cancer treatment strategy that combines the specificity of tumor-targeting antibodies with toxicity caused by a photoabsorber (IR700Dye) after exposure to NIR light. The efficacy of NIR-PIT has been reported using various antibodies; however, only a few reports have shown the therapeutic effect of this strategy in an orthotopic model. In the present study, we demonstrate an example of efficacy evaluation of the pleural disseminated lung cancer model, which was treated using NIR-PIT.

Near-infrared photoimmunotherapy (NIR-PIT) is an emerging cancer therapeutic strategy that utilizes an antibody-photoabsorber (IR700Dye) conjugate and NIR light to destroy cancer cells. Here, we present a method to evaluate the antitumor effect of NIR-PIT in a mouse model of pleural disseminated lung cancer and malignant pleural mesothelioma using bioluminescence imaging.

The efficacy of photoimmunotherapy can be evaluated more accurately with an orthotopic mouse model than with a subcutaneous one. A pleural dissemination ...

In Vitro Phototoxicity Assay: A PDT-based Method to Evaluate the Phototoxic Potential of a Photosensitizer in Lung Cancer Cells

Source: Chang, J. E., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/54865/anticancer-efficacy-photodynamic-therapy-with-lung-cancer-targeted">Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles.</a>&#160;J. Vis. Exp.&#160;(2016).
Photodynamic therapy (PDT) is a non-invasive and non-surgical method 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. In this protocol video, PDT-based method is used to evaluate the phototoxic potential of a potential photosensitizer in lung cancer cells.

Source: Chang, J. E., et al. Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles.&#160;J. Vis. Exp.&#160;(2016).
...

JoVE Encyclopedia of Experiments

Lung Cancer

In vitro study

In Vivo Antitumor Efficacy Analysis of PDT: A Technique to Determine the Phototoxic Potential of a Photosensitizer in Tumor Bearing Mice

Source: Chang, J. E., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/54865/anticancer-efficacy-photodynamic-therapy-with-lung-cancer-targeted">Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles.</a> J. Vis. Exp. (2016).
Photodynamic therapy (PDT) is a non-invasive and non-surgical method 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. This video describes the in vivo anti-tumor efficacy analysis using photosensitizer containing nanoparticles in a tumor-bearing mouse, to determine the phototoxic potential of the photosensitizer.

Source: Chang, J. E., et al. Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles. J. Vis. Exp. (2016).
Photodynamic ...

Anticancer Efficacy of Photodynamic Therapy with Lung Cancer-Targeted Nanoparticles

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