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 2...

<ol>
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(98), e52462 (2015).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematoxylin+and+eosin+staining+of+tissue+and+cell+sections.">Hematoxylin and eosin staining of tissue and cell sections.</a> Cold Spring Harbor Protocols. (5), (2008).</li><li>Lam, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+of+lung+cancer.">Photodynamic therapy of lung cancer.</a> Semin Oncol. 21 (6 Suppl 15), 15-19 (1994).</li><li>Simone, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photodynamic+therapy+for+the+treatment+of+non-small+cell+lung+cancer.">Photodynamic therapy for the treatment of non-small cell lung cancer.</a> J Thorac Dis. 4 (1), 63-75 (2012).</li></ol>

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,...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>oligo hyaluronic acid</td>
			<td>Bioland Co., Ltd.</td>
			<td>_</td>
			<td></td>
		</tr>
		<tr>
			<td>DS-Y30 (ceramide 3B; mainly N-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>N-(3-dimethylaminopropyl)-N&#8242;-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 &#181;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&#8211;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 cytot...

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. ...

The overall goal of this experiment is to increase the therapeutic effect of photodynamic therapy in treating lung cancer by using both photosensitizer and anticancer drug-loaded, lung cancer-targeted nanoparticles. This method can help answer key questions in the photodynamic therapy field such as what are the proper agent conditions for in vitro and in vivo studies. Since photodynamic therapies are widely used in the cancer fields, pre-cancer studies with normal photosensitizers or new indications may be performed with this technique.The implications of this technique extended towards therapy of various diseases such as tuberculosis, ulcers and dermatology fields using different annual modules. To begin, synthesize hyaluronic acid ceramide as described in the accompanying text protocol. Purify the product using a 3.5 kilodalton dialysis membrane and myophalize it.Next, prepare hyaluronic acid-ceramide nanoparticles by themselves. Hyaluronic acid-ceramide nanoparticles along with a photosensitizer and hyaluronic acid-ceramide nanoparticles with an anticancer agent and the photosensitizer. Seed the lung cancer cell line, A549, into the wells of a 24-well cell culture plate at a density of one time 10 to the fifth cells per well and incubate the cells for 24 hours.The next day, remove the medium and wash the cells with one milliliter of PBS. Then, dissolve the prepared nanoparticles in PBS to a final concentration of two micromoles per milliliter of the photosensitizer Hypocrellin B by vortexing. Into four wells each and one milliliter of PBS, one milliliter of the empty hyaluronic acid-ceramide nanoparticles, one milliliter of the nanoparticles containing the photosensitizer and one milliliter of the nanoparticles containing both the photosensitizer and the anticancer drug.When finished, cover the plate and incubate it at 37 degrees Celsius for four hour in the dark to allow for nanoparticle uptake into the cells. Then, remove all of the solution and wash the cells by adding one milliliter of cold PBS. Repeat the washing step once more and then add fresh culture medium.Next, place the 24-well culture plate under a photodynamic therapy fiber and position the fiber one centimeter from the first well. The distensae from the photodynamic therapy fiber to the cells was the critical factor in this study. It was important to find out the optimal distance which can cover the whole cell with the light.Then, put on safety glasses and turn out the lights. Illuminate the cells with the laser in the dark for between five and 40 seconds to activate highly reactive singlet oxygen and other free radicals in the photosensitizer containing nanoparticles. Following light exposure, incubate the cells in the dark for 24 hours.Next, aspirate the culture medium and wash the cells with one milliliter of cold PBS. Repeat the wash step once more and then add 100 microliters of media and 10 microliters of cytotoxicity measuring solution to each well. Incubate cells in the solution for two hours in the dark and then move all the samples to a 96-well plate.Measure the absorbance at 450 nanometers using a microplate reader. Harvest A549 cells and re-suspend one million cells in 0.1 milliliters of RPMI-1640 medium. Place the re-suspended cells on ice.Next, prepare a BALB/C male nude mouse for injection. Then, load the cells into a one-milliliter syringe equipped with a 26-gauge needle and inject them into the left flank of the mouse. Measure the tumor size with calipers every day.When the tumor size reaches approximately 200 cubed millimeters in volume, start the experiment by first weighting each mouse. Then, dissolve the nanoparticles in PBS to a final concentration of two milligrams per milliliter and load one microliter of the suspension per gram of mouse into a one-milliliter syringe equipped with a 26-gauge needle. Inject the nanoparticles via tail vein injection on day zero and seven.24 hours after each injection, anesthetize the mouse and place the tumor site under the photodynamic therapy fiber so that it is one centimeter from the fiber. It is important to cover the entire tumor surface with the light in order to achieve the best results. Put on the laser safety glasses, turn off the room switch and illuminate the tumor with a photodynamic therapy laser for 500 seconds on both days one and eight.Following laser treatment, maintain the mice in their cages in the dark for 24 hours. Visually monitor the tumor volume and the changes of the tumor site every day. Use calipers to measure the tumor's size and take pictures of the tumor sites every day to check for surface alterations.Lung cancer cell viability is shown here after four hours of incubation with the nanoparticles followed by light irradiation. Without light, the cells exposed to the nanoparticles containing only photosensitizer showed no sign of toxicity at all, while cells exposed to nanoparticles containing both the photosensitizer and paclitaxel showed about an 81%cell viability. With increasing irradiation time, the cell viability was decreased in both treatment groups with the photosensitizer.The treatment group also containing paclitaxel showed the greatest amount of phototoxicity. This was also reflected in the tumor model study as the tumor volume in mice treated with these two treatment groups were lower than the controls. In addition, the group treated with the paclitaxel and photosensitizer containing nanoparticles was significantly lower than all the other groups.Although the tumor size was significantly smaller, the mice treated with nanoparticles having both the photosensitizer and paclitaxel showed severe hemorrhaging and the fastest development of necrosis. After the development, this technique paved the way for laser treats in the field of photodynamic therapy to explore cancer treatment in in vitro and in vivo systems. After watching this video, you should have a good understanding of how to purport photodynaic therapy in vitro and in vivo systems.

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8.9K Views.  Seoul National University Bundang Hospital. The overall goal of this experiment is to increase the therapeutic effect of photodynamic therapy in treating lung cancer by using both photosensitizer and anticancer drug-loaded, lung cancer-targeted nanoparticles. This method can help answer key questions in the photodynamic therapy field such as what are the proper agent conditions for in vitro and in vivo studies. Since photodynamic therapies are widely used in the cancer fields, pre-cancer studies with normal photosensitizers or new indi...