Name: anticancer efficacy of photodynamic therapy with lung cancer-targeted nanoparticles
Uploaded: 2016-12-01T13:00:00
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 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 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. ...

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