The authors gratefully acknowledge the National Science Foundation (NSF) for financial support of this work through a CAREER award (CBET-0746210) and through award CBET-1159500. We would like to thank Dr. Turkson (Univ. of Hawaii Cancer Center) and Dr. Altomare (Univ. of Central Florida College of Medicine) for assistance with cell culture.

1. Culturing Cell Lines
<ol>
	<li>Thaw TE 71 (Mouse thymic epithelial cells), MDA-MB-231 (Human breast cancer cells), A549 (Human lung cancer cells) and OVCAR3 (Human ovarian tumor cells) by holding the cryogen vials in warm water for less than 2 min. Add 10 ml DMEM media supplemented with 10% FBS to each cell line and centrifuge for 6 min at 106 x g.</li>
	<li>Aspirate the suspension and add 3ml media to the pellet. Mix the cells properly by pipetting several times. Add this cell solution to pre-warmed 7 ml DMEM media supplemented with 10% FBS in T75 flasks and keep the flasks in humidified atmosphere of 95% air/ 5% CO2 at 37 &#176;C. Label this flask as Passage 0.</li>
	<li>When the confluency of the cells reaches 80%, harvest the cells by incubating them with 0.05% trypsin for 10 min. Neutralize the trypsin by adding equal amount of media. Centrifuge this solution for 6 min at 106 x g. Remove the suspension and add 3 ml fresh media to it. Mix well and transfer small amount (100 &#181;l) to a culture flask containing 7 ml media. Incubate the culture flask in incubator. Label the flask as Passage 1.</li>
	<li>Culture the cell lines until passage 11 or 12.</li>
</ol>
2. Fabrication of Nanoparticles
<ol>
	<li>Preparation of ~10-6 M (adjusted) undiluted MEH-PPV stock solution
	<ol>
		<li>In a vial add 1mg Poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) with molecular weight (average Mn) 150,000-250,000 g/mol and 3 ml tetrahydrofuran (THF). Stir the mixture for 2 hr while heating at 80 &#176;C on a hot plate.</li>
		<li>Filter the above solution in a new vial using a 0.2 &#181;m syringe filter. Label this solution as &#8216;undiluted MEH-PPV stock solution&#8217;. This solution will be involved in nanoparticle preparation after fine tuning of the concentration as described in steps 2.1.3 and 2.1.4.</li>
		<li>Add 50 &#181;l of the undiluted MEH-PPV stock solution into 3 ml THF. Label this solution as &#8216;diluted MEH-PPV stock solution&#8217;. Transfer to a 1 cm quartz cuvette and measure the absorbance at 495 nm by UV-vis spectroscopy.</li>
		<li>If the absorbance of the diluted MEH-PPV stock solution is higher than 0.17, dilute the undiluted MEH-PPV stock solution by adding more THF 1 ml at a time, and repeat step 2.1.3 until the measured absorbance at 495 nm is in the range 0.13-0.17.</li>
		<li>Calculate the molarity of the diluted MEH-PPV stock solution by using Lambert-Beer law as shown below. Here 0.15 absorbance is used as an example for the remainder of the protocol (i.e., adjust all following calculations based on observed absorbance), the MEH-PPV extinction coefficient used is 107 M-1 cm-1 and the path length used is 1 cm. 
		A = &#949; x b x c (Eqn 1) 
		(&#949; - the molar extinction coefficient, A &#8211; absorbance, b &#8211; path length, c &#8211; concentration of the solution) 
		0.15 = 107 M-1 cm-1 x 1 cm x c (Eqn 2) 
		c = 1.5 x 10-8 M (Eqn 3) 
		Use this concentration to find the concentration of the undiluted MEH-PPV stock solution as follows: 
		M1V1 = M2V2 (Eqn 4) 
		0.15 x 10-7 M x 3050 &#181;l = M2 x 50 &#181;l (Eqn 5) 
		M2 = 9.15 x 10-7 M (Eqn 6) 
		This is the concentration of the &#8216;adjusted undiluted MEH-PPV stock solution&#8217;.</li>
	</ol>
	</li>
	<li>Calculating mass of MEH-PPV in the adjusted undiluted MEH-PPV stock solution
	<ol>
		<li>Calculate the mass of MEH-PPV in 1 ml of the adjusted undiluted stock solution as shown below using the molarity obtained in section 2.1.4 and the molecular weight (Mw) of MEH-PPV, which is 106 g/mol. 
		M = n/V (n &#8211; no. of moles, V &#8211; volume in L) (Eqn 7) 
		Thus, the mass of MEH-PPV in 1 ml of adjusted undiluted stock solution is 9.15 x 10-4 g.</li>
	</ol>
	</li>
	<li>Blending PCBM in MEH-PPV
	<ol>
		<li>Calculate the mass of Phenyl-C61-butyric acid methyl ester (PCBM) to be added into the stock MEH-PPV solution to make 50 wt% PCBM doped MEH-PPV solution, where 50 wt% PCBM is defined with respect to the mass of MEH-PPV (i.e., half the mass of MEH-PPV). By using the weight of MEH-PPV obtained in section 2.2. 
		Mass of PCBM/9.15 x 10-4 g of MEH-PPV x 100% = 50 wt% PCBM (Eqn 8) 
		Mass of PCBM = 4.57 x 10-4 g (Eqn 9)</li>
		<li>Weigh 1 mg PCBM in a vial and add 500 &#181;l THF. Calculate the concentration of PCBM in this solution using the molecular weight of PCBM as 910.88 g/mol 
		Molarity of PCBM solution = 0.001 g/(910.88 g/mol * 500 &#181;l)(Eqn 10) 
		Molarity of PCBM solution = 2.19 x 10-9 mol/&#181;l(Eqn 11)</li>
		<li>Calculate the volume of PCBM solution needed to add to the adjusted undiluted MEH-PPV stock solution to obtain the 50 wt% PCBM doped MEH-PPV solution in THF by using the molarity calculated in step 2.3.2 
		4.57 x 10-4 g of PCBM x 1 mol/910.88 g x 1 &#181;l/2.19 x 10-9 mol = 229 &#181;l (Eqn 12) 
		Add 229 &#181;l of the PCBM solution into 1 ml of adjusted undiluted MEH-PPV stock solution and mix well.</li>
	</ol>
	</li>
	<li>Preparation of nanoparticles by reprecipitation method
	<ol>
		<li>Transfer 1 ml of the blended MEH-PPV/PCBM solution into the 1 ml syringe with the needle attached to it.</li>
		<li>Rapidly inject 1 ml of the blended MEH-PPV/PCBM solution into 4 ml of DI water stirring at 1,200 rpm. Stop stirring immediately after injection. Use these nanoparticles without further processing.</li>
	</ol>
	</li>
</ol>
3. Incubation of Cell Lines with Nanoparticles for Imaging
NOTE: All the imaging experiments were completed in 35 mm petri dishes
<ol>
	<li>Uptake of nanoparticles in cell lines
	<ol>
		<li>Culture cell lines up to the 12th passage. At the 12th passage culture cells in 35 mm Petri dishes. Adjust concentration of cells added to the 35 mm Petri dishes such that after 24 hr the cells are 40% confluent.</li>
		<li>At this stage remove the DMEM media supplemented with 10% FBS from the petri dishes, wash the cells with 1x DPBS twice, and add 100 &#181;l of the nanoparticles suspension into 2 ml DMEM to the Petri dishes.</li>
		<li>After 24 hr remove the DMEM/nanoparticles suspension from the petri dishes and wash the cells with 1x DPBS 3 times. Then fix the cells by incubating with 4% paraformaldehyde for 10 min. Wash twice with DPBS. Stain the cells with 300 nM DAPI by incubating with the dye for 2 min. Wash twice with DPBS. Then keep the cells in DPBS for imaging.</li>
	</ol>
	</li>
	<li>Detection of ROS
	<ol>
		<li>Culture A549 and OVCAR3 cell lines in Petri dishes, 6 per cell line, as explained in section 3.1.1. Label the Petri dishes as shown in the Table 1.</li>
		<li>Add 100 &#181;l of the nanoparticle suspension into 2 ml DMEM to three of the petri dishes as shown in the Table 1 and incubate for 24 hr. For the Petri dishes that will receive light doses, wash the cells after 24 hr and suspend the cells in HBSS (Hank&#8217;s Balanced Salt Solution) dye free media.</li>
		<li>Warm up the lamp of the solar simulator for 15 min. Place UV filter in front of the lamp to filter out UV light. Calibrate the lamp with a reference solar cell by adjusting the lamp power to obtain 0.5 sun (50 mW/cm2) intensity at the surface of the petri dish. In this particular setup that condition was achieved with 218 W power supplied to the lamp.</li>
		<li>Place the petri dishes under the lamp (lid open) for 60 min, which results in a light dosage of 180 J/cm2 as shown in the calculations below: 
		50 mW/cm2 x 3,600 sec/1,000 = 180 J/cm2</li>
		<li>Remove HBSS and without washing further add 2 ml DMEM media supplemented with 10% FBS to the petri dishes. Incubate the cells for another 2 hr.</li>
		<li>For the positive control incubate the cells with 100 &#181;M hydrogen peroxide (H2O2) for 30 min.</li>
		<li>Stain the cells in all the petri dishes with a final concentration of 5 &#181;M of the ROS detecting reagent by incubating the cells with the dye for 30 min at 37 &#176;C.</li>
		<li>Fix the cells by incubating with 4% paraformaldehyde for 10 min. Wash twice with DPBS. Stain the cells with 300 nM DAPI by incubating with the dye for 2 min. Wash twice with DPBS. Then keep the cells in DPBS for imaging.</li>
	</ol>
	</li>
	<li>Apoptosis and necrosis by PI and annexin V FITC
	<ol>
		<li>Culture each cell line in 5 Petri dishes. Three of these Petri dishes will be for experiment while the remaining 2 petri dishes will be control samples. Incubate the 3 Petri dishes for experiment with nanoparticles as explained in section 3.1.2. The control samples are not incubated with nanoparticles.</li>
		<li>After 24 hr incubation follow the step 3.2.3.</li>
		<li>Place the 3 experimental petri dishes under the lamp (lid open) and remove one at 20 min, one at 40 min and one at 60 min. This yields three samples that were exposed to light doses of 60, 120 and 180 J/cm2, respectively (calculation in section 3.2.4). Next, administer a 180 J/cm2 light dosage to one of the control petri dishes. This is the control with light dose applied in the absence of nanoparticles. Do not apply light dose to the remaining control sample (no nanoparticles and no light dose applied).</li>
		<li>Replace the HBSS with DMEM media supplemented with 10% FBS and keep the petri dishes in the incubator for 4 hr.</li>
		<li>Stain the cells in the experimental and the control petri dishes with 20 &#181;l of annexin V FITC by incubating the cells with the dye for 15 min. Wash twice with DPBS. Stain the cells with 300 nM DAPI as well as 300 nM PI (propidium iodide) by incubating with the dyes for 2 min. Wash twice with DPBS. Then keep the cells in DPBS for imaging.</li>
	</ol>
	</li>
</ol>
4. Intrinsic Cytotoxicity of Nanoparticles
<ol>
	<li>Counting cells and culturing in 96-well plates
	<ol>
		<li>Harvest the cells from culture flasks by removing media and washing the cells twice with DPBS followed by incubation of the cells with 0.05% trypsin for 10 min. Add 2 ml DMEM media supplemented with 10% FBS to the resulting cell solution. Mix the solution properly to separate cell clusters into singlets.</li>
		<li>Take 100 &#181;l of the cell suspension and add to 900 &#181;l DMEM media supplemented with 10% FBS. Mix well. Place 10 &#181;l of this suspension onto a hemocytometer. Count the cells using the hemocytometer and adjust the concentration of the cell suspension in 4.1.1 to 5 x 104 cells/ml.</li>
		<li>Take 5 96-well plates labeled as 0 hr, 24 hr, 48 hr, 72 hr and 96 hr. Add 50 &#181;l of the 5 x 104 cells/ml cell solutions (TE 71 and A549) into the wells as shown in Table 2, thus seeding 2,500 cells/well. Do the same procedure for OVCAR3 and MDA-MB-231 cell lines.</li>
		<li>After 24 hr wash the wells with 1x DPBS and add 50 &#181;l of increasing concentrations of nanoparticles into the wells as shown in Table 2. Prepare different nanoparticle concentrations by adding 20, 100, and 180 &#181;l of nanoparticle suspension from 2.4.2 to DMEM to obtain a final volume of 2 ml, which yields nanoparticle concentrations of 0.4 x 10-4 mg/ml, 2.0 x 10-4 mg/ml, and 3.6 x 10-4 mg/ml, respectively. Each cell line has triplicates for each concentration of nanoparticles.</li>
	</ol>
	</li>
	<li>Measuring the cell viability in dark
	<ol>
		<li>Add 10 &#181;l MTT to the 0 hr plate immediately after adding nanoparticles. Incubate the plate for 4 hr for formazan crystals to form. Add 50 &#181;l solubilization solution into the wells. Incubate the plate for 6 hr to dissolve the formazan crystals.</li>
		<li>Measure the cell viability of the 0 hr plate by recording the absorbance at 570 nm with microplate reader.</li>
		<li>For 24 hr plate, wash the cells with 1x DPBS and add 50 &#181;l media in each well after 24 hr incubation with nanoparticles. Add MTT and read the plate as explained in 4.2.1 and 4.2.2.</li>
		<li>For 48 hr, 72 hr, and 96 hr plates, wash the cells with 1x DPBS and add 50 &#181;l media into the wells after 24 hr incubation with nanoparticles. Incubate the cells for the remaining time periods.</li>
		<li>4.2.5) Measure the cell viability at the particular time points as explained in 4.2.1 and 4.2.2.</li>
		<li>Repeat the complete experiment for 3 times (n = 3)</li>
	</ol>
	</li>
</ol>
5. Measuring Cell Viability After PDT
<ol>
	<li>Counting cells and culturing in 96-well plates.
	<ol>
		<li>Label a set of 96-well plates as shown in Table 3, both for the TE 71 + A549 plates and OVCAR3 + MDA-MB-231 plates. The layout of the plates will be the same as shown in 4.1.3.</li>
		<li>Seed the 96-well plates as explained in section 4.1 with the same layout as shown in Table 2 in section 4.1.2.</li>
		<li>After 24 hr add nanoparticles to the plates as explained in 4.1.4.</li>
		<li>24 hr after addition of nanoparticles wash the cells with 1x DPBS and add 50 &#181;l HBSS to each well.</li>
		<li>Irradiate the plates with the respective light doses as explained in 3.2.3.</li>
		<li>Replace the HBSS with 50 &#181;l DMEM media supplemented with 10% FBS and incubate the plates for the respective time periods after PDT.</li>
		<li>After each time period measure the cell viability as explained in 4.2.1 and 4.2.2.</li>
	</ol>
	</li>
</ol>
6. Fluorescence Microscopy
<ol>
	<li>Uptake of nanoparticles
	<ol>
		<li>Turn on the lamps of the microscope and the laser 30 min before imaging. Put the petri dish containing the fixed cells (as explained in section 3.1) on the stage of the microscope.</li>
		<li>Collect the fluorescence from nanoparticles and DAPI by using filters as shown in Table 4. Overlay the phase contrast, nanoparticle and DAPI images in ImageJ software.</li>
	</ol>
	</li>
	<li>Detection of ROS
	<ol>
		<li>Turn on the lamps of the microscope 30 min before imaging. Put the petri dish containing the cells (as explained in section 3.2) on the stage of the microscope.</li>
		<li>Collect the fluorescence from nanoparticles, DAPI, and the ROS detecting reagent by using filters as shown in Table 4. Overlay the phase contrast, nanoparticle, DAPI and ROS detecting reagent images in ImageJ software.</li>
	</ol>
	</li>
	<li>Apoptosis and necrosis by PI and annexin V FITC
	<ol>
		<li>Turn on the lamps of the microscope 30 min before imaging. Put the Petri dish containing the cells (as explained in section 3.3) on the stage of the microscope.</li>
		<li>Collect the fluorescence from nanoparticles, DAPI, PI, and Annexin V FITC by using filters as shown in Table 4. Overlay the phase contrast, nanoparticle, DAPI, PI and Annexin V FITC images in ImageJ software.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

To achieve nanoparticle uptake it was necessary to maintain some critical measures while fabricating the nanoparticles. A 10-6 M MEH-PPV solution (blended with 50 wt% PCBM) in THF was prepared to inject into DI water, as it was observed that the concentration of this solution plays an important role in determining the size of nanoparticles being formed. Concentration was checked by UV-vis spectroscopy. Note that in protocol step 2.1.3 it was necessary to dilute the initially prepared MEH-PPV solution (undilute...

In Photodynamic Therapy (PDT) photosensitizers are administered to target tissue, and upon exposure to light the photosensitizer generates Reactive Oxygen Species (ROS). ROS species such as singlet oxygen and superoxide can induce oxidative stress and subsequent structural damage to cells and tissue1-4. Due to its ease of application this method has been actively investigated and clinical trials have taken place5,6. However, significant issues such as dark toxicity of the sensitizers, patient sensitivity to light (due to non-selective distribution of the sensitizer), and hydrophobicity of the sensitizers (which leads to reduced bioavailability and potential acute toxicity) remain.
Here we report a method for the fabrication and in-vitro evaluation of conducting polymer nanoparticles blended with fullerene as the next generation photosensitizers for PDT. The nanoparticles are formed by self-aggregation 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) when these materials dissolved in a compatible solvent are rapidly injected into a non-compatible solvent (Figure 1A). The choice of MEH-PPV as the host polymer is motivated by its high extinction coefficient that leads to high rates of triplet formation, and both efficient and ultrafast charge and energy transfer to the fullerene PCBM7. These properties are ideal for sensitization of singlet oxygen and superoxide formation in PDT.
Fullerene has in fact been applied in PDT in both molecular and nanoparticle form8-13. However, severe cytotoxicity has hampered further development12. Here we show that encapsulating the fullerene in a host matrix of MEH-PPV to yield composite MEH-PPV/PCBM nanoparticles results in a PDT sensitizing material that is not intrinsically cytotoxic, shows specificity towards cancer cells due to nanoparticle size and surface charge, and yields highly effective PDT treatment at low light doses due to the aforementioned photophysical properties.

<table><tbody><tr><td>Poly[2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV)</td><td>Sigma Aidrich</td><td>536512-1G</td><td>average M&lt;sub&gt;n&lt;/sub&gt;&amp;nbsp;150,000-250,000</td></tr><tr><td>[6,6]-Phenyl C61&amp;nbsp;butyric acid methyl ester (PCBM)</td><td>Sigma Aidrich</td><td>684449-500MG</td><td>&gt;99.5%</td></tr><tr><td>Tetrahydrofuran (THF)</td><td>EMD</td><td>TX0284-6</td><td>Drisolv</td></tr><tr><td>1 ml syringe</td><td>National Scientific Company</td><td>37510-1</td><td>For filtration of MEH-PPV solution</td></tr><tr><td>Syringe filter</td><td>VWR</td><td>28145-495</td><td>25 mm, 0.2 &amp;micro;m, PTFE</td></tr><tr><td>1 ml syringe</td><td>Hamilton Company</td><td>81320</td><td>For injection of MEH-PPV solution into water to make nanoparticles</td></tr><tr><td>Dulbecco&#039;s Modification of Eagle&#039;s Medium/Ham&#039;s F-12 50/50 Mix (DMEM)</td><td>Corning (VWR)</td><td>45000-350</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution without phenol red (HBSS)</td><td>Quality Biological (VWR)</td><td>10128-740</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline, 1x without calcium and magnesium (DPBS)</td><td>Corning (VWR)</td><td>45000-436</td><td></td></tr><tr><td>Fetal Bovine Serum, Regular (Heat Inactivated) (FBS)</td><td>Corning (VWR)</td><td>45000-736</td><td></td></tr><tr><td>Trypsin EDTA 1x 0.25%</td><td>Corning (VWR)</td><td>45000-664</td><td>Trypsin/2.21 mM EDTA in HBSS without sodium bicarbonate, calcium and magnesium Porcine Parvovirus Tested</td></tr><tr><td>16% Paraformaldehyde</td><td>Electron Microscopy Sciences&amp;nbsp;</td><td>15710</td><td>16% paraformaldehyde is diluted to 4% by adding PBS</td></tr><tr><td>DAPI</td><td>&amp;nbsp;Biotium VWR</td><td>89139-054</td><td>Nuclear stain</td></tr><tr><td>5 ml pipettes</td><td>VWR</td><td>82050-478</td><td></td></tr><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; culture flask</td><td>VWR</td><td>82050-856</td><td>for culturing cells</td></tr><tr><td>96-well plates</td><td>VWR</td><td>82050-771</td><td>for MTT assays</td></tr><tr><td>Tissue Culture Dishes with Vents</td><td>Greiner Bio-One (VWR)</td><td>82050-538</td><td></td></tr><tr><td>Propidium iodide</td><td>Molecular probes</td><td>P3566</td><td></td></tr><tr><td>Annexin V FITC</td><td>Invitrogen</td><td>A13199</td><td>dye for apoptosis</td></tr><tr><td>Celltiter 96 non-R 1000 assays</td><td>Promega (VWR)</td><td>&lt;a href=&quot;https://www.vwrsp.com/catalog/product/index.cgi?catalog_number=PAG4000&quot;&gt;PAG4000&lt;/a&gt;</td><td>MTT</td></tr><tr><td>CellROX Green Reagent, for oxidative stress detection</td><td>Invitrogen</td><td>C10444</td><td>For ROS detection</td></tr><tr><td>UV-vis spectrometer</td><td>Agilent 8453</td><td></td><td></td></tr><tr><td>Fluorescence spectrometer</td><td>NanoLog HoribaJobin Yvon</td><td></td><td></td></tr><tr><td>Dynamic light scattering</td><td>PD2000DLS, Precision detector</td><td></td><td></td></tr><tr><td>Incubator</td><td>NuAir DH Autoflow</td><td></td><td></td></tr><tr><td>Confocal microscope</td><td>Zeiss Axioskop2</td><td></td><td>63X oil immersion objective lens</td></tr><tr><td>Epiluminescence microscope</td><td>Olympus IX71</td><td></td><td>60X water immersion objective lens, Andor Zyla sCMOS camera</td></tr><tr><td>Solar Simulator</td><td>Newport 67005 Oriel Instruments</td><td></td><td></td></tr><tr><td>Reference solar cell</td><td>Oriel&amp;nbsp;</td><td></td><td>VLSI Standards Incorporated</td></tr><tr><td>Microplate reader</td><td>BioTek Ex808</td><td></td><td></td></tr><tr><td>Hemocytometer</td><td>Hausser Scientific Partnership</td><td>3200</td><td>For counting cells</td></tr></tbody></table>

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.

Uptake and intrinsic cytotoxicity of nanoparticles
The 50 wt% blended MEH-PPV/PCBM nanoparticles were incubated with TE 71, MDA-MB-231, A549 and OVCAR3 cell lines. The PCBM blending level was chosen as 50 wt% PCBM, which has been shown to provide ideal charge and energy transfer properties between conjugated polymers and fullerenes14. Fluorescence images of nanoparticle uptake are shown in Figure 1B. Cells were incubated for 24 hr with nanoparticles to ensure nanopa...

Watch this Scientific Journal Video about Photodynamic Therapy with Blended Conducting Polymer/Fullerene Nanoparticle Photosensitizers at JoVE.com

Photodynamic Therapy with Blended Conducting Polymer/Fullerene Nanoparticle Photosensitizers

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 ...

photodynamic-therapy-with-blended-conducting-polymerfullerene

Research

JoVE Journal

Chemistry

8.5K Views.  University of Central Florida. 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). 

Video: Photodynamic Therapy with Blended Conducting Polymer/Fullerene Nanoparticle Photosensitizers

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.

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. ...

Cancer Research

A 'Plug and Play' Method to Create Water-dispersible Nanoassemblies Containing an Amphiphilic Polymer, Organic Dyes and Upconverting Nanoparticles

In this protocol, we first describe a procedure to synthesize lanthanide doped upconverting nanoparticles (UCNPs). We then demonstrate how to generate amphiphilic polymers in situ, and describe a protocol to encapsulate the prepared UCNPs and different organic dye molecules (porphyrins and diarylethenes) using polymer shells to form stable water-dispersible nanoassemblies. The nanoassembly samples containing both the UCNPs and the diarylethene organic dyes have interesting photochemical and photophysical properties. Upon 365 nm UV irradiation, the diarylethene group undergoes a visual color change. When the samples are irradiated with visible light of another specific wavelength, the color fades and the samples return to the initial colorless state. The samples also emit visible light from the UCNPs upon irradiation with 980 nm near-infrared light. The emission intensity of the samples can be tuned through alternate irradiation with UV and visible light. Modulation of fluorescence can be performed for many cycles without observable degradation of the samples. This versatile encapsulation procedure allows for the transfer of hydrophobic molecules and nanoparticles from an organic solvent to an aqueous medium. The polymer helps to maintain a lipid-like microenvironment for the organic molecules to aid in preservation of their photochemical behavior in water. Thus this method is ideal to prepare water-dispersible photoresponsive systems. The use of near-infrared light to activate upconverting nanoparticles allows for lower energy light to be used to activate photoreactions instead of more harmful ultraviolet light.

Organic dye molecules and oleic acid coated upconverting nanoparticles are not water-soluble. This protocol describes a &#8216;plug and play&#8217; method that enables the transfer of organic dye molecules and upconverting particles from their initial hydrophobic solvent to water.

In this protocol, we first describe a procedure to synthesize lanthanide doped upconverting nanoparticles (UCNPs). We then demonstrate how to generate ...

Electroactive Polymer Nanoparticles Exhibiting Photothermal Properties

A method for the synthesis of electroactive polymers is demonstrated, starting with the synthesis of extended conjugation monomers using a three-step process that finishes with Negishi coupling. Negishi coupling is a cross-coupling process in which a chemical precursor is first lithiated, followed by transmetallation with ZnCl2. The resultant organozinc compound can be coupled to a dibrominated aromatic precursor to give the conjugated monomer. Polymer films can be prepared via electropolymerization of the monomer and characterized using cyclic voltammetry and ultraviolet-visible-near infrared (UV-Vis-NIR) spectroscopy. Nanoparticles (NPs) are prepared via emulsion polymerization of the monomer using a two-surfactant system to yield an aqueous dispersion of the polymer NPs. The NPs are characterized using dynamic light scattering, electron microscopy, and UV-Vis-NIR-spectroscopy. Cytocompatibility of NPs is investigated using the cell viability assay. Finally, the NP suspensions are irradiated with a NIR laser to determine their effectiveness as potential materials for photothermal therapy (PTT).

A protocol is presented for the synthesis and preparation of nanoparticles consisting of electroactive polymers.

A method for the synthesis of electroactive polymers is demonstrated, starting with the synthesis of extended conjugation monomers using a three-step ...

Engineering

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 ...

Optical Control of Living Cells Electrical Activity by Conjugated Polymers

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated polymers display several optimal properties as substrates for biological systems, such as good biocompatibility, excellent mechanical properties, cheap and easy processing technology, and possibility of deposition on light, thin and flexible substrates. These materials have been employed for cellular interfaces like neural probes, transistors for excitation and recording of neural activity, biosensors and actuators for drug release. Recent experiments have also demonstrated the possibility to use conjugated polymers for all-optical modulation of the electrical activity of cells. Several in-vitro study cases have been reported, including primary neuronal networks, astrocytes and secondary line cells. Moreover, signal photo-transduction mediated by organic polymers has been shown to restore light sensitivity in degenerated retinas, suggesting that these devices may be used for artificial retinal prosthesis in the future. All in all, light sensitive conjugated polymers represent a new approach for optical modulation of cellular activity.
In this work, all the steps required to fabricate a bio-polymer interface for optical excitation of living cells are described. The function of the active interface is to transduce the light stimulus into a modulation of the cell membrane potential. As a study case, useful for in-vitro studies, a polythiophene thin film is used as the functional, light absorbing layer, and Human Embryonic Kidney (HEK-293) cells are employed as the biological component of the interface. Practical examples of successful control of the cell membrane potential upon stimulation with light pulses of different duration are provided. In particular, it is shown that both depolarizing and hyperpolarizing effects on the cell membrane can be achieved depending on the duration of the light stimulus. The reported protocol is of general validity and can be straightforwardly extended to other biological preparations.

A method for stimulation of in-vitro cell cultures electrical activity with visible light, based on the use of organic semiconducting polymers is described.

Hybrid interfaces between organic semiconductors and living tissues represent a new tool for in-vitro and in-vivo applications. In particular, conjugated ...

Bioengineering

An Integrated System to Remotely Trigger Intracellular Signal Transduction by Upconversion Nanoparticle-mediated Kinase Photoactivation

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper tissue penetration. However, the existing instrumentation on the market is not readily compatible with upconversion application. Therefore, modifying the commercially available instrument is essential for this research. In this paper, we first illustrate the modifications of a conventional fluorimeter and fluorescence microscope to make them compatible for photon upconversion experiments. We then describe the synthesis of a near-infrared (NIR)-triggered caged protein kinase A catalytic subunit (PKA) immobilized on a UCNP complex. Parameters for microinjection and NIR photoactivation procedures are also reported. After the caged PKA-UCNP is microinjected into REF52 fibroblast cells, the NIR irradiation, which is significantly superior to conventional UV irradiation, efficiently triggers the PKA signal transduction pathway in living cells. In addition, positive and negative control experiments confirm that the PKA-induced pathway leading to the disintegration of stress fibers is specifically triggered by NIR irradiation. Thus, the use of protein-modified UCNP provides an innovative approach to remotely control light-modulated cellular experiments, in which direct exposure to UV light must be avoided.

In this protocol, caged protein kinase A (PKA), a cellular signal transduction bioeffector, was immobilized on a nanoparticle surface, microinjected into the cytosol, and activated by the upconverted UV light from near-infrared (NIR) irradiation, inducing downstream stress fiber disintegration in the cytosol.

Upconversion nanoparticle (UCNP)-mediated photoactivation is a new approach to remotely control bioeffectors with much less phototoxicity and with deeper ...

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

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce cell apoptosis. The Federal Drug Administration has approved PDT for the treatment of actinic keratosis, and clinical guidelines recommend PDT as a treatment for certain non-melanoma skin cancers and acne vulgaris. PDT is an advantageous therapeutic modality as it is low cost, non-invasive, and associated with minimal adverse events and scaring. In the first step of PDT, a PS is applied and allowed to accumulate intracellularly. Subsequent light irradiation induces reactive oxygen species formation, which may ultimately lead to cell apoptosis, membrane disruption, mitochondrial damage, immune modulation, keratinocyte proliferation, and collagen turnover. Herein, we present an in vitro method to study PDT in an adherent cell line. This treatment protocol is designed to simulate PDT and may be adjusted to studying the use of PDT with various cell lines, photosensitizers, incubation temperatures, or photoactivation wavelengths. Squamous cell carcinoma cells were incubated with 0, 0.5, 1.0, and 2 mM 5-aminolevulinic acid (5-ALA) for 30 min and photoactivated with 417 nm blue light for 1,000 s. The primary outcome measure was apoptosis and necrosis, as measured by annexin-V and 7-aminoactinomycin D flow cytometry. There was a dose-dependent increase in cell apoptosis following thirty-minute incubation of 5-ALA. To achieve high inter-test validity, it is important to maintain consistent incubation and light parameters when performing in vitro PDT experiments. PDT is a useful clinical procedure and in vitro research may allow for the development of novel PSs, optimization of protocols, and new indications for PDT.

An In Vitro Approach to Photodynamic Therapy

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light photoactivation to induce apoptosis. We present an in vitro PDT protocol designed to simulate PDT that may be used to study differences in PS incubation and light treatment parameters.

Photodynamic therapy (PDT) is a medical procedure that involves incubation of an exogenously applied photosensitizer (PS) followed by visible light ...

Medicine

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, mouth, vagina, and skin. Despite the availability of antifungal medication, the mortality rate of invasive candidiasis remains ~50%. Unfortunately, the incidence of drug-resistant C. albicans is increasing globally. Antimicrobial photodynamic therapy (aPDT) may offer an alternative or adjuvant treatment to inhibit C. albicans biofilm formation and overcome drug resistance. Rose bengal (RB)-mediated aPDT has shown effective cell killing of bacteria and C. albicans. In this study, the efficacy of RB-aPDT on multidrug-resistant C. albicans is described. A homemade green light-emitting diode (LED) light source is designed to align with the center of a well of a 96-well plate. The yeasts were incubated in the wells with different concentrations of RB and illuminated with varying fluences of green light. The killing effects were analyzed by the plate dilution method. With an optimal combination of light and RB, 3-log growth inhibition was achieved. It was concluded that RB-aPDT might potentially inhibit drug-resistant C. albicans.

Rose Bengal-Mediated Photodynamic Therapy to Inhibit Candida albicans

The growing incidence of drug-resistant Candida albicans is a serious health issue worldwide. Antimicrobial photodynamic therapy (aPDT) may offer a strategy to fight against drug-resistant fungal infections. The present protocol describes Rose bengal-mediated aPDT efficacy on a multidrug-resistant C. albicans strain in vitro.

Invasive Candida albicans infection is a significant opportunistic fungal infection in humans because it is one of the most common colonizers of the gut, ...

Immunology and Infection

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.

Photodynamic Therapy with Blended Conducting Polymer/Fullerene Nanoparticle Photosensitizers

Summary

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