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>

<ol>
	<li>Dolmans, D., Fukumura, D., Jain, R. K. <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+cancer.">Photodynamic therapy for cancer.</a> Nat Rev Cancer. 3 (5), 380-387 (2003).</li><li>Dougherty, T. J., 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.">Photodynamic therapy.</a> J Natl Cancer Inst. 90 (12), 889-905 (1998).</li><li>Ferrari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+nanotechnology:+Opportunities+and+challenges.">Cancer nanotechnology: Opportunities and challenges.</a> Nat Rev Cancer. 5 (3), 161-171 (2005).</li><li>Oleinick, N. L., Morris, R. L., Belichenko, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+apoptosis+in+response+to+photodynamic+therapy:+what,+where,+why,+and+how.">The role of apoptosis in response to photodynamic therapy: what, where, why, and how.</a> Photochem Photobiol Sci. 1 (1), 1-21 (2002).</li><li>Ormond, A., Freeman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dye+Sensitizers+for+Photodynamic+Therapy.">Dye Sensitizers for Photodynamic Therapy.</a> Materials. 6 (3), 817-840 (2013).</li><li>Pass, H. I. <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+in+Oncology+-+Mechanisms+and+Clinical+Use.">Photodynamic Therapy in Oncology - Mechanisms and Clinical Use.</a> J Natl Cancer Inst. 85 (6), 443-456 (1993).</li><li>Sariciftci, N. S., Smilowitz, L., Heeger, A. J., Wudl, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoinduced+electron+transfer+from+a+conducting+polymer+to+buckminsterfullerene.">Photoinduced electron transfer from a conducting polymer to buckminsterfullerene.</a> Science. 258 (5087), 1474-1476 (1992).</li><li>Sperandio, F. F., 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=Photoinduced+electron-transfer+mechanisms+for+radical-enhanced+photodynamic+therapy+mediated+by+water-soluble+decacationic+C-70+and+C84O2+Fullerene+Derivatives.">Photoinduced electron-transfer mechanisms for radical-enhanced photodynamic therapy mediated by water-soluble decacationic C-70 and C84O2 Fullerene Derivatives.</a> Nanomed-Nanotechnol. 9 (4), 570-579 (2013).</li><li>Fan, J. Q., Fang, G., Zeng, F., Wang, X. D., Wu, S. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Water-Dispersible+Fullerene+Aggregates+as+a+Targeted+Anticancer+Prodrug+with+both+Chemo-+and+Photodynamic+Therapeutic+Actions.">Water-Dispersible Fullerene Aggregates as a Targeted Anticancer Prodrug with both Chemo- and Photodynamic Therapeutic Actions.</a> Small. 9 (4), 613-621 (2013).</li><li>Grynyuk, I., 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=Photoexcited+fullerene+C-60+disturbs+prooxidant-antioxidant+balance+in+leukemic+L1210+cells.">Photoexcited fullerene C-60 disturbs prooxidant-antioxidant balance in leukemic L1210 cells.</a> Materialwiss Werkstofftech. 44 (2-3), 139-143 (2013).</li><li>Liu, X. M., 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=Separately+doped+upconversion-C-60+nanoplatform+for+NIR+imaging-guided+photodynamic+therapy+of+cancer+cells.">Separately doped upconversion-C-60 nanoplatform for NIR imaging-guided photodynamic therapy of cancer cells.</a> Chem Commun. 49 (31), 3224-3226 (2013).</li><li>Trpkovic, A., Todorovic-Markovic, B., Trajkovic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxicity+of+pristine+versus+functionalized+fullerenes:+mechanisms+of+cell+damage+and+the+role+of+oxidative+stress.">Toxicity of pristine versus functionalized fullerenes: mechanisms of cell damage and the role of oxidative stress.</a> Arch Toxicol. 86 (12), 1809-1827 (2012).</li><li>Chen, Z. Y., MA, L. J., Liu, Y., Chen, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+Functionalized+Fullerenes+in+Tumor+Theranostics.">Applications of Functionalized Fullerenes in Tumor Theranostics.</a> Theranostics. 2 (3), 238-250 (2012).</li><li>Park, S. H., 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=Bulk+heterojunction+solar+cells+with+internal+quantum+efficiency+approaching+100%.">Bulk heterojunction solar cells with internal quantum efficiency approaching 100%.</a> Nat Photonics. 3 (5), 297-302 (2009).</li></ol>

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 border="0" cellpadding="0" cellspacing="0">
	<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 Mn&#160;150,000-250,000</td>
		</tr>
		<tr>
			<td>[6,6]-Phenyl C61&#160;butyric acid methyl ester (PCBM)</td>
			<td>Sigma Aidrich</td>
			<td>684449-500MG</td>
			<td>&#62; 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 &#181;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&#39;s Modification of Eagle&#39;s Medium/Ham&#39;s F-12 50/50 Mix (DMEM)</td>
			<td>Corning (VWR)</td>
			<td>45000-350</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#39;s Balanced Salt Solution without phenol red (HBSS)</td>
			<td>Quality Biological (VWR)</td>
			<td>10128-740</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;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&#160;</td>
			<td>15710</td>
			<td>16% paraformaldehyde is diluted to 4% by adding PBS</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>&#160;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 cm2 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><a href="https://www.vwrsp.com/catalog/product/index.cgi?catalog_number=PAG4000">PAG4000</a></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&#160;</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 ...

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8.4K 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

Conducting Miller-Urey Experiments

In 1953, Stanley Miller reported the production of biomolecules from simple gaseous starting materials, using an apparatus constructed to simulate the primordial Earth&#39;s atmosphere-ocean system. Miller introduced 200 ml of water, 100 mmHg of H2, 200 mmHg of CH4, and 200 mmHg of NH3 into the apparatus, then subjected this mixture, under reflux, to an electric discharge for a week, while the water was simultaneously heated. The purpose of this manuscript is to provide the reader with a general experimental protocol that can be used to conduct a Miller-Urey type spark discharge experiment, using a simplified 3 L reaction flask. Since the experiment involves exposing inflammable gases to a high voltage electric discharge, it is worth highlighting important steps that reduce the risk of explosion. The general procedures described in this work can be extrapolated to design and conduct a wide variety of electric discharge experiments simulating primitive planetary environments.

The Miller-Urey experiment was a pioneering study regarding the abiotic synthesis of organic compounds with possible relevance to the origins of life. Simple gases were introduced into a glass apparatus and subjected to an electric discharge, simulating the effects of lightning in the primordial Earth&#8217;s atmosphere-ocean system. The experiment was conducted for one week, after which, the samples collected from it were analyzed for the chemical building blocks of life.

In 1953, Stanley Miller reported the production of biomolecules from simple gaseous starting materials, using an apparatus constructed to simulate the ...

A Technique to Functionalize and Self-assemble Macroscopic Nanoparticle-ligand Monolayer Films onto Template-free Substrates

This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles1,2. The simple, robust and scalable technique efficiently functionalizes metallic nanoparticles with thiol-ligands in a miscible water/organic solvent mixture allowing for rapid grafting of thiol groups onto the gold nanoparticle surface. The hydrophobic ligands on the nanoparticles then quickly phase separate the nanoparticles from the aqueous based suspension and confine them to the air-fluid interface. This drives the ligand-capped nanoparticles to form monolayer domains at the air-fluid interface.&#160; The use of water-miscible organic solvents is important as it enables the transport of the nanoparticles from the interface onto template-free substrates.&#160; The flow is mediated by a surface tension gradient3,4 and creates macroscopic, high-density, monolayer nanoparticle-ligand films.&#160; This self-assembly technique may be generalized to include the use of particles of different compositions, size, and shape and may lead to an efficient assembly method to produce low-cost, macroscopic, high-density, monolayer nanoparticle films for wide-spread applications.

A simple, robust and scalable technique to functionalize and self-assemble macroscopic nanoparticle-ligand monolayer films onto template-free substrates is described in this protocol.

This protocol describes a self-assembly technique to create macroscopic monolayer films composed of ligand-coated nanoparticles1,2. The simple, robust and ...

Synthesis and Testing of Supported Pt-Cu Solid Solution Nanoparticle Catalysts for Propane Dehydrogenation

A convenient method for the synthesis of bimetallic Pt-Cu catalysts and performance tests for propane dehydrogenation and characterization are demonstrated here. The catalyst forms a substitutional solid solution structure, with a small and uniform particle size around 2 nm. This is realized by careful control over the impregnation, calcination, and reduction steps during catalyst preparation and is identified by advanced in situ synchrotron techniques. The catalyst propane dehydrogenation performance continuously improves with increasing Cu:Pt atomic ratio.

A convenient method for the synthesis of 2 nm supported bimetallic nanoparticle Pt-Cu catalysts for propane dehydrogenation is reported here. In situ synchrotron X-ray techniques allow for the determination of the catalyst structure, which is typically unobtainable using laboratory instruments.

A convenient method for the synthesis of bimetallic Pt-Cu catalysts and performance tests for propane dehydrogenation and characterization are ...

Liquid-cell Transmission Electron Microscopy for Tracking Self-assembly of Nanoparticles

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully understood. We have traced the trajectories of individual nanoparticles using liquid-cell transmission electron microscopy (TEM) to investigate the mechanism of the assembly process. Herein, we present the protocols used for liquid-cell TEM studies of the self-assembly mechanism. First, we introduce the detailed synthetic protocols used to produce uniformly sized platinum and lead selenide nanoparticles. Next, we present the microfabrication processes used to produce liquid cells with silicon nitride or silicon windows and then describe the loading and imaging procedures of the liquid-cell TEM technique. Several notes are included to provide helpful tips for the entire process, including how to manage the fragile cell windows. The individual motions of nanoparticles tracked by liquid-cell TEM revealed that changes in the solvent boundaries caused by evaporation affected the self-assembly process of nanoparticles. The solvent boundaries drove nanoparticles to primarily form amorphous aggregates, followed by flattening of the aggregates to produce a 2-dimensional (2D) self-assembled structure. These behaviors are also observed for different nanoparticle types and different liquid-cell compositions.

Here we introduce experimental protocols for the real-time observation of a self-assembly process using liquid-cell transmission electron microscopy.

Drying a nanoparticle dispersion is a versatile way to create self-assembled structures of nanoparticles, but the mechanism of this process is not fully ...

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good&#39;s buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

In this work, a protocol for signal enhancement of nanoprobe-based biosensing is presented. The protocol is based on the reduction of chloroauric acid onto the surface of existing nanoprobes that consist of gold, silver, silica or iron-oxide nanoparticles.

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...

Methionine Functionalized Biocompatible Block Copolymers for Targeted Plasmid DNA Delivery

Reversible addition-fragmentation chain transfer (RAFT) polymerization integrates the advantages of radical polymerization and living polymerization. This work presents the preparation of methionine functionalized biocompatible block copolymers via RAFT polymerization. Firstly, N,N-bis(2-hydroxyethyl)methacrylamide-b-N-(3-aminopropyl)methacrylamide (BNHEMA-b-APMA, BA) was synthesized via RAFT polymerization using 4,4&#8217;-azobis(4-cyanovaleric acid) (ACVA) as an initiating agent and 4-cyanopentanoic acid dithiobenzoate (CTP) as the chain transfer agent. Subsequently, N,N-bis(2-hydroxyethyl)methacrylamide-b-N-(3-guanidinopropyl)methacrylamide (methionine grafted BNHEMA-b-GPMA, mBG) was prepared by modifying amine groups in APMA with methionine and guanidine groups. Three kinds of block polymers, mBG1, mBG2, and mBG3, were synthesized for comparison. A ninhydrin reaction was used to quantify the APMA content; mBG1, mBG2, and mBG3 had 21%, 37%, and 52% of APMA, respectively. Gel permeation chromatography (GPC) results showed that BA copolymers possess molecular weights of 16,200 (BA1), 20,900(BA2), and 27,200(BA3) g/mol. The plasmid DNA (pDNA) complexing ability of the obtained block copolymer gene carriers was also investigated. The charge ratios (N/P) were 8, 16, and 4 when pDNA was complexed completely with mBG1, mBG2, mBG3, respectively. When the N/P ratio of mBG/pDNA polyplexes was higher than 1, the Zeta potential of mBG was positive. At an N/P ratio between 16 and 32, the average particle size of mBG/pDNA polyplexes was between 100-200 nm. Overall, this work illustrates a simple and convenient protocol for the block copolymer carrier synthesis.

This work presents the preparation of methionine functionalized biocompatible block copolymers (mBG) via the reversible addition-fragmentation chain transfer (RAFT) method. The plasmid DNA complexing ability of the obtained mBG and their transfection efficiency were also investigated. The RAFT method is very beneficial for polymerizing monomers containing special functional groups.

Reversible addition-fragmentation chain transfer (RAFT) polymerization integrates the advantages of radical polymerization and living polymerization. This ...

Gene-therapy Inspired Polycation Coating for Protection of DNA Origami Nanostructures

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in physiological fluids induce denaturation and degradation of self-assembled DNA nanostructures. In non-viral gene delivery, enzymatic degradation of DNA is overcome by the encapsulation of the negatively charged DNA in a cationic shell. Herein, inspired by gene delivery advancements, a simple, one-step and robust methodology is presented for the stabilization of DNA origami nanostructures by coating them with chitosan and linear polyethyleneimine. The polycation coating efficiently protects DNA origami nanostructures in Mg-depleted and nuclease-rich media. This method also preserves the full addressability of enzyme- and aptamer-based functionalization of DNA nanostructures.

Here, a protocol for the protection of DNA origami nanostructures in Mg-depleted and nuclease-rich media using natural cationic polysaccharide chitosan and synthetic linear polyethyleneimine (LPEI) coatings is presented.

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in ...

Gold Nanoparticle Modified Carbon Fiber Microelectrodes for Enhanced Neurochemical Detection

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into glass capillaries, pulled to a fine taper, and then sealed using an epoxy to create electrode materials that are used for fast scan cyclic voltammetry testing. The use of bare CFMEs has several limitations, though. First and foremost, the carbon fiber contains mostly basal plane carbon, which has a relatively low surface area and yields lower sensitivities than other nanomaterials. Furthermore, the graphitic carbon is limited by its temporal resolution, and its relatively low conductivity. Lastly, neurochemicals and macromolecules have been known to foul at the surface of carbon electrodes where they form non-conductive polymers that block further neurotransmitter adsorption. For this study, we modify CFMEs with gold nanoparticles to enhance neurochemical testing with fast scan cyclic voltammetry. Au3+ was electrodeposited or dipcoated from a colloidal solution onto the surface of CFMEs. Since gold is a stable and relatively inert metal, it is an ideal electrode material for analytical measurements of neurochemicals. Gold nanoparticle modified (AuNP-CFMEs) had a stability to dopamine response for over 4 h. Moreover, AuNP-CFMEs exhibit an increased sensitivity (higher peak oxidative current of the cyclic voltammograms) and faster electron transfer kinetics (lower &#916;EP or peak separation) than bare unmodified CFMEs. The development of AuNP-CFMEs provides the creation of novel electrochemical sensors for detecting fast changes in dopamine concentration and other neurochemicals at lower limits of detection. This work has vast applications for the enhancement of neurochemical measurements. The generation of gold nanoparticle modified CFMEs will be vitally important for the development of novel electrode sensors to detect neurotransmitters in vivo in rodent and other models to study neurochemical effects of drug abuse, depression, stroke, ischemia, and other behavioral and disease states.

In this study, we modify carbon-fiber microelectrodes with gold nanoparticles to enhance the sensitivity of neurotransmitter detection.

For over 30 years, carbon-fiber microelectrodes (CFMEs) have been the standard for neurotransmitter detection. Generally, carbon fibers are aspirated into ...

Nanoparticle Tracking Analysis of Gold Nanoparticles in Aqueous Media through an Inter-Laboratory Comparison

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). Characterization needs to be thorough and the technique chosen should be well-suited to the property to be determined, the material being analyzed and the medium in which it is present. Furthermore, the instrument operation and methodology need to be well-developed and clearly understood by the user to avoid data collection errors. Any discrepancies in the applied method or procedure can lead to differences and poor reproducibility of obtained data. This paper aims to clarify the method to measure the hydrodynamic diameter of gold nanoparticles by means of Nanoparticle Tracking Analysis (NTA). This study was carried out as an inter-laboratory comparison (ILC) amongst seven different laboratories to validate the standard operating procedure&#8217;s performance and reproducibility. The results obtained from this ILC study reveal the importance and benefits of detailed standard operating procedures (SOPs), best practice updates, user knowledge, and measurement automation.

The protocol described here aims to measure the hydrodynamic diameter of spherical nanoparticles, more specifically gold nanoparticles, in aqueous media by means of Nanoparticle Tracking Analysis (NTA). The latter involves tracking the movement of particles due to Brownian motion and implementing the Stokes-Einstein equation to obtain the hydrodynamic diameter.

In the field of nanotechnology, analytical characterization plays a vital role in understanding the behavior and toxicity of nanomaterials (NMs). ...

Gold Nanoparticle Synthesis

Gold nanoparticles (Au nanoparticles) that are ~12 nm in diameter were synthesized by rapidly injecting a solution of 150 mg (0.15 mmol) of tetrachloroauric acid in 3.0 g (3.7 mmol, 3.6 mL) of oleylamine (technical grade) and 3.0 mL of toluene into a boiling solution of 5.1 g (6.4 mmol, 8.7 mL) of oleylamine in 147 mL of toluene. While boiling and mixing the reaction solution for 2 hours, the color of the reaction mixture changed from clear, to light yellow, to light pink, and then slowly to dark red. The heat was then turned off, and the solution was allowed to gradually cool down to room temperature for 1 hour. The gold nanoparticles were then collected and separated from the solution using a centrifuge and washed three times; by vortexing and dispersing the gold nanoparticles in 10 mL portions of toluene, and then precipitating the gold nanoparticles by adding 40 mL portions of methanol and spinning them in a centrifuge. The solution was then decanted to remove any remaining byproducts and unreacted starting materials. Drying the gold nanoparticles in a vacuum environment produced a solid black pellet; which could be stored for long periods of time (up to one year) for later use, and then redissolved in organic solvents such as toluene.

A protocol for synthesizing ~12 nm diameter gold nanoparticles (Au nanoparticles) in an organic solvent is presented. The gold nanoparticles are capped with oleylamine ligands to prevent agglomeration. The gold nanoparticles are soluble in organic solvents such as toluene.