This work was supported by the Princess Margaret Hospital Foundation, the National Science and Engineering Research Council of Canada, the Canadian Institute for Health Research, the Canadian Foundation of Innovation, and the Joey and Toby Tanenbaum/Brazilian Ball Chair in Prostate Cancer Research, a grant from the SUNY Research Foundation and startup support from University at Buffalo.

1. Synthesis of Pyropheophorbide-lipid
<ol>
	<li>Combine 200 mmol pyropheophorbide (prepared from Spirulina Pacifica algae as described previously; Zheng et al., Bioconj. Chem., 2002, 13-392)4, 98.7 mg 16:0 lysophophatidylcholine (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine, Avanti Polar Lipids #855675), 76.3 mg of EDC , 48.7 mg DMAP (4-(dimethylamino)pyridine) in 5 ml amylene stabilized chloroform for a ratio of 1:1:2:2 Pyro:Lipid:EDC:DMPA.</li>
	<li>Stir the reaction mixtur...

<ol>
	<li>Lovell, J. 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=Porphysome+nanovesicles+generated+by+porphyrin+bilayers+for+use+as+multimodal+biophotonic+contrast+agents.">Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents.</a> Nat. Mater. 10, 324-332 (2011).</li><li>Lovell, J. 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=Enzymatic+regioselection+for+the+synthesis+and+biodegradation+of+porphysome+nanovesicles.">Enzymatic regioselection for the synthesis and biodegradation of porphysome nanovesicles.</a> Angew Chem Int Ed Engl. 51, 2429-2433 (2012).</li><li>Liu, T. W., MacDonald, T. D., Shi, J., Wilson, B. C., Zheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsically+Copper-64-Labeled+Organic+Nanoparticles+as+Radiotracers.">Intrinsically Copper-64-Labeled Organic Nanoparticles as Radiotracers.</a> Angewandte Chemie. , (2012).</li><li>Zheng, G., 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=Low-density+lipoprotein+reconstituted+by+pyropheophorbide+cholesteryl+oleate+as+target-specific+photosensitizer.">Low-density lipoprotein reconstituted by pyropheophorbide cholesteryl oleate as target-specific photosensitizer.</a> Bioconjug Chem. 13, 392-396 (2002).</li><li>Chen, B., Pogue, B. W., Hasan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+delivery+of+photosensitising+agents.">Liposomal delivery of photosensitising agents.</a> Expert Opin Drug Deliv. 2, 477-487 (2005).</li><li>Jin, C. S., Zheng, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+nanostructures+for+photosensitizer+delivery.">Liposomal nanostructures for photosensitizer delivery.</a> Lasers Surg Med. 43, 734-748 (2011).</li><li>Olson, F., Hunt, C. A., Szoka, F. C., Vail, W. J., Papahadjopoulos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+liposomes+of+defined+size+distribution+by+extrusion+through+polycarbonate+membranes.">Preparation of liposomes of defined size distribution by extrusion through polycarbonate membranes.</a> Biochim Biophys Acta. 557, 9-23 (1979).</li><li>Berger, N., Sachse, A., Bender, J., Schubert, R., Brandl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filter+extrusion+of+liposomes+using+different+devices:+comparison+of+liposome+size,+encapsulation+efficiency,+and+process+characteristics.">Filter extrusion of liposomes using different devices: comparison of liposome size, encapsulation efficiency, and process characteristics.</a> Int J Pharm. 223, 55-68 (2001).</li><li>Jesorka, A., Orwar, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomes:+technologies+and+analytical+applications.">Liposomes: technologies and analytical applications.</a> Annu Rev Anal Chem (Palo Alto Calif). 1, 801-832 (2008).</li><li>Lapinski, M. M., Castro-Forero, A., Greiner, A. J., Ofoli, R. Y., Blanchard, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+liposomes+formed+by+sonication+and+extrusion:+rotational+and+translational+diffusion+of+an+embedded+chromophore.">Comparison of liposomes formed by sonication and extrusion: rotational and translational diffusion of an embedded chromophore.</a> Langmuir. 23, 11677-11683 (2007).</li><li>Liu, D., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+Homogeneity+of+a+Liposome+Preparation+is+Crucial+for+Liposome+Biodistribution+In+Vivo.">Size Homogeneity of a Liposome Preparation is Crucial for Liposome Biodistribution In Vivo.</a> Journal of Liposome Research. 2, 57-66 (1992).</li><li>Zhang, Y., Lovell, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Porphyrins+as+Theranostic+Agents+from+Prehistoric+to+Modern+Times.">Porphyrins as Theranostic Agents from Prehistoric to Modern Times.</a> Theranostics. 2, 905-915 (2012).</li></ol>

In the development of drug delivery technologies, multifunctional nanoparticles are currently under wide investigations for accurate tracking of the drug delivery vehicle while maintaining a high drug payload. Porphyrin-loaded liposomes have been developed for better pharmacokinetic properties and more efficient delivery than direct administration of porphyrin, but obstacles exist, including the restricted loading amount of porphyrin and rapid redistribution of porphyrin from liposomes to plasma proteins5,6. I...

Porphysomes are novel multifunctional nanovesicles that we recently developed which are capable of multimodal imaging and therapy1. They are formed from self-assembled porphyrin bilayers and contain an extremely high density of porphyrin (over 83, 000/porphysome particle), which generates large extinction coefficient and results in unique structure-dependent fluorescence self-quenching. Porphysomes have good in vivo pharmacokinetic and biodistribution properties: they exhibit a blood half-life of 12 hr following systematic administration, and passively accumulate in xenograft tumors with 7.5% ID/g at 24 hr post-injection2.
Their unique structure and physiochemical properties make porphysome a good candidate for multimodal imaging and image-guided therapy. First of all, containing porphyrin, porphysomes are intrinsically suitable for fluorescence imaging of tumors upon the tumor accumulation1. In addition, each porphyrin has a stable site for chelating radioisotopes, therefore, porphysomes can be easily labeled with radioisotopes such as 64Cu for PET imaging3. Furthermore, the absorbed light energy is dissipated thermally under laser irradiation exposure when porphysome structure is intact, so porphysomes also exhibit unique photoacoustic imaging and PTT capabilities. It has been shown that 24 hr after intravenous injection of porphysomes, laser irradiation of the porphysome-accumulated tumor induced a rapid temperature increase and strong photothermal tumor ablation. This demonstrated that porphysomes are efficient photothermal enhancers with extinction coefficient as high as gold nanoparticles (AuNPs)1. On the other hand, in comparison with other inorganic photothermal agents, including AuNPs, porphysomes show an outstanding advantage in biosafety due to their organic nature. Porphysomes are enzymatically biodegradable and induce minimal acute toxicity in mice with intravenous doses as high as 1,000 mg/kg1. Furthermore, similar to liposomes, the large aqueous core of porphysomes could be passively or actively loaded with therapeutic or imaging agents. The optical properties and biocompatibility of porphysomes demonstrate the multimodal potential of organic nanoparticles for biophotonic imaging and therapy.
In this paper, we introduce the synthesis method of pyropheophorbide-lipid conjugates, the manufacturing and the characterization method of porphysomes using high-pressure extrusion. In vivo PTT on mice is conducted as well to demonstrate the efficiency of porphysome-enabled PTT in the tumor treatment using a subcutaneous xenograft tumor model.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>Rotary evaporator</td>
			<td>Thermo-Savant</td>
			<td>SPD131DDA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Votex</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td>Model number: G560</td>
		</tr>
		<tr>
			<td>High pressure extruder</td>
			<td>LIPEX, Northern Lipids Inc.</td>
			<td>T.001</td>
			<td>10 ml Thermobarrel Extruder</td>
		</tr>
		<tr>
			<td>Heated bath circulators (Thermostatted circulator)</td>
			<td>Thermo SCIENTIFIC</td>
			<td>SC100-S5P</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Polycarbonate filters</td>
			<td>Avanti Polar Lipids</td>
			<td>610005</td>
			<td>Pore size 100 nm, and membrane diameter 19 mm</td>
		</tr>
		<tr>
			<td>Zetasizer</td>
			<td>Malvern Instruments</td>
			<td>ZS90</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>UV/Visible Spectrophototmeter</td>
			<td>Varian Australia</td>
			<td>Cary 50 Bio UV/ Visible Spectrophotometer</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Spectrofluorometer</td>
			<td>HORIBA Scientific</td>
			<td>FluoroMax-4</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Transmission Electron Microscopy (TEM)</td>
			<td>Hitachi</td>
			<td>H-7000</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>SANYO</td>
			<td>MCO-18AIC</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Powermeter</td>
			<td>Thorlabs</td>
			<td>PM100D</td>
			<td>with sensor S142C</td>
		</tr>
		<tr>
			<td>671nm Laser</td>
			<td>LaserGlow Technologies</td>
			<td>LRS-0671_PFN-02000-05</td>
			<td>S/N:10097270</td>
		</tr>
		<tr>
			<td>Infrared Thermometer</td>
			<td>Mikroshot, LUMASENSE Technologies</td>
			<td>9102409</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Laser protective eye goggles</td>
			<td>LaserGlow Technologies</td>
			<td>AGF6602XX</td>
			<td>Optical Density: 1.5+ at 630-700 nm</td>
		</tr>
	</tbody>
</table>

one minute, sub-one-watt photothermal tumor ablation using porphysomes, intrinsic multifunctional nanovesicles

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a phospholipid and then self-assembled to liposome-like spherical vesicles. Due to the extremely high density of porphyrin in the porphyrin-lipid bilayer, porphysomes generated large extinction coefficients, structure-dependent fluorescence self-quenching, and excellent photothermal efficacy. In our formulation, porphysomes were synthesized using high pressure extrusion, and displayed a mean particle size around 120 nm. Twenty-four hr post-intravenous injection of porphysomes, the local temperature of the tumor increased from 30 &deg;C to 62 &deg;C rapidly upon one minute exposure of 750 mW (1.18 W/cm2), 671 nm laser irradiation. Following the complete thermal ablation of the tumor, eschars formed and healed within 2 weeks, while in the control groups the tumors continued to grow and all reached the defined end point within 3 weeks. These data show how porphysomes can be used as potent photothermal therapy (PTT) agents.

Pyropheophorbide is conjugated to the phospholipid as a stable lipid monomer (Figure 1a) and the conjugates self-assemble to form porphysomes by membrane extrusion method using a high pressure extruder. It is usually difficult to extrude porphysome-lipid suspension during the first 3 cycles of extrusion with relatively slow flow rate. As more extrusions are repeated, the flow rate of extruded solution gradually increases, and pressure can be slightly decreased if needed. Porphysome size, concentration, a...

Watch this Scientific Journal Video about One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles at JoVE.com

One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

We developed novel intrinsic multifunctional nanovesicles called porphysomes, which have structure-dependent fluorescence self-quenching and unique photothermal properties, thus functioning as potent photothermal therapy agents. We formulated porphysomes using high pressure extrusion and investigated their photothermal therapy efficacy in a xenograft tumor model.

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a ...

one-minute-sub-one-watt-photothermal-tumor-ablation-using-porphysomes

Research

JoVE Journal

Bioengineering

10.7K visualizações.  University of Toronto.  Nós desenvolvemos novas nanovesículas multifuncionais intrínsecas chamados porphysomes, que têm dependentes de estrutura fluorescência auto-têmpera e propriedades fototérmicos únicas, assim funcionando agentes terapia fototérmica como potentes. Formulamos porphysomes utilizando extrusão de alta pressão e investigou sua eficácia terapia fototérmica em um modelo de tumor xenoenxerto. 