Name: synthesis and characterization of multi-modal phase-change porphyrin droplets
Uploaded: 2021-10-15T14:10:00
Duration: 7 min 59 s
Description: Watch this Scientific Journal Video about Synthesis and Characterization of Multi-Modal Phase-Change Porphyrin Droplets at JoVE.com

The authors would like to thank Dr. Brandon Helfield for helping build the gas exchanger and Dr. Miffy Hok Yan Cheng for technical discussions. The authors would like to thank the following funding sources: Ontario Graduate Scholarship, Canadian Institutes of Health Research, Terry Fox Research Institute, and Princess Margaret Cancer Foundation.

<ol>
	<li>Gramiak, R., Shah, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Echocardiography+of+the+aortic+root.">Echocardiography of the aortic root.</a> Investigative Radiology. 3 (5), 356-366 (1968).</li><li>Ferrara, K., Pollard, R., Borden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+microbubble+contrast+agents:+fundamentals+and+application+to+gene+and+drug+delivery.">Ultrasound microbubble contrast agents: fundamentals and application to gene and drug delivery.</a> Annual Review of Biomedical Engineering. 9, 415-447 (2007).</li><li>Bing, K. F., Howles, G. P., Qi, Y., Palmeri, M. L., Nightingale, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood-brain+barrier+(BBB)+disruption+using+a+diagnostic+ultrasound+scanner+and+Definity+in+mice.">Blood-brain barrier (BBB) disruption using a diagnostic ultrasound scanner and Definity in mice.</a> Ultrasound in Medicine and Biology. 35 (8), 1298-1308 (2009).</li><li>Helfield, B. L., 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=Investigating+the+accumulation+of+submicron+phase-change+droplets+in+tumors.">Investigating the accumulation of submicron phase-change droplets in tumors.</a> Ultrasound in Medicine and Biology. 49 (10), 2891 (2020).</li><li>Sheeran, P. S., Luois, S. H., Mullin, L. B., Matsunaga, T. O., Dayton, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+ultrasonically-activatable+nanoparticles+using+low+boiling+point+perfluorocarbons.">Design of ultrasonically-activatable nanoparticles using low boiling point perfluorocarbons.</a> Biomaterials. 33 (11), 3262-3296 (2012).</li><li>Sheeran, P. S., 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=Methods+of+generating+submicrometer+phase-shift+perfluorocarbon+droplets+for+applications+in+medical+ultrasonography.">Methods of generating submicrometer phase-shift perfluorocarbon droplets for applications in medical ultrasonography.</a> IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control. 64 (1), 252-263 (2016).</li><li>Yoo, K., 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=Impact+of+encapsulation+on+in+vitro+and+in+vivo+performance+of+volatile+nanoscale+phase-shift+perfluorocarbon+droplets.">Impact of encapsulation on in vitro and in vivo performance of volatile nanoscale phase-shift perfluorocarbon droplets.</a> Ultrasound in Medicine and Biology. 44 (8), 1836-1852 (2018).</li><li>Sheeran, P. S., 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=Image-guided+ultrasound+characterization+of+volatile+sub-micron+phase-shift+droplets+in+the+20-40+MHz+frequency+range.">Image-guided ultrasound characterization of volatile sub-micron phase-shift droplets in the 20-40 MHz frequency range.</a> Ultrasound in Medicine and Biology. 42 (3), 795-807 (2016).</li><li>Sheeran, P. S., 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=More+than+bubbles:+creating+phase-shift+droplets+from+commercially+available+ultrasound+contrast+agents.">More than bubbles: creating phase-shift droplets from commercially available ultrasound contrast agents.</a> Ultrasound in Medicine and Biology. 43 (2), 531-540 (2017).</li><li>Chen, C. C., 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=Targeted+drug+delivery+with+focused+ultrasound-induced+blood-brain+barrier+opening+using+acoustically-activated+nanodroplets.">Targeted drug delivery with focused ultrasound-induced blood-brain barrier opening using acoustically-activated nanodroplets.</a> Journal of Controlled Release. 172 (3), 795-804 (2013).</li><li>Wu, S. Y., 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=Focused+ultrasound-facilitated+brain+drug+delivery+using+optimized+nanodroplets.">Focused ultrasound-facilitated brain drug delivery using optimized nanodroplets.</a> Physics in Medicine and Biology. 63 (3), 035002 (2018).</li><li>Lee, J. Y., 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=Ultrasound-enhanced+siRNA+delivery+using+magnetic+nanoparticle-loaded+chitosan-deoxycholic+acid+nanodroplets.">Ultrasound-enhanced siRNA delivery using magnetic nanoparticle-loaded chitosan-deoxycholic acid nanodroplets.</a> Advanced Healthcare Materials. 6 (8), 1601246 (2017).</li><li>Cao, Y., 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=Drug+release+from+phase-changeable+nanodroplets+triggered+by+low-intensity+focused+ultrasound.">Drug release from phase-changeable nanodroplets triggered by low-intensity focused ultrasound.</a> Theranostics. 8 (5), 1327-1339 (2018).</li><li>Huynh, E., Jin, C. S., 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=Aggregate+enhanced+trimodal+porphyrin+shell+microbubbles+for+ultrasound,+photoacoustic,+and+fluorescence+imaging.">Aggregate enhanced trimodal porphyrin shell microbubbles for ultrasound, photoacoustic, and fluorescence imaging.</a> Bioconjugate Chemistry. 25 (4), 796-801 (2014).</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> Bioconjugate Chemistry. 13 (3), 392-396 (2002).</li><li>Dhaliwal, A., 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=Improving+accessibility+of+EPR-insensitive+tumor+phenotypes+using+EPR-adaptive+strategies:+Designing+a+new+perspective+in+nanomedicine+delivery.">Improving accessibility of EPR-insensitive tumor phenotypes using EPR-adaptive strategies: Designing a new perspective in nanomedicine delivery.</a> Theranostics. 9 (26), 8091-8108 (2019).</li><li>Goertz, D. E., de Jong, N., vander Steen, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuation+and+size+distribution+measurements+of+Definity+and+manipulated+Definity+populations.">Attenuation and size distribution measurements of Definity and manipulated Definity populations.</a> Ultrasound in Medicine and Biology. 33 (9), 1376 (2007).</li><li>Pellow, C., Acconcia, C., Zheng, G., Goertz, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Threshold-dependent+nonlinear+scattering+from+porphyrin+nanobubbles+for+vascular+and+extravascular+applications.">Threshold-dependent nonlinear scattering from porphyrin nanobubbles for vascular and extravascular applications.</a> Physics in Medicine and Biology. 63 (21), 215001 (2018).</li><li>Kwan, J. J., Borden, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+monolayer+collapse+and+microbubble+stability.">Lipid monolayer collapse and microbubble stability.</a> Advances in Colloid and Interface Science. 183, 82-99 (2012).</li><li>Overchuk, 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=Subtherapeutic+Photodynamic+Treatment+Facilitates+Tumor+Nanomedicine+Delivery+and+Overcomes+Desmoplasia.">Subtherapeutic Photodynamic Treatment Facilitates Tumor Nanomedicine Delivery and Overcomes Desmoplasia.</a> Nano Letters. 21 (1), 344-352 (2021).</li><li>Feshitan, J., Chen, C. C., Kwan, J. J., Borden, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbubble+size+isolation+by+differential+centrifugation.">Microbubble size isolation by differential centrifugation.</a> Journal of Colloid and Interface Science. 329 (2), 316-324 (2009).</li><li>Paproski, R. 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=Porphyrin+nanodroplets:+Sub-micrometer+ultrasound+and+photoacoustic+contrast+imaging+agents.">Porphyrin nanodroplets: Sub-micrometer ultrasound and photoacoustic contrast imaging agents.</a> Small. 12 (3), 371-380 (2016).</li><li>Periyasamy, P. C., Leijten, J. C. H., Dijkstra, P. J., Karperien, M., Post, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanomaterials+for+the+local+and+targeted+delivery+of+osteoarthritis+drugs.">Nanomaterials for the local and targeted delivery of osteoarthritis drugs.</a> Journal of Nanomaterials. 2021, 1-13 (2012).</li><li>Matsumura, Y., Maeda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+concept+for+macromolecular+therapeutics+in+cancer+chemotherapy:+mechanism+of+tumoritropic+accumulation+of+proteins+and+the+antitumor+agent+smancs.">A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs.</a> Cancer Research. 46 (12), 6387-6392 (1986).</li></ol>

The authors have nothing to disclose.

Ultrasound imaging is a non-invasive, non-ionizing form of medical imaging that utilizes acoustic waves. While ultrasound scanners are more portable and can provide real-time images, ultrasound imaging can suffer from low contrast, making it difficult for sonographers to reliably distinguish similarly echogenic pathological features. To counteract this limitation, microbubbles can be injected into the host to improve vascular contrast. Microbubbles are micron-sized gas filled contrast agents that are highly echogenic to acoustic waves and can provide enhanced vessel contrast1,2. The shells and gas cores of microbubbles can be tailored for different applications, such as imaging, thrombolysis, cell membrane permeabilization, or transient vascular opening2.
A drawback of microbubbles is their short circulation half-lives. For example, clinically available perflutren lipid microspheres only have a half-life of 1.3 minutes3. For long imaging sessions, multiple injections of microbubbles are needed. Another drawback of microbubbles is their large diameters. While perflutren lipid microspheres are around 1 to 3 &#181;m in diameter, small enough to circulate in vasculature, they are too large to extravasate and passively accumulate into tissues of interest, such as tumors4. One strategy to overcome these limitations is to condense the gas-core microbubbles into smaller, liquid-core droplets5,6. While droplets are not echogenic in their liquid state, they can be vaporized into microbubbles upon exposure to ultrasound with sufficiently high peak negative pressure, regaining their ability to provide contrast. This allows for the droplet to take advantage of the more favorable pharmacokinetics&#160;of a small liquid-core, while retaining the ability to provide contrast when insonated and without changing the chemical composition4,7.
Decafluorobutane is an ideal perfluorocarbon compound for phase-shifting between gaseous and liquid states5,6,7. Decafluorobutane allows for condensation of microbubbles into droplets with temperature reduction alone, whereas less dense perfluorocarbons require additional pressurization5. This gentle method minimizes destruction of bubbles during condensation7,8,9. As their cores are liquid, droplets are non-echogenic and invisible to ultrasound. However, with the application of sufficient acoustic or thermal energy, the liquid cores can vaporize back into a gaseous state, generating echogenic microbubbles8. This vaporization allows for control of when and where to generate microbubbles.
While droplets are useful for passive accumulation, in situ vaporization, or improving cell permeability4, droplets (and their fragments) cannot be imaged or quantified ex vivo. Therefore, quantifiable companion diagnostic agent, such as fluorescent4,10,11, magnetic&#160;particles12, optically absorbent agents13, are utilized as an analogue to gauge droplet delivery to tissues of interest. For example, Helfield et al. used a co-injection of fluorescent nano-beads for histology image quantification of mouse organs&#160;as droplets could not be detected fluorescently4. The disadvantage of companion diagnostic agents is the trackable component may act independently from the droplet depending on its individual pharmacokinetic profile.
Fortunately, the shell of microbubbles and droplets can be customized. For example, Huynh et al. demonstrated ultrasound contrast agents with porphyrin-lipid shells, creating multi-modal microbubbles14. Porphyrins are a class of organic compounds with an aromatic macrocylic structure14,15. They are optically absorbent, fluorescent, and can be chelated to a wide variety of metals for radiotherapy, radionuclide-based imaging, or trace metal-based quantification14. One example of porphyrin is pyropheophorbide (Pyro). By conjugating Pyro onto lipids, incorporating Pyro-lipids in microbubbles or droplets allow them to be imaged and tracked through multiple modalities: acoustically, fluorescently, and through absorbance14. This multi-modal contrast agent could be used to track and quantify accumulation. This could eliminate the need for companion diagnostic agents as the quantifiable component is now conjugated onto the shell, enabling more accurate delivery quantification16.
Herein, a protocol for creating multi-modal phase-change porphyrin droplets is outlined. As ultrasound contrasts agents can be used as a platform for drug delivery to tissues of interest, such as tumors2,4, extending their detectability beyond ultrasound could prove useful for delivery efficacy quantification. The purpose of these droplets is to provide trackable ultrasound contrast agents capable of passive accumulation in vivo, in situ vaporization and acoustics, and with the potential to quantify bio-distribution or accumulation from ex vivo organs without the reliance on secondary sensors. Characterization methods are also outlined to showcase porphyrin droplets&#39; potential as bio-distribution sensors. The effects of Pyro-lipid loading in the shell (0% to 50% by molar ratio) are also discussed.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-5000] (ammonium salt)</td>
			<td >Avanti Polar Lipids</td>
			<td >880220</td>
			<td >Also known as &#34;DSPE-PEG5K&#34;</td>
		</tr>
		<tr >
			<td >1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine</td>
			<td >Avanti Polar Lipids</td>
			<td >855775</td>
			<td>Also known as &#34;DSPC&#34;</td>
		</tr>
		<tr >
			<td >Aluminum Foil</td>
			<td >Any brand</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Aluminum Seals, Tear-Off</td>
			<td >VWR</td>
			<td >16171-840</td>
			<td>Standard Aluminum, 13 mm outer diameter</td>
		</tr>
		<tr >
			<td >Bath Sonicator</td>
			<td >Any brand</td>
			<td ></td>
			<td>Capable of&#160; sonicating and heating up to 70 &#176;C,</td>
		</tr>
		<tr >
			<td >Bio-Stor Screw Cap Vials</td>
			<td >National Scientific</td>
			<td >BS20NABP</td>
			<td>Plastic, 2 mL Skirted, with O-ring</td>
		</tr>
		<tr >
			<td >Borosilicate glass clear serum vials</td>
			<td >VWR</td>
			<td >16171-285</td>
			<td>3 mL, 7 mm inner mouth diameter, 13 mm outer mouth diameter</td>
		</tr>
		<tr >
			<td >Borosilicate Glass Sample Vial with Phenolic Screw Cap</td>
			<td >VWR</td>
			<td >66011-020</td>
			<td>1.85 mL, Short Form Style, 12 mm outer diameter, 35 mm height, 8-425 cap size</td>
		</tr>
		<tr >
			<td >Borosilicate Glass Vial with Screw-On cap</td>
			<td >Any brand</td>
			<td ></td>
			<td>Sizes will depend on desired volumes</td>
		</tr>
		<tr >
			<td >Chloroform&#160;</td>
			<td >Any brand</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Coulter Counter Elctrolyte Diluent</td>
			<td >Any brand</td>
			<td ></td>
			<td>Compatible with Coulter Counter</td>
		</tr>
		<tr >
			<td >Decafluorobutane (C4F10)</td>
			<td >FluoroMed</td>
			<td >355-25-9</td>
			<td></td>
		</tr>
		<tr >
			<td >Deionized Water</td>
			<td >Any brand</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Dry Ice (Carbon Dioxide)</td>
			<td >Any brand</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Dynamic Light Scattering (DLS) Particle Analyzer</td>
			<td >Any brand</td>
			<td ></td>
			<td>Capable of temperature control</td>
		</tr>
		<tr >
			<td >E-Z Crimper, 13 mm</td>
			<td >Wheaton</td>
			<td >W225302</td>
			<td>13 mm Standard Aluminum Seals</td>
		</tr>
		<tr >
			<td >E-Z Decapper, 13 mm</td>
			<td >Wheaton</td>
			<td >W225352</td>
			<td>13 mm Standard Aluminum Seals</td>
		</tr>
		<tr >
			<td >Fluorescent Spectrophotometer</td>
			<td >Any brand</td>
			<td ></td>
			<td>Capable of 400 to 600 excitation and 300 to 800 nm emission detection, detector perpendicular to laser source</td>
		</tr>
		<tr >
			<td >Fluorescent Spectrophotometer Compatible Cuvette</td>
			<td >Any brand</td>
			<td ></td>
			<td>Can hold at least 2 mL, capable of 300 to 800 nm, all four sides are&#160; optical windows&#160;</td>
		</tr>
		<tr >
			<td >Gas Exchanger</td>
			<td >Made in-house</td>
			<td ></td>
			<td>Refer to Supplementary Information - &#34;Other Protocols and Data&#34; for assembly instructions.</td>
		</tr>
		<tr >
			<td >Glass syringes</td>
			<td >Any brand</td>
			<td ></td>
			<td>Sizes will depend on desired volumes</td>
		</tr>
		<tr >
			<td >GLWR Custom Aperture Tube 10 um</td>
			<td >Beckman Coulter</td>
			<td >B42812</td>
			<td>10 &#181;m aperture, compatible with Beckman Coulter MultiSizer 4e</td>
		</tr>
		<tr >
			<td >Glycerol</td>
			<td >Any brand</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Insulated Styrofaom containers with lids</td>
			<td >Any brand</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Isopropanol</td>
			<td >Any brand</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Lyophilization-Style Rubber Stoppers</td>
			<td >VWR</td>
			<td >71000-060</td>
			<td>7 mm inner mouth diameter, 13 mm outer mouth diameter, 2-leg, Chlorobutyl</td>
		</tr>
		<tr >
			<td >Membrane Diaphram Vacuum Pump</td>
			<td >Sartorius Stedim</td>
			<td >16694-1-60-06</td>
			<td>Adjustable pressure</td>
		</tr>
		<tr >
			<td >Metal Tongs</td>
			<td >Any brand</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Methanol</td>
			<td >Any brand</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >MS250 21 MHz Linear Array Ultrasound Transducer</td>
			<td >VisualSonics</td>
			<td ></td>
			<td>21 MHz, Capable of B-mode and non-linear imaging.</td>
		</tr>
		<tr >
			<td >MultiSizer 4e</td>
			<td >Beckman Coulter</td>
			<td ></td>
			<td>Capable of sizing from 0.2&#181;m to 6 &#181;m</td>
		</tr>
		<tr >
			<td >Nalgene Rapid-Flow Sterile Single Use Vacuum Filter Units</td>
			<td >Thermo Scientific</td>
			<td >567-0010</td>
			<td>Polyethersulfone (PES) membrane, 0.1&#956;m pore size, 1000 mL volume. As Isoton II is non-sterile, can use Filter units multiple times</td>
		</tr>
		<tr >
			<td >Needles, Conventional</td>
			<td >BD</td>
			<td >305176</td>
			<td>20 gauge, 1.5 inch length</td>
		</tr>
		<tr >
			<td >Nitrogen Gas</td>
			<td >Any brand</td>
			<td ></td>
			<td>Make sure there are regulator valves and tubes to direct the flow. Setup will be dependend on brand and source.</td>
		</tr>
		<tr >
			<td >Parafilm</td>
			<td >Any brand</td>
			<td ></td>
			<td>Called &#34;wax film&#34; in the protocol.</td>
		</tr>
		<tr >
			<td >Phosphate Buffered Saline (PBS)</td>
			<td >Any brand</td>
			<td ></td>
			<td>1X, 7.4 pH</td>
		</tr>
		<tr >
			<td >Pipette</td>
			<td >Any brand</td>
			<td ></td>
			<td>Sizes will depend on desired volumes</td>
		</tr>
		<tr >
			<td >Pipette Tips</td>
			<td >Any brand</td>
			<td ></td>
			<td>Sizes will depend on desired volumes</td>
		</tr>
		<tr >
			<td >Plastic Syringes</td>
			<td >Any brand</td>
			<td ></td>
			<td>1 mL, 3 mL, and 30 mL. With Luer Lock connections</td>
		</tr>
		<tr >
			<td >Polyethersulfone (PES) Membrane Filter</td>
			<td >Any brand</td>
			<td ></td>
			<td>0.2 &#181;m pore size</td>
		</tr>
		<tr >
			<td >Propylene Glycol</td>
			<td >Any brand</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Pyropheophorbide conjugated 1-stearoyl-2-hydroxy-sn-glycero-3-phosphocholine</td>
			<td >Made in-house</td>
			<td ></td>
			<td>Also known as &#34;Pyro-SPC&#34;. Refer to &#34;Supplementary Information - Other Protocols and Data&#34; for synthesis.</td>
		</tr>
		<tr >
			<td >Thermometer</td>
			<td >Any brand</td>
			<td ></td>
			<td>(-20 to 100 &#176;C)</td>
		</tr>
		<tr >
			<td >Triton X-100</td>
			<td >Any brand</td>
			<td ></td>
			<td>Also known as &#34;2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol&#34;</td>
		</tr>
		<tr >
			<td >Ultrapure Water</td>
			<td >Any brand</td>
			<td ></td>
			<td>Type 1 Purity</td>
		</tr>
		<tr >
			<td >Ultraviolet&#8211;Visible (UV-Vis) Spectrophotometer</td>
			<td >Any brand</td>
			<td ></td>
			<td>Capable of absorbance from 300 to 800 nm, at least 0.5 nm resolution</td>
		</tr>
		<tr >
			<td >Ultraviolet&#8211;Visible (UV-Vis) Spectrophotometer Compatible Cuvette, 1 cm Path Length</td>
			<td >Any brand</td>
			<td ></td>
			<td>Can hold at least 2 mL, capable of 300 to 800 nm</td>
		</tr>
		<tr >
			<td >Vacuum Desiccator</td>
			<td >Any brand</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Vevo 2100 Ultrasound Imaging Platform</td>
			<td >VisualSonics</td>
			<td ></td>
			<td>Pre-clinical ultrasound imaging system</td>
		</tr>
		<tr >
			<td >Vialmix</td>
			<td >Bristol-Myers-Squibb</td>
			<td ></td>
			<td>Called &#34;mechanical agitator&#34; in the protocol. Agitates for 45 s.</td>
		</tr>
		<tr >
			<td >Vortex Mixer</td>
			<td >Any brand</td>
			<td ></td>
			<td></td>
		</tr>
	</tbody>
</table>

synthesis and characterization of multi-modal phase-change porphyrin droplets

Phase-change droplets are a class of ultrasound contrast agents that can convert into echogenic microbubbles in situ with the application of sufficient acoustic energy. Droplets are smaller and more stable than their microbubble counterparts. However, traditional ultrasound contrast agents are not trackable beyond acoustic feedback measurements, which makes quantifying contrast agent bio-distribution or accumulation ex vivo difficult. Researchers may have to rely on fluorescent or optically absorbent companion diagnostic particles to infer bio-distribution. The purpose of this protocol is to detail steps for creating multi-modal phase-change porphyrin droplets using a condensation method. Porphyrins are fluorescent molecules with distinct absorbance bands that can be conjugated onto lipids and incorporated into droplets to extend droplet versatility, enabling more robust bio-distribution while retaining acoustic properties. Seven formulations with varying porphyrin-lipid and base lipid contents were made to investigate microbubble and droplet size distributions. Characterizations suited to porphyrin-containing structures are also described in the protocol to demonstrate their analytic versatility in-solution. Sizing showed that the post-condensed mean diameters were 1.72 to 2.38 times smaller than precursor populations. Absorbance characterization showed intact assemblies had a Q-band peak of 700 nm while disrupted samples had an absorbance peak at 671 nm. Fluorescence characterization showed intact 30% porphyrin-lipid assemblies were fluorescently quenched (&gt;97%), with fluorescence recovery achieved upon disruption. Acoustic vaporization showed that porphyrin droplets were non-echogenic at lower pressures and could be converted into echogenic microbubbles with sufficient pressures. These characterizations show the potential for porphyrin droplets to eliminate the need for absorbance or fluorescence-based companion diagnostic strategies to quantify ultrasound contrast agent bio-distribution for delivery or therapeutic applications in vivo or ex vivo.

Pre-condensed, size-selected microbubble samples (n = 3) and post-condensed droplet samples (n = 3) were sized on a Coulter Counter (CC) with a 10 &#181;m aperture. A limitation of the 10 &#181;m aperture is it cannot measure particles smaller than 200 nm, which can bias the mean size and concentration. Figure 4 shows the sizing data for each of the Pyro-lipid content formulations. Table 1 shows statistics based on the sizing data. Using a ratio of pre- and post-condensed mean diameters, the results showed that the 0% Pyro-lipid formulation had the smallest mean diameter shift at 1.72 &#177; 0.02. The 50% Pyro-lipid formulation had the greatest mean diameter at 2.38 &#177; 0.08. The 1% Pyro-lipid droplet sample had the highest observed concentration at (2.71 &#177; 0.13) &#215; 1010 /mL while the 40% Pyro-lipid droplet sample had the lowest observed concentration at (7.36 &#177; 0.81) &#215; 109 /mL. Sizing data showed the 10% Pyro-lipid droplet sample had the smallest peak dimeter at 261 &#177; 13 nm while the 50% Pyro-lipid droplet sample had the largest at 390 &#177; 55 nm. Generally, as the Pyro-lipid content increased, the concentration decreased and the mean diameter increased. As the post-condensed samples are based on the precursor microbubble sample, the trend occurred for both types of ultrasound contrast agents. As the Pyro-lipid content increased, a microbubble subpopulation (with a peak size at approximately 2000 &#181;m) started to form. This secondary peak was not present in the 0% Pyro-lipid microbubble sample and most apparent in the 40% and 50% Pyro-lipid populations.
Figure 5&#160;shows representative absorbance measurements of the 30% Pyro-lipid droplet sample. The peak of the intact sample in PBS was 700 nm while the disrupted sample in Triton shifted the peak to 671 nm. This showed that the intact assembles have different optical properties compared to the individual, unassembled lipid components.
Figure 6A shows representative fluorescence measurements of the pre-condensed microbubble sample while Figure 6B shows post-condensed droplet sample with 30% Pyro-lipid. The intact sample in PBS had a fluorescence peak at 704 nm while the disrupted form had a peak at 674 nm. Subtracting the disrupted area under the curve with the intact area under the curve, and dividing the difference by the disrupted area under the curve gives the quenching efficiency, which works out to be 98.61% and 98.07% for the 30% Pyro-lipid microbubble sample and droplet sample, respectively.
To demonstrate droplets converting to microbubbles, diluted droplets were imaged and vaporized in a 37 &#176;C flow phantom with an ultrasound system. Figure 7 shows representative ultrasound images of the 30% Pyro-lipid droplet sample imaged at different pressures. At low pressures (Figure 7A), there was very little signal, only background signal from air bubbles stuck from the agar synthesis. This is because droplets are non-echogenic and do not scatter ultrasound. At a slightly high power, a few microbubbles were generated (Figure 7B) as shown by the appearance of bright speckles. As the pressure increased, more microbubbles were generated (Figure 7C and 7D). This also demonstrated that the droplets will not spontaneously vaporize at 37 &#176;C.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62665/62665fig1v2.jpg" /> 
Figure 1: Images of the steps to form the 30% Pyro-lipid solution. A) Lipid powder plus Pyro-SPC in chloroform. B) Dissolving solution added. C) Lipid film dried and coated onto interior wall of vial. D) Lipid vial wrapped in aluminum foil (exterior foil taped for reuse). E) Hydrated lipid solution. F) Lipid solution in serum vial. <a href="https://www.jove.com/files/ftp_upload/62665/62665fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62665/62665fig2v2.jpg" /> 
Figure 2: The 10-manifold gas exchanger. The valves referenced in the protocol are labelled. See Supplemental&#160;File &#34;Other Protocols and Data&#34; for instructions on how to assemble the Gas Exchanger. <a href="https://www.jove.com/files/ftp_upload/62665/62665fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62665/62665fig03.jpg" /> 
Figure 3: A) The lipid solutions of the 7 formulations (0% to 50% Pyro-SPC) in sample vials. Figures B to D show images of the steps taken to make 30% Pyro-lipid droplets. B) 30% Pyro-lipid solution in a sample vial. C) Post-agitation. D) 15 min size-selected. E) Bottom partition transferred to decafluorobutane vial. D) Post-condensation. <a href="https://www.jove.com/files/ftp_upload/62665/62665fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62665/62665fig04.jpg" /> 
Figure 4: Coulter Counter (CC) sizing data of the size-selected microbubble and droplet samples with different Pyro-lipid shell content (n = 3). The solid green lines represent microbubbles and the dotted cyan lines represent droplets. A) 0% Pyro-SPC. B) 1% Pyro-SPC. C) 10% Pyro-SPC. D) 20% Pyro-SPC. E) 30% Pyro-SPC. F) 40% Pyro-SPC. G) 50% Pyro-SPC. H) The total observed concentrations of microbubble and droplet samples from the CC based on Pyro-SPC content in the shell. All error bars indicate standard deviation. All measurements were performed using a 10 &#181;m aperture which has a size range of 200 nm to 6000 nm. <a href="https://www.jove.com/files/ftp_upload/62665/62665fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62665/62665fig05.jpg" /> 
Figure 5: Representative ultraviolet-visible (UV-Vis) spectroscopy absorbance measurements from 300 to 800 nm of the post-condensed 30% Pyro-lipid droplet sample diluted in PBS and in 1% Triton. <a href="https://www.jove.com/files/ftp_upload/62665/62665fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62665/62665fig06.jpg" /> 
Figure 6: Representative fluorescence emission from 600 to 750 nm excited at 410 nm. A) Size-selected, pre-condensed 30% Pyro-lipid microbubble sample in PBS and in 1% Triton. B) Post-condensed 30% Pyro-lipid droplet sample in PBS and in 1% Triton. <a href="https://www.jove.com/files/ftp_upload/62665/62665fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62665/62665fig07.jpg" /> 
Figure 7: Representative ultrasound images of a 37 &#176;C agar flow phantom taken with a pre-clinical 21 MHz linear array transducer in B-mode (see Table of Materials). The left column (Figures A, C, E,&#38; G) shows PBS controls. The right column (Figures B, D, F,&#38; H) shows 20 &#181;L of post-condensed 30% Pyro-lipid droplet sample diluted into 50 mL of 37 &#176;C PBS. Each row represents free-field peak negative pressures, which were estimated from the work done by Sheeran et al.8 The yellow triangles indicate focus depth. <a href="https://www.jove.com/files/ftp_upload/62665/62665fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Method</td>
			<td>Agent</td>
			<td>Pyro Shell %</td>
			<td>0</td>
			<td>1</td>
			<td>10</td>
			<td>20</td>
			<td>30</td>
			<td>40</td>
			<td>50</td>
		</tr>
		<tr>
			<td>CC</td>
			<td>Bubbles</td>
			<td>Conc. [/mL]</td>
			<td>(2.76 &#177; 0.28) &#215; 10^10</td>
			<td>(3.04 &#177; 0.15) &#215; 10^10</td>
			<td>(2.02 &#177; 0.11) &#215; 10^10</td>
			<td>(1.91 &#177; 0.22) &#215; 10^10</td>
			<td>(1.47 &#177; 0.05) &#215; 10^10</td>
			<td>(8.47 &#177; 0.95) &#215; 10^9</td>
			<td>(9.89 &#177; 0.15) &#215; 10^9</td>
		</tr>
		<tr>
			<td>CC</td>
			<td>Bubbles</td>
			<td>Peak [nm]</td>
			<td>329 &#177; 6</td>
			<td>297 &#177; 15</td>
			<td>305 &#177; 21</td>
			<td>273 &#177; 14</td>
			<td>310 &#177; 40</td>
			<td>266 &#177; 33</td>
			<td>393 &#177; 89</td>
		</tr>
		<tr>
			<td>CC</td>
			<td>Bubbles</td>
			<td>Mean [nm]</td>
			<td>609 &#177; 2</td>
			<td>603 &#177; 15</td>
			<td>635 &#177; 6</td>
			<td>690 &#177; 8</td>
			<td>812 &#177; 1</td>
			<td>935 &#177; 22</td>
			<td>950 &#177; 55</td>
		</tr>
		<tr>
			<td>CC</td>
			<td>Bubbles</td>
			<td>Median [nm]</td>
			<td>450 &#177; 6</td>
			<td>421 &#177; 6</td>
			<td>414 &#177; 6</td>
			<td>432 &#177; 5</td>
			<td>490 &#177; 2</td>
			<td>596 &#177; 37</td>
			<td>695 &#177; 41</td>
		</tr>
		<tr>
			<td>CC</td>
			<td>Droplets</td>
			<td>Conc. [/mL]</td>
			<td>(2.18 &#177; 0.07) &#215; 10^10</td>
			<td>(2.71 &#177; 0.13) &#215; 10^10</td>
			<td>(1.75 &#177; 0.18) &#215; 10^10</td>
			<td>(1.72 &#177; 0.13) &#215; 10^10</td>
			<td>(1.09 &#177; 0.01) &#215; 10^10</td>
			<td>(7.36 &#177; 0.81) &#215; 10^9</td>
			<td>(7.38 &#177; 0.28) &#215; 10^9</td>
		</tr>
		<tr>
			<td>CC</td>
			<td>Droplets</td>
			<td>Peak [nm]</td>
			<td>292 &#177; 0</td>
			<td>297 &#177; 17</td>
			<td>261 &#177; 13</td>
			<td>280 &#177; 9</td>
			<td>268 &#177; 17</td>
			<td>287 &#177; 38</td>
			<td>390 &#177; 55</td>
		</tr>
		<tr>
			<td>CC</td>
			<td>Droplets</td>
			<td>Mean [nm]</td>
			<td>353 &#177; 5</td>
			<td>350 &#177; 5</td>
			<td>347 &#177; 1</td>
			<td>347 &#177; 4</td>
			<td>397 &#177; 1</td>
			<td>399 &#177; 6</td>
			<td>400 &#177; 7</td>
		</tr>
		<tr>
			<td>CC</td>
			<td>Droplets</td>
			<td>Median [nm]</td>
			<td>318 &#177; 4</td>
			<td>318 &#177; 4</td>
			<td>310 &#177; 1</td>
			<td>315 &#177; 2</td>
			<td>340 &#177; 0</td>
			<td>367 &#177; 5&#160;</td>
			<td>370 &#177; 9</td>
		</tr>
	</tbody>
</table>
Table 1: Sizing data statistics of the microbubble and droplet samples with different Pyro-SPC content from Coulter Counter (CC) (n = 3). All errors indicate standard deviation.
Supplementary Information - Lipid Formula Sheet: <a href="https://www.jove.com/files/ftp_upload/62665/Supplementary Information - Lipid Formula Sheet.xlsx" target="_blank">Please click here to download this File.</a>
Supplementary Information - Other Protocols and Data: <a href="https://www.jove.com/files/ftp_upload/62665/Supplementary_Information-Other_Protocols_and_Data.docx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Synthesis and Characterization of Multi-Modal Phase-Change Porphyrin Droplets at JoVE.com

Synthesis and Characterization of Multi-Modal Phase-Change Porphyrin Droplets

In this protocol, methods for synthesizing and characterizing multi-modal phase-change porphyrin droplets are outlined.

Phase-change droplets are a class of ultrasound contrast agents that can convert into echogenic microbubbles in situ with the application of sufficient ...

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