Name: Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration (Video) | JoVE Journal
Uploaded: 2014-05-26T14:00:00.000+00:00
Duration: 13 min 17 s
Description: Learn to manufacture lipid-based oxygen microbubbles for intravenous oxygen delivery using high-shear homogenization and serial concentration. This scalable method produces two liters of 90% oxygen microbubbles for therapeutic applications.

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Funding: U.S. Army Medical Research &#38; Materiel Command (USAMRMC) and managed by the Telemedicine &#38; Advanced Technology Research Center.&#160; Shunxi Ji contributed the modification of the syringes as described here.

1. System Set-up
The system consists of a holding and concentrating tank (HCT) fitted with a single stage mixer, an in-line high shear homogenizer, a roller pump to move fluid between the HCT and the homogenizer, and a heat-exchanger (Figure 1).
<ol>
	<li>Place a sterilized, wide-mouthed 4 L glass collection vessel fitted with 2 base ports and 3 side ports beneath the single stage mixer. Lower the mixer head to the mouth of the vessel and ensure a gas-tight fitting using rubber seals or tape (in order to prevent ambient air from contaminating the head space).</li>
	<li>Fit one of the base ports (Figure 1, port #1) of the HCT with sterile 3/8&#8221; (ID) clear tubing, approximately 10&#8221; long, fitted with a 3-way stopcock at the tip for collection of the concentrated emulsion.</li>
	<li>Fit the second base port (Figure 1, port #2) with sterile 3/8&#8221; (ID) tubing, approximately 36&#8221; in length. Feed this tubing through a roller pump. Fit the inlet of the high shear homogenizer with a T-piece including two ports and connect as follows: connect tubing from port #2, through the roller pump and connect to the side port of the T-piece. Attach the other port to an oxygen tank using a low-flow oxygen gas flowmeter.</li>
	<li>Connect the outlet port of the high shear homogenizer to the inlet port of an in-line heat exchanger maintained at 4 &#176;C. Connect the outlet port of the heat exchanger to the return port of the HCT (Figure 1, port #3), creating a closed loop system.</li>
	<li>Attach an oxygen tank (via a flowmeter) to the HCT (Figure 1, port #4). Attach a gas composition monitor that is open to atmosphere to the top port of the HCT (Figure 1, port #5).</li>
	<li>If sterility is desired, sterilize the glass and metal components before each use by autoclave. Sterilize the tubing components and plastic connectors by ethylene oxide before each use. This is especially important if the product is to be tested in vivo.</li>
</ol>
2. LOM Manufacture
<ol>
	<li>Place 20 g of GMP 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and 10 g of cholesterol in the base of the HCT. Add 1 L of Plasma-Lyte A to the HCT and hand stir for 1 min, integrating as much lipid as possible into aqueous phase.</li>
	<li>Lower the single stage mixer into the aqueous phase, ensuring that the entire mixer head is covered by the aqueous phase. Ensure that the top of the HCT is gas tight (see Step 1.1 above) and that there are no open side ports. Turn on the gas source attached to port #4 and wait until the oxygen fraction of the HCT headspace reaches &#62;95%. At 10 L/min&#160;(LPM), this should take ~10 min.</li>
	<li>Using the single stage mixer, mix the precursor emulsion for 5 min at 5,000 rpm. The resulting mixture should appear pale white and contain no visible lipid clumps. Once mixed, unused lipid-water mixture can be stored at 4 &#186;C for up to 30 days before single use.</li>
	<li>Prime the entire closed loop system with the precursor emulsion by turning on the roller pump at 1.3 LPM. Once the system is primed, keep the pump on at 1.3 LPM.</li>
	<li>To begin manufacture of the LOMs, turn on the in-line high shear homogenizer to 7,500 rpm. Immediately thereafter, turn on oxygen flow to the inlet portion of the homogenizer at 0.5 LPM. Keep the single stage mixer (in the HCT) on at 3,500 rpm. LOMs are formed at the interface of rotor blades and the emulsor screens within the in-line homogenizer (Figure 2). Within&#160;minutes, fluid should become visibly more viscous. A more rigorous approach is to determine viscosity as a function of time, which can be done by removing aliquots from Port 1 during fabrication and analyzing with a viscometer. 
	Note: If visible air bubbles are present in the tubing exiting the mixer, oxygen flow to the in-line homogenizer is too high. Titrate down gas flow until fluid is opaque and contains no visible gas bubbles.</li>
	<li>Run the system for 15 min, and then turn off the high shear homogenizer and the oxygen inlet to it. Continue to run the single stage mixer in the HCT until the emulsion is removed; this mitigates phase separation and keeps the product relatively uniform within the HCT. 
	Note: The volume of the gas-filled emulsion should increase approximately 2-3x&#160;during the serial concentration phase. If it does not, check to ensure that oxygen is flowing into the high shear homogenizer and that lipid concentrations in the precursor emulsion are correct. Efficiency of production decreases as lipid concentration decreases.</li>
</ol>
3. Collection, Concentration, Assessment, and Storage of LOMs
<ol>
	<li>Attach a sterile, modified 140 ml luer-lock syringe to the stopcock attached to base port #1 on the collection vessel. Draw up 100 ml of fluid. Tightly cap syringe and repeat until all fluid has been removed.
	<ol>
		<li>Modify syringes by withdrawing 100 ml of air into the syringe and then sawing off the excess plunger and syringe material above the 140 ml mark. Fill and empty syringes using toothed forceps to draw up the plunger. This modification allows easier centrifugation.</li>
	</ol>
	</li>
	<li>Centrifuge syringes with the capped end oriented downward in a refrigerated (4 &#186;C) bucket centrifuge at 225 x g for 10 min.</li>
	<li>Three layers of material will appear after centrifugation. Expel the bottom layer of excess cloudy aqueous phase and discard. The second layer is bright white and contains concentrated LOMs. Transfer concentrated foam to a gas-impermeable syringe using a three-way stopcock to prevent ambient gas contamination. Discard the final layer, which contains free oxygen gas from ruptured LOMs.</li>
	<li>Foam quality can be assessed by reaching &#8805;90% gas of concentrated foam. Calculate gas concentration as follows: 
	Volume % gas = [(Foam weight/ Foam volume)- 1] x 100
	<ol>
		<li>As a second quality control, size microbubbles by light obscuration to determine if particle size is within the expected range. It should be noted that a change in homogenization time or formulation might alter bubble size.</li>
	</ol>
	</li>
	<li>Tightly cap the glass syringe with a luer-lock fitting. Concentrated LOMs can be diluted with Plasma-Lyte A at the time of use. Syringes can be stored at 22, 4, or -20 &#186;C; colder temperatures may provide enhanced shelf-life stability7.</li>
</ol>

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<li>Ren, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Novel+Ultrasound+Microbubble+Carrying+Gene+and+Tat+Peptide%3A+Preparation+and+Characterization%5BTitle%5D)+AND+%22Academic+Radiology%22%5BJournal%5D)">A Novel Ultrasound Microbubble Carrying Gene and Tat Peptide: Preparation and Characterization.</a> Academic Radiology. 16 (12), 1457-1465 (2009).</li>
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<li>Kheir, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Oxygen+Gas-Filled+Microparticles+Provide+Intravenous+Oxygen+Delivery%5BTitle%5D)+AND+%22Science+Translational+Medicine%22%5BJournal%5D)">Oxygen Gas-Filled Microparticles Provide Intravenous Oxygen Delivery.</a> Science Translational Medicine. 4 (140), (2012).</li>
<li>Lindner, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microvascular+rheology+of+definity+microbubbles+after+intra-arterial+and+intravenous+administration%5BTitle%5D)+AND+%22Journal+of+the+American+Society+of+Echocardiography%22%5BJournal%5D)">Microvascular rheology of definity microbubbles after intra-arterial and intravenous administration.</a> Journal of the American Society of Echocardiography. 15 (5), 396-403 (2002).</li>
<li>Zhao, Y. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Preparation%2C+characterization+and+in+vivo+observation+of+phospholipid-based+gas-filled+microbubbles+containing+hirudin%5BTitle%5D)+AND+%22Ultrasound+in+Medicine+and+Biology%22%5BJournal%5D)">Preparation, characterization and in vivo observation of phospholipid-based gas-filled microbubbles containing hirudin.</a> Ultrasound in Medicine and Biology. 31 (9), 1237-1243 (2005).</li>
<li>Seo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microfluidic+Assembly+of+Monodisperse%2C+Nanoparticle-Incorporated+Perfluorocarbon+Microbubbles+for+Medical+Imaging+and+Therapy%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">Microfluidic Assembly of Monodisperse, Nanoparticle-Incorporated Perfluorocarbon Microbubbles for Medical Imaging and Therapy.</a> Langmuir. 26 (17), 13855-13860 (2010).</li>
<li>Wan, J. D., Stone, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coated+Gas+Bubbles+for+the+Continuous+Synthesis+of+Hollow+Inorganic+Particles%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">Coated Gas Bubbles for the Continuous Synthesis of Hollow Inorganic Particles.</a> Langmuir. 28 (1), 37-41 (2012).</li>
<li>Duncanson, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monodisperse+Gas-Filled+Microparticles+from+Reactions+in+Double+Emulsions%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">Monodisperse Gas-Filled Microparticles from Reactions in Double Emulsions.</a> Langmuir. 28 (17), 6742-6745 (2012).</li>
<li>Kendall, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Scaled-Up+Production+of+Monodisperse%2C+Dual+Layer+Microbubbles+Using+Multi-Array+Microfluidic+Module+for+Medical+Imaging+and+Drug+Delivery%5BTitle%5D)+AND+%22Bubble+Science+Engineering+and+Technology%22%5BJournal%5D)">Scaled-Up Production of Monodisperse, Dual Layer Microbubbles Using Multi-Array Microfluidic Module for Medical Imaging and Drug Delivery.</a> Bubble Science Engineering and Technology. 4 (1), 12-20 (2012).</li>
<li>Szijjarto, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+Perfluorocarbon+Gases+on+the+Size+and+Stability+Characteristics+of+Phospholipid-Coated+Microbubbles%3A+Osmotic+Effect+versus+Interfacial+Film+Stabilization%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">Effects of Perfluorocarbon Gases on the Size and Stability Characteristics of Phospholipid-Coated Microbubbles: Osmotic Effect versus Interfacial Film Stabilization.</a> Langmuir. 28 (2), 1182-1189 (2012).</li>
<li>Rossi, S., Waton, G., Krafft, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phospholipid-Coated+Gas+Bubble+Engineering%3A+Key+Parameters+for+Size+and+Stability+Control%2C+as+Determined+by+an+Acoustical+Method%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">Phospholipid-Coated Gas Bubble Engineering: Key Parameters for Size and Stability Control, as Determined by an Acoustical Method.</a> Langmuir. 26 (3), 1649-1655 (2010).</li>
<li>Swanson, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phospholipid-Stabilized+Microbubble+Foam+for+Injectable+Oxygen+Delivery%5BTitle%5D)+AND+%22Langmuir%22%5BJournal%5D)">Phospholipid-Stabilized Microbubble Foam for Injectable Oxygen Delivery.</a> Langmuir. 26 (20), 15726-15729 (2010).</li>
<li>Kvåle, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Size+fractionation+of+gas-filled+microspheres+by+flotation%5BTitle%5D)+AND+%22Separations+Technology%22%5BJournal%5D)">Size fractionation of gas-filled microspheres by flotation.</a> Separations Technology. 6 (4), 219-226 (1996).</li>
<li>Dressaire, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interfacial+polygonal+nanopatterning+of+stable+microbubbles%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Interfacial polygonal nanopatterning of stable microbubbles.</a> Science. 320 (5880), 1198-1201 (2008).</li>
<li>Kheir, J. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bulk+Manufacture+of+Concentrated+Oxygen+Gas-Filled+Microparticles+for+Intravenous+Oxygen+Delivery%5BTitle%5D)+AND+%22Advanced+Healthcare+Materials%22%5BJournal%5D)">Bulk Manufacture of Concentrated Oxygen Gas-Filled Microparticles for Intravenous Oxygen Delivery.</a> Advanced Healthcare Materials. , (2013).</li>
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The authors declare that they have no competing financial interests.

The most important steps to creating concentrated, highly oxygenated LOMs include: 1) ensuring that the headspace within the HCT remains fully oxygenated; 2) ensuring that the purity of the lipid excipients is optimal (including storage conditions and use of GMP products); 3) ensuring that the powdered lipids mix completely with the aqueous phase prior to priming the system; and 4) paying close attention to the increase in gas fraction within the HCT to ensure that the volume fraction of gas does not exceed 70%.
<p c...

Microbubbles composed of protein, polymer, and lipid shells have been developed as vectors for drug delivery, gene therapy, and ultrasound contrast agents1-5. Because these therapeutic uses require intravascular microbubble persistence, such microbubbles are commonly filled with inert, high molecular-weight gases such as perfluorocarbons6, which have low solubility in blood and stabilize the bubble3,4.
Recently, lipid-based oxygen microbubbles (LOMs) have been designed to deliver therapeutic doses of oxygen, which may preserve end-organ oxygen delivery and prevent hemodynamic instability during periods of airway obstruction or hypoxemia7. Emulsions designed for intravenous gas delivery require different design features than those used for ultrasound contrast agents or targeted drug delivery. First, because the body consumes large volumes of oxygen gas (~200 ml/min), LOMs must be produced and injected on a large scale. This requires that the manufacturing process be efficient. Second, the manufacturing process should be closed-loop in order to avoid nitrogen contamination through the exposure of LOMs (which should be filled with 100% oxygen) to ambient air. Third, because the purpose of LOMs is intravenous gas delivery, the gas fraction of LOMs should be maximized, recognizing the limitations imposed by emulsion viscosity7. Finally, as with any intravenous injectable, precise control over particle size distribution is essential to avoiding microvascular obstruction8.
There are several established methods for microbubble manufacture. Sonication utilizes high intensity, low-frequency ultrasound applied to the air-fluid interface of an emulsion which includes a surfactant, such as an amphipathic phospholipid, in the presence of a gas headspace to produce microbubbles7,9. This process is controllable by varying ultrasound frequency, power and pulse duration, and the resulting size distribution can be tailored to produce microbubbles of a specific size distribution, though sonication is rarely used in the manufacture of clinically-used microbubbles. Amalgamation is the intense mechanical agitation of a surfactant and gas in a closed system, which is also difficult to scale up to accommodate large volumes2. Droplet-based microfluidics allows precise control of microbubble size distribution10-13. Though traditionally difficult to scale up, multi-channel, high-speed microfluidics have been described which increase microbubble production efficiency13. Microbubbles manufactured using any of these methods may require post-manufacture size-reduction processes, such as centrifugal fractionation14,15 and microbubble flotation16,17.
Another established method for the manufacture of highly stable microbubbles is shear homogenization6, which can result in a stabilizing hexagonal phospholipid pattern on the microbubble surface18. Building on this concept, we describe the incorporation of an in-line high shear homogenizer to create self-assembling LOMs19. In this process, the homogenizer utilizes rapidly rotating blades in close proximity to dual fine mesh emulsor screens, creating high mechanical and hydraulic shear for the creation of microbubbles. Serial concentration of the lipid emulsion through this system yields an increasingly concentrated gas fraction, which can be even further concentrated by centrifugation.

<table><tbody><tr><td>1,2-distearoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phosphocholine (DSPC)</td><td>Avanti Polar Lipids</td><td>770365</td><td>Alternate product: non-GMP from NOF America (Coatsome MC-8080)</td></tr><tr><td>Cholesterol</td><td>Sigma Aldrich</td><td>C75209</td><td></td></tr><tr><td>Plasma-Lyte A</td><td>VWR</td><td>80089-818</td><td>Alternatively can use NaCl</td></tr><tr><td>Glass collection vessel</td><td>Specialty Glass, Inc.</td><td>Custom</td><td>Contact: Pam Zurbrick - 281-595-2210</td></tr><tr><td>Gas composition (oxygen) monitor</td><td>Precision Medical</td><td>PM5900L</td><td></td></tr><tr><td>Sarns 8000 roller pump</td><td>Calicut Medical</td><td>16407</td><td>Part of a modular perfusion system</td></tr><tr><td>BIOtherm Heat Exchanger</td><td>Medtronic</td><td>ECMOtherm-II</td><td></td></tr><tr><td>Verso laboratory in-line mixer</td><td>Silverson Machines, Inc</td><td>TH-IL-102-VERSO</td><td>Use multistage workheads and front-end extension with T piece</td></tr><tr><td>T-piece for Silverson Verso inlet port</td><td>Process Innovations</td><td>Custom</td><td>Contact: Brian Leavitt - 508-423-2266</td></tr><tr><td>L5M-A laboratory mixer</td><td>Silverson Machines, Inc</td><td>NC0136483</td><td>Use mesh emulsor screen (fine)</td></tr><tr><td>Rochester-Ochsner toothed forceps</td><td>Fisher Scientific</td><td>13-812-18</td><td></td></tr><tr><td>140 ml syringe</td><td>Kendall Healthcare Monoject</td><td>8881114030</td><td>Ensure there is a luer lock.</td></tr><tr><td>IX71 Inverted light microscope</td><td>Olympus</td><td>IX71</td><td></td></tr><tr><td>Retiga-2000R microscope camera</td><td>QImaging</td><td>RET-2000R-F-M-12</td><td></td></tr><tr><td>Accusizer 780A Autodilution</td><td>PSS-NICOMP Particle Sizing Systems</td><td>Out of production</td><td></td></tr></tbody></table>

manufacture of concentrated, lipid-based oxygen microbubble emulsions by high shear homogenization and serial concentration

Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using sonication, mechanical agitation, microfluidic devices, or homogenization. Recently, lipid-based oxygen microbubbles (LOMs) have been designed to deliver oxygen intravenously during medical emergencies, reversing life-threatening hypoxemia, and preventing subsequent organ injury, cardiac arrest, and death. We present methods for scaled-up production of highly oxygenated microbubbles using a closed-loop high-shear homogenizer. The process can produce 2 L of concentrated LOMs (90% by volume) in 90 min. Resulting bubbles have a mean diameter of ~2 &#956;m, and a rheologic profile consistent with that of blood when diluted to 60 volume %. This technique produces LOMs in high capacity and with high oxygen purity, suggesting that this technique may be useful for translational research labs.

High shear homogenization enables the efficient (i.e. within one afternoon) production of sufficient LOMs for an animal study and does not require technical expertise. Once proficient, up to 2 L of concentrated LOMs can be manufactured in 90 min.
Microbubble size and morphology was assessed by light microscopy and by light obscuration. When a 10 μl sample of LOMs was visualized, spherical LOMs were noted, as well as a relative paucity of lipid debris (Figure 3A). This...

We describe methods for the manufacture of large volumes of lipid-based oxygen microbubbles (LOMs) designed for intravenous oxygen delivery using high-shear homogenization and serial concentration.

Gas-filled microbubbles have been developed as ultrasound contrast and drug delivery agents. Microbubbles can be produced by processing surfactants using ...

The overall goal of this procedure is to manufacture a large volume of self assembling, lipid-based oxygen, microbubbles or OMS for intravenous oxygen delivery. This is accomplished using a high shear homogenizer and a gas tight tank to manufacture and serially concentrate oms. The second step is to use modified syringes to extrude and LOM emulsion containing 70 volume percent oxygen.Next, the emulsion is concentrated by centrifugation to yield a 90 volume percent oxygen foam. The final step is to perform quality control by calculating volume percent oxygen and assessing the particle size distribution using light scattering technology. Ultimately, high shear homogenization produces two liters of OMS containing 90%oxygen by volume.The main advantage of this technique over existing manufacturing methods like sonication and amalgamation, is that homogenization produces self assembling microbubbles on the order of liters, a volume necessary for large animal testing of therapeutic oxygen delivery. One of the uses of this technique is to treat patients with high hypoxemia because oxygen gas filled microbubbles transfer oxygen to deoxyhemoglobin within seconds and carry three times the oxygen content of blood Visual. Demonstration of this method is important as the depth of the homogenizer head.The maintenance of a purely oxidated environment and the discernment of concentrated LMS versus the aqueous phase can be difficult to appreciate. The system for manufacturing. Lipid-based oxygen, microbubbles or OMS consists of a holding and concentrating tank or HCT fitted with a single stage mixer, an inline high shear homogenizer, a roller pump to move fluid between the HCT and the homogenizer and a heat exchanger.To begin the system setup, place a sterilized wide mouthed four liter glass collection vessel fitted with two base ports and three side ports beneath the single stage mixer fit one of the base ports of the HCT port number one with a sterile three eighth inch inner diameter clear tubing, approximately 10 inches long, fitted with a three-way stop cock at the tip for collection of the concentrated emulsion. Next, fit the second base port, port number two with sterile three eighth inch inner diameter tubing, approximately 36 inches in length. Feed this tubing through the roller pump.The inlet of the high shear homogenizer is fitted with a tee piece that includes two ports. Connect tubing from port number two through the roller pump and connect to the side port of the tee piece. Attach the other port to an oxygen tank using a low flow oxygen gas flow meter.Connect the outlet port of the high shear homogenizer to the inlet port of an inline heat exchanger maintained at four degrees Celsius. Connect the outlet port of the heat exchanger to the return port of the HCT creating a closed loop system. Attach the oxygen tank via a flow meter to the HCT via port number four.Lastly, attach a gas composition monitor that is open to atmosphere to the top port of the HCT. To begin this procedure, place 20 grams of GMP one two DYS steer oil, SN glycerol three PHOSPHOCHOLINE or DSPC and 10 grams of cholesterol in the base of the HCT. Add one liter of plasma light A to the HCT and hand stir for one minute, integrating as much lipid as possible into aqueous phase.Lower the single stage mixer into the aqueous phase, ensuring that the entire mixer head is covered by the aqueous phase To prevent ambient air from contaminating the head space, use rubber seals or tape to ensure that the top of the HCT is gas tight and there are no open side ports. Turn on the gas source attached to port number four and wait until the oxygen fraction of the HCT Headspace reaches greater than 95%Using the single stage mixer mix, the precursor emulsion for five minutes. At 5, 000 RPM, the resulting mixture should appear pale white and contain no visible lipid clumps.Once mixed, unused lipid water mixture can be stored at four degrees Celsius for up to 30 days before single. Use prime the entire closed loop system with the precursor emulsion by turning on the roller pump at 1.3 liters per minute or LPM. Once the system is primed, keep the pump on at 1.3 LPM to begin manufacture of the LO's.Turn on the inline high shear homogenizer to 7, 500 RPM. Immediately thereafter, turn on oxygen flow to the inlet portion of the homogenizer at 0.5 LPM. Keep the single stage mixer in the HCT on at 3, 500 RPM.Oms are formed at the interface of rotor blades and the emulsion screens within the inline homogenizer. Within the first few minutes, the fluid should become visibly more viscous. Run the system for 15 minutes and then turn off the high shear homogenizer and the oxygen inlet to it.This mitigates phase separation and keeps the product relatively uniform within the HCT. Note that the volume of the gas filled emulsion should increase approximately two to threefold during the serial concentration phase. The 140 milliliter lure lock syringes for collecting the manufactured OMS have previously been modified by withdrawing 100 milliliters of air into the syringe and then sawing off the excess plunger and syringe material above the 140 milliliter mark.Attach a sterile modified syringe to the stop cock attached to base port number one on the collection vessel used toothed forceps to drop the plunger and draw up 100 milliliters of fluid, tightly capped the syringe repeat until all fluid has been removed. Centrifuge syringes with the capped end oriented downward in a refrigerated bucket centrifuge at 225 times G for 10 minutes. Three layers of material will be apparent after centrifugation, expel and discard the bottom layer of excess cloudy aqueous phase.The second layer is bright white and contains concentrated oms. Transfer the concentrated foam to a gas impermeable glass syringe using a three-way stop cock to prevent ambient gas contamination. Discard the final layer, which contains free oxygen gas from ruptured oms.Foam quality can be assessed by calculating gas concentration as follows. A high quality foam will reach a gas concentration of greater than or equal to 90 volume percent as a second quality control size microbubbles by optical light scatter to determine if particle size is within the expected range. It should be noted that a change in homogenization time or formulation might alter bubble size, tightly capped the glass syringes containing concentrated oms.With a lure lock fitting, syringes can be stored at 22 degrees Celsius, four degrees Celsius or negative 20 degrees Celsius. Colder temperatures may provide enhanced shelf life stability. Using this protocol up to two liters of concentrated OMS can be manufactured in 90 minutes.Visualization of a 10 microliter sample of OMS by light microscopy revealed spherical OMS as well as a relative posity of lipid debris. When the same sample of OMS was assessed by optical scatter, the mean particle diameter was 2.624 plus or minus 0.332 micrometers. Greater than 90%of OMS were less than 10 micrometers in diameter and the population was poly disperse.Emulsion viscosity was heavily dependent on gas fraction and therefore on microbubble concentration. Two milliliter aliquots of LOM emulsion at 60, 70 and 90. Volume percent gas were studied using a steady state flow sweep using a 40 millimeter parallel plate.Geometry as stress was varied from 0.1 to 1000 past gass OMS containing 60 volume percent gas exhibited a radiological profile similar to human blood. The oxygen content of OMS was tested by adding varying volumes of oxygen contained within 60 volume percent OMS to aliquots of desaturated human blood with a known oxygen deficit. The relationship between volume of oxygen added within OMS and the volumetric increase in oxygen content of the blood was 1.053 plus or minus 0.03025.Suggesting that the OMS tested contain nearly 100%oxygen exhibited few trapped gas pockets, and effectively transfer their entire oxygen payload to human blood in vitro Once mastered, this technique can be used to manufacture two liters of LMS in 90 minutes if it is performed properly and efficiently. While attempting this procedure, it is important to remember to maintain visual inspection of accumulating OMS and to perform quality control upon concentration of LOM foam. Following this manufacturing procedure, OMS can be used for large scale intravenous injection and delivery of therapeutic gases.

manufacture-concentrated-lipid-based-oxygen-microbubble-emulsions

Research

JoVE Journal

Bioengineering

Combining monodisperse drop generation with inertial ordering of cells and particles, we describe a method to encapsulate a desired number of cells or particles in a single drop at kHz rates.  We demonstrate efficiencies twice exceeding those of unordered encapsulation for single- and double-particle drops.

High Throughput Single-cell and Multiple-cell Micro-encapsulation

This protocol describes an extrusion method for preparing lipid vesicles of sub-micron sizes with a high degree of homogeneity. This method uses a pressure-controlled system with controlled nitrogen flow rates for liposome preparation. The lipid preparation1,2, liposome extrusion, and size characterization will be presented herein.

Constant Pressure-controlled Extrusion Method for the Preparation of Nano-sized Lipid Vesicles

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Microfluidic double emulsions generation typically involves devices with patterned wettability or custom-fabricated glass components. Here we describe the fabrication and testing of an all polydimethylsiloxane (PDMS) double emulsion generator that does not require surface treatment or complicated fabrication processes, and is capable of producing double emulsions down to 14 µm.

Double Emulsion Generation Using a Polydimethylsiloxane (PDMS) Co-axial Flow Focus Device

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The protocol presents the optimized parameters for preparing thermosensitive liposomes using the staggered herringbone micromixer microfluidics device. This also allows co-encapsulation of doxorubicin and indocyanine green into the liposomes and the photothermal-triggered release of doxorubicin for controlled/triggered drug release.

Microfluidic Production of Lysolipid-Containing Temperature-Sensitive Liposomes

This protocol describes a method of generating large volumes of lipid encapsulated decafluorobutane microbubbles using probe-tip sonication and subsequently condensing them into phase-shift nanodroplets using high-pressure extrusion and mechanical filtration.

Production of Membrane-Filtered Phase-Shift Decafluorobutane Nanodroplets from Preformed Microbubbles

The present protocol describes octanol-assisted liposome assembly (OLA), a microfluidic technique to generate biocompatible liposomes. OLA produces monodispersed, micron-sized liposomes with efficient encapsulation, allowing immediate on-chip experimentation. This protocol is anticipated to be particularly suitable for synthetic biology and synthetic cell research.

On-Chip Octanol-Assisted Liposome Assembly for Bioengineering

This protocol outlines the fabrication of lipid microbubbles and a compatible one-pot microbubble radiolabeling method with purification-free &gt;95% labeling efficiency that conserves microbubble physicochemical properties. This method is effective across diverse lipid microbubble formulations and can be tailored to generate radioactive and/or fluorescent microbubbles.

Fabricating and Labeling Microbubbles with Fluorescent and Radioactive Tracers

Ultrasound Targeted Microbubble Destruction  (UTMD) can be used to direct site-specific delivery of bioactive molecules, including therapeutic genes, to target organs accessible to ultrasound, such as the heart and liver1-6.

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

Learn to manufacture lipid-based oxygen microbubbles for intravenous oxygen delivery using high-shear homogenization and serial concentration. This scalable method produces two liters of 90% oxygen microbubbles for therapeutic applications.

Manufacture of Concentrated, Lipid-based Oxygen Microbubble Emulsions by High Shear Homogenization and Serial Concentration

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