pubs.acs.org/Langmuir © 2010 American Chemical Society
Phospholipid-Stabilized Microbubble Foam for Injectable Oxygen Delivery Edward J. Swanson,† Vickram Mohan,† John Kheir,‡ and Mark A. Borden*,†,§ †
Department of Chemical Engineering, Columbia University, New York, New York 10027, United States, Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts 02115, United States, and § Department of Mechanical Engineering, University of Colorado, Boulder, Colorado 80309, United States ‡
Received July 23, 2010. Revised Manuscript Received September 17, 2010 A detailed study is presented on the synthesis and characterization of purely oxygen-filled microbubbles (OMBs) stabilized by phospholipids. Microbubbles with a diameter of less than 10 μm were generated and concentrated to >50 vol % in saline. The lipid acyl chain length had little effect on the size distribution but profoundly affected the foam stability. For example, OMBs stabilized by dipalmitoyl phosphatidylcholine (DPPC) degraded over 3 weeks, but OMBs stabilized with distearoyl phosphatidylcholine (DSPC) retained over half of their initially encapsulated gas. Interestingly, the polydisperse size distribution remained nearly constant as the foam slowly broke down. Injection into an undersaturated solution led to the immediate release of the oxygen gas core. Injectable gas delivery by OMBs may find use in a variety of medical and industrial fields.
Introduction Microbubbles (MBs) are micrometer-sized spherical gas-filled particles stabilized by an organic coating at the gas-liquid interface. Phospholipid MBs have been used clinically as ultrasound contrast agents for almost a decade;1-3 however, the high surface area-to-volume ratio (105 to 106 m-1) of these structures also makes them ideal vehicles for the rapid delivery of gases. There are a number of biomedical applications that can be envisioned when this gas payload is oxygen. The systemic delivery of metabolic oxygen from purely oxygen-filled microbubbles to essential organs could significantly improve the outcome of a variety of situations in critical care.4-6 Perfluorocarbon-based oxygen carrying MBs is already being used,7-10 for example, to counter the resistance of hypoxic cancer tumors to radiation and chemotherapy,11 and purely oxygen-filled MBs (without fluorocarbons) could further improve tumor sensitization while simultaneously reducing immunogenic and renal side effects. Wound healing12 and organ preservation13 are examples of biomedical applications that would be improved by the application of a fluid containing a readily available source of oxygen. Finally, industrial applications *Current address: Department of Mechanical Engineering, University of Colorado, 1111 Engineering Drive, Boulder, Colorado 80309-0427. E-mail:
[email protected]. (1) Unger, E.; Lund, P.; Shen, D.; Fritz, T.; Yellowhair, D.; New, T. Radiology 1992, 185, 453–456. (2) Feinstein, S. B. Am. J. Physiol. 2004, 287, H450–H457. (3) Lindner, J. R. Nat. Rev. Drug Discovery 2004, 3, 527–532. (4) Vishnevskii, R. E.; Karichev, Z. R.; Muler, A. L.; Gerasimchuk, A. A.; Itkin, G. P. Theor. Found. Chem. Eng. 1998, 32, 444–448. (5) Karichev, Z. R.; Muler, A. L.; Itkin, G. P. Theor. Found. Chem. Eng. 1998, 32, 235–240. (6) Kheir, J.; Zurakowski, D.; McGowan, F.; Borden, M. Crit. Care Med. 2007, 35, 61. (7) Burkard, M. E.; Van Liew, H. D. J. Appl. Physiol. 1994, 77, 2874–2878. (8) Van Liew, H. D.; Burkard, M. E. In Oxygen Transport to Tissue XVIII; Nemoto E. M., LaManna J. C., Eds.; Advances in Experimental Medicine and Biology; Plenum Press: New York, 1997; Vol. 411, pp 395-401. (9) Gerber, F.; Waton, G.; Krafft, M. P.; Vandamme, T. F. Artif. Cells, Blood Substitutes, Biotechnol. 2007, 35, 119–124. (10) Bisazza, A.; Giustetto, P.; Rolfo, A.; Caniggia, I.; Balbis, S.; Guiot, C.; Cavalli, R. , 30th Annual International Conference of the IEEE; Vancouver, British Columbia, Canada, August, 2008; Engineering in Medicine and Biology Society, 2008. (11) Yu, M.; Dai, M.; Liu, Q.; Xiu, R. Cancer Treat. Rev. 2007, 33, 757–761. (12) Sen, C. K. Wound Repair Regen. 2009, 17, 1–18. (13) McLaren, A.; Friend, P. Transplant Int. 2003, 15, 834-844.
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such as fermentation,14 remediation,15 sonochemistry,16,17 and other solution-phase reactions of gaseous species could benefit from the high transfer rates of MBs. Herein, we describe the synthesis and characterization of a highly concentrated suspension of purely oxygen-filled microbubbles (OMBs) that are stable during storage yet readily release their gas payload upon introduction into a desaturated solution, enabling many of the above applications. The stability, release, and biocompatibility of OMBs can be controlled through the rational design of the coating material. Stablility, which is essential to the storage of OMBs, can be achieved using a variety of coating materials.18 In this work, the OMBs are encapsulated using mixtures of saturated diacyl phosphatidylcholine (DPPC or DSPC) and polyoxyethylene-40 stearate (PEG40S). This self-assembled coating is on the order of 10 nm thick, leaving virtually all of the internal volume free for gas storage and presenting a minor barrier to gas release.19-22 In addition, the coating materials are expected to be biocompatible because the phospholipids are biologically derived and PEG40S is a commonly used excipient.23
Experimental Methods The phospholipid liposomal precursor solution was prepared as follows. A concentrated (10) phosphate-buffered saline (PBS) solution was purchased from Sigma-Aldrich (St. Louis, MO) and diluted to normal concentration with filtered water (Milli-Q, 18 MΩ cm). The solution was then vacuum filtered through a 0.2 μm latex filter (Whatman, Kent, U.K.). DPPC and DSPC were purchased from NOF (Tokyo, Japan), and PEG40S was purchased from Sigma-Aldrich (St. Louis, MO). Phospholipid and PEG40S were weighed and combined in a 9:1 molar ratio and (14) Bredwell, M. D.; Srivastava, P.; Worden, R. M. Biotechnol. Prog. 1999, 15, 834–844. (15) Choi, Y. J.; Kim, Y. J.; Nam, K. Environ. Pollut. 2009, 157, 2197–2202. (16) Adewuyi, Y. G. Environ. Sci. Technol. 2005, 39, 8557–8570. (17) Beckett, M. A.; Hua, I. J. Phys. Chem. A 2001, 105, 3796–3802. (18) Sirsi, S.; Borden, M. Bubble Sci. Eng. Technol. 2009, 1, 3–17. (19) Borden, M.; Longo, M. Langmuir 2002, 18, 9225–9233. (20) Duncan, P. B.; Needham, D. Langmuir 2004, 20, 2567–2578. (21) Borden, M. A.; Longo, M. L. J. Phys. Chem. B 2004, 108, 6009–6016. (22) Pu, G.; Longo, M.; Borden, M. J. Am. Chem. Soc. 2005, 127, 6524–6525. (23) Strickley, R. Pharm. Res. 2004, 21, 201–230-230.
Published on Web 09/28/2010
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Scheme 1. Synthesis of Oxygen Microbubbles
then mixed with PBS to create a final concentration of 5 mg mL-1. The mixture was heated in a water bath (VWR, Batavia, IL) to 5 °C above the lipid’s main phase-transition temperature and dispersed using a Branson 450A sonifier (Danbury, CT) with an output power varying between 2 and 5 until the solution was translucent. The resulting solution was stored under partial vacuum in a refrigerator until preparation of the bubbles. At least two separate lipid solutions were prepared for stability and oxygen release experiments. The reactor, designed for synthesizing large volumes of concentrated OMBs, consisted of an ultrasonic horn enclosed in a water-cooled continuous flow chamber (Branson, Danbury, CT) (Scheme 1). The vacuum-desaturated phospholipid solution was combined with oxygen gas in the flow chamber at approximately equal volumetric flow rates, where they were emulsified at full sonifier power output into a mixture of foam and OMBs. No fluorocarbons were used in the generation, processing, or testing of the OMB suspensions. Both gas and liquid feeds entered the reactor at room temperature, and the produced microbubble suspension was less than 5 °C warmer. The reactor was capable of handling flow rates in excess of 100 mL min-1 lipid solution, and there was no detectable change in the product properties over a relatively wide operational space (Scheme 1, inset). After synthesis, OMBs were separated from foam and large bubbles in a glass column by flotation for 20-30 min. Solution from the bottom of the column, containing OMBs below 10 μm, was removed using modified 40 mL syringes. OMBs were concentrated by centrifuging (Eppendorf, Hauppauge, NY) at 100 relative centrifugal force (RCF) units for 5 min. The infranatant was collected and degassed under vacuum for recycling into the system, and the cakes were consolidated through syringe-syringe transfers via 12 mL syringes. OMBs were further concentrated by centrifuging at 80 RCF for 3 min. The centrifugation process was repeated until the cake was approximately 50 vol %, and microbubble synthesis and concentration were repeated until an 8 mL sample of a 50 vol % gas cake was obtained. The OMBs were stored in capped 12 mL syringes under an oxygen atmosphere at 4 °C. Samples were removed from storage every 2 to 3 days, and OMBs were found to disperse easily in the aqueous suspension. Size measurements were taken using an Accusizer 280A (Particle Sizing Systems, Santa Barbara, CA), which operates on the principle of laser light obscuration and scattering. The sizing capabilities and limitations of this instrument were described previously.24 The mass and volume of each sample was recorded on testing days to determine the percent volume gas contained in each syringe. The release of oxygen from the microbubble core was measured using two Clark-type microcathode electrodes equipped with fastresponse FEP membranes (Strathkelvin Instruments, North Lanarkshire, Scotland). The electrodes were separated by 60 cm (24) Feshitan, J.; Chen, C.; Kwan, J.; Borden, M. J. Colloid Interface Sci. 2009, 329, 316–324.
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Figure 1. DPPC (left column) and DSPC (right column) OMB size distribution and stability: number-weighted size distributions (row A); volume-weighted size distributions (row B); volume- and number-weighted mean diameters (row C); and percent of the total suspension volume for this gas (row D) over time. Error bars (dotted lines in A and B, vertical bars in C and D) represent the standard deviation for n = 2 preparations for DPPC and n = 3 preparations for DSPC. Vertical dotted lines in rows A and B represent the 99th percentiles. For number-weighted distributions (row A), the line denotes the diameter above which only 1% of the bubbles are larger. For volume-weighted distributions (row B), the line denotes the diameter above which only 1% of the gas is encapsulated. The arrows and shaded gray regions denote the change in 99th percentile diameters over 3 weeks. of 1.57 mm i.d. tubing, and the temperature of the test fluid was monitored using 0.9-mm-diameter thermistors (TE Technology, Traverse City, MI) mounted immediately after each electrode (Figure 2A). This assembly was immersed in a temperaturecontrolled bath held at 37 °C. Injection experiments were performed using a 60 mL syringe of microbubbles in PBS, prepared as described above. The syringe was prepared with ∼20% volume gas and contained a 1-cm-long magnetic stir bar. The syringe was connected to a 16 gauge needle via 50 cm of 0.75 mm i.d. Tygon tubing. Most of this tubing was placed in the water bath to allow heat transfer to the microbubble solution before mixing with the flowing PBS. The injection needle was mounted in a luerlock T-fitting such that the needle tip was in the flow path of the PBS. The flow rate of the microbubble solution was controlled using a syringe pump (Kent Scientific, Litchfield, CT), and the microbubble solution was mixed using a magnetic stir plate to ensure consistent delivery. The microbubble suspension was injected at flow rates of 1.0, 2.5, 5.0, and DOI: 10.1021/la1029432
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Figure 2. OMB oxygen release. (A) Schematic of the oxygen release testing apparatus. (B) Typical experimental results showing the steps in the flow rate. Error bars show the standard deviation between electrodes. (C) Comparison of measured dissolved oxygen (points) with the equilibrium model prediction (dotted lines). 10 mL min-1. The flow of PBS was adjusted to ensure that the total flow rate was always 40 mL min-1. Assuming plug flow, the microbubble suspension reached the first electrode less than a second after injection, and the two electrodes were separated by about 2 s.
Results and Discussion Despite having different acyl chain lengths, DPPC and DSPC formulations generated OMBs with similar initial size distributions (Figure 1A,B). The diameter of the OMBs yielded by both formulations was polydisperse, as expected from a sonicationbased synthesis process. Both formed bubbles with a series of preferred diameters, indicated by the peaks in the size distribution at 1-2, 4-5, 6-8, and 8-10 μm, as previously observed.24 Most importantly from a biomedical point of view, the initial numberweighted 99th percentiles (Figure 1A) showed that all but 1% of the MBs are smaller than 10 μm for DPPC and 6 μm for DSPC. The initial volume-weighted 99th percentiles (Figure 1B) showed that only 1% of the gas volume is encapsulated in particles above 16 μm for DPPC and 10 μm for DSPC. The small size will allow OMBs to clear the capillary beds of the body, ensuring minimal risk of occlusion or embolism. The percentiles also changed very little over time, indicating a level of stability that is sufficient for the advance preparation and storage of OMBs. Although the particle size distributions of the highly concentrated OMBs remained relatively constant over time, there was a slight shift during the first week, as illustrated by the numberweighted and volume-weighted mean diameters (Figure 1C). Linear regression revealed that the change in diameter was significant for the volume-weighted mean (p = 0.0002) but not for the number-weighted mean for DPPC and both means for DSPC (p < 0.0001). However, the means remained constant after the first week of storage (p > 0.05). Thus, aside from a slight initial increase, the mean bubble diameter of DPPC- and DSPC-coated OMBs did not change. It is normally accepted that microbubble stability is related to the main phase-transition temperature (rigidity) of the phospholipids contained in their monolayer coating.19,21,25,26 Phospholipids with longer acyl chains tend to provide a higher mass-transfer resistance to gas molecules into solution.19,21,22 Although DPPC (25) Kim, D. H.; Costello, M. J.; Duncan, P. B.; Needham, D. Langmuir 2003, 19, 8455–8466. (26) Borden, M. A.; Kruse, D.; Caskey, C.; Zhao, S.; Dayton, P.; Ferrara, K. IEEE Trans. Ultrason., Ferroelectr., Freq. Control 2005, 52, 1992–2002.
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contains a shorter acyl chain and a lower main phase-transition temperature than DSPC (41 vs 55 °C), DPPC-coated OMBs also exhibited a smaller shift in both the mean diameter and 99th percentiles over time. This apparent discrepancy could be explained by the higher initial polydispersity of DPPC, ostensibly starting closer to a more stable size distribution. This stable distribution could be related to the packing of OMBs in the dense foam formed during storage, with a higher polydispersity leading to more point contacts between bubbles and less deformation from their spherical shape. Although the particle size distributions changed very little, the percent volume gas of both formulations decreased linearly with time and followed the expected trend based on the phase-transition temperature (rigidity) of the coating material (Figure 1D).19-22 The DPPC-based MBs lost almost all of their gas over the 3 week testing period; however, DSPC retained its gas for a far longer period of time and still contained more than 30 vol % gas at the end of testing. This lost gas accumulated in the syringes as a gas pocket above the MB cake and was easily expelled each time the suspensions were remixed for sampling. The fact that this gas was continuously lost from the microbubbles without significant changes in the size distribution indicates that the conventional mechanisms of bubble breakdown (namely, dissolution, coalescence, and Ostwald ripening) are acting only on a small subset of the total MB population. The factors that select which OMBs participate in this phase-separation process appear to be entirely random because gas is lost continuously but the OMB distribution appears to be unaffected. Although the OMBs were stable under storage conditions, their gas core was readily available upon introduction into an undersaturated solution. Figure 2B shows the data obtained from a typical injection experiment. As the flow ratio of the OMB suspension was increased in discrete steps, the measured oxygen concentration increased accordingly. The error bars, represented by dotted lines, indicated the standard deviation of the two measurement locations. Because one of these measurements was made soon after mixing (50 vol % gas. These novel and biocompatible particles were shown to have remarkable stability, retaining over half of their original gas payload for over 3 weeks while exhibiting a minimal change in the particle size distribution. When introduced into an undersaturated solution, the gas payload was released rapidly into the surrounding fluid, enabling a wide range of biomedical and industrial gas delivery applications. Acknowledgment. This work was supported by NYSTAR C020028 and NSF 0952681 to M.A.B. and NSERC to E.J.S. Special thanks goes to Givi Basishvili, Xavier Marrero, and Nathan Lee for help in developing the microbubble synthesis apparatus. Supporting Information Available: Mass balance model for OMB delivery to a flowing solution. This material is available free of charge via the Internet at http://pubs.acs.org.
DOI: 10.1021/la1029432
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