, Volume 16, Issue 3, pp 878–890 | Cite as

Characterization and Imaging of Lipid-Shelled Microbubbles for Ultrasound-Triggered Release of Xenon

  • Himanshu ShekharEmail author
  • Arunkumar Palaniappan
  • Tao Peng
  • Maxime Lafond
  • Melanie R. Moody
  • Kevin J. Haworth
  • Shaoling Huang
  • David D. McPherson
  • Christy K. Holland
Original Article


Xenon (Xe) is a bioactive gas capable of reducing and stabilizing neurologic injury in stroke. The goal of this work was to develop lipid-shelled microbubbles for xenon loading and ultrasound-triggered release. Microbubbles loaded with either xenon (Xe-MB) or xenon and octafluoropropane (Xe-OFP-MB) (9:1 v/v) were synthesized by high-shear mixing. The size distribution and the frequency-dependent attenuation coefficient of Xe-MB and Xe-OFP-MB were measured using a Coulter counter and a broadband acoustic attenuation spectroscopy system, respectively. The Xe dose was evaluated using gas chromatography/mass spectrometry. The total Xe doses in Xe-MB and Xe-OFP-MB were 113.1 ± 13.5 and 145.6 ± 25.5 μl per mg of lipid, respectively. Co-encapsulation of OFP increased the total xenon dose, attenuation coefficient, microbubble stability (in an undersaturated solution), and shelf life of the agent. Triggered release of gas payload was demonstrated with 6-MHz duplex Doppler and 220-kHz pulsed ultrasound. These results constitute the first step toward the use of lipid-shelled microbubbles for applications such as neuroprotection in stroke.

Key Words

Bioactive gas delivery xenon delivery lipid-shelled microbubbles ultrasound cytoprotection. 



This work was supported by the National Institutes of Health/National Institute of Neurological Disorders and Stroke through grant R01 NS047603. Kevin Haworth, Ph.D., was supported by the National Institutes of Health/National Heart, Lung, and Blood Institute through grant K25HL133452. The authors thank Prof. Jack Rubinstein, Dr. Sheryl Koch, and Michelle Nieman for their help with in vivo imaging and Dr. Karla Mercado-Shekhar for assistance with in vitro imaging. Prof. Dong Zhang and Prof. Xiasheng Guo are acknowledged for providing the 220-kHz transducer used in this study, and Robert Kleven for calibrating it. The authors are grateful to Prof. Kenneth Setchell for sharing his expertise on gas chromatography/mass spectroscopy measurements.

Required Author Forms

Disclosure forms provided by the authors are available with the online version of this article.

Supplementary material

13311_2019_733_MOESM1_ESM.pdf (515 kb)
ESM 1 (PDF 514 kb)


  1. 1.
    Benjamin EJ, Virani SS, Callaway CW, et al. Heart disease and stroke statistics—2018 update: a report from the American Heart Association. Circulation. 2018;137:e67-e492.CrossRefGoogle Scholar
  2. 2.
    Majid A. Neuroprotection in stroke: past, present, and future. ISRN Neurol 2014;2014:515716.CrossRefGoogle Scholar
  3. 3.
    Moskowitz MA, Lo EH, Iadecola C. The science of stroke: mechanisms in search of treatments. Neuron. 2010;67:181–198.CrossRefGoogle Scholar
  4. 4.
    Hwang BY, Appelboom G, Ayer A, et al. Advances in neuroprotective strategies: potential therapies for intracerebral hemorrhage. Cerebrovasc Dis 2011;31:211–222.CrossRefGoogle Scholar
  5. 5.
    Laskowitz DT, Kolls BJ. Neuroprotection in subarachnoid hemorrhage. Stroke. 2010;41:S79-S84.CrossRefGoogle Scholar
  6. 6.
    Lai TW, Zhang S, Wang YT. Excitotoxicity and stroke: identifying novel targets for neuroprotection. Prog Neurobiol 2014;115:157–188.CrossRefGoogle Scholar
  7. 7.
    Rajah GB, Ding Y. Experimental neuroprotection in ischemic stroke: a concise review. Neurosurg Focus 2017;42:E2.Google Scholar
  8. 8.
    Fan X, Kavelaars A, Heijnen CJ, Groenendaal F, van Bel F. Pharmacological neuroprotection after perinatal hypoxic-ischemic brain injury. Curr Neuropharmacol 2010;8:324–334.CrossRefGoogle Scholar
  9. 9.
    Fisher M. New approaches to neuroprotective drug development. Stroke. 2011;42:S24–S27.CrossRefGoogle Scholar
  10. 10.
    Preckel B, Weber NC, Sanders RD, Maze M, Schlack W. Molecular mechanisms transducing the anesthetic, analgesic, and organ-protective actions of xenon. Anesthesiology. 2006;105:187–197.CrossRefGoogle Scholar
  11. 11.
    Sanders RD, Ma D, Maze M. Anaesthesia induced neuroprotection. Best Pract Res Clin Anaesthesiol 2005;19:461–474.CrossRefGoogle Scholar
  12. 12.
    Dickinson R, Franks NP. Bench-to-bedside review: molecular pharmacology and clinical use of inert gases in anesthesia and neuroprotection. Crit Care 2010;14.Google Scholar
  13. 13.
    Ma D, Hossain M, Pettet GK, et al. Xenon preconditioning reduces brain damage from neonatal asphyxia in rats. J Cereb Blood Flow Metab 2006;26:199–208.CrossRefGoogle Scholar
  14. 14.
    Koerner IP, Brambrink AM. Brain protection by anesthetic agents. Curr Opin Anaesthesiol 2006;19:481–486.CrossRefGoogle Scholar
  15. 15.
    Adibhatla RM, Hatcher JF. Tissue plasminogen activator (tPA) and matrix metalloproteinases in the pathogenesis of stroke: therapeutic strategies. CNS Neurol Disord Drug Targets 2008;7:243–253.CrossRefGoogle Scholar
  16. 16.
    Dingley J, Findlay GP, Foex BA, et al. A closed xenon anesthesia delivery system. Anesthesiology. 2001;94:173–176.CrossRefGoogle Scholar
  17. 17.
    Miao YF, Peng T, Moody MR, et al. Delivery of xenon-containing echogenic liposomes inhibits early brain injury following subarachnoid hemorrhage. Sci Rep 2018;8:450.CrossRefGoogle Scholar
  18. 18.
    Peng T, Britton GL, Kim H, et al. Therapeutic time window and dose dependence of xenon delivered via echogenic liposomes for neuroprotection in stroke. CNS Neurosci Ther 2013;19:773–784.Google Scholar
  19. 19.
    Britton GL, Kim H, Kee PH, et al. In vivo therapeutic gas delivery for neuroprotection with echogenic liposomes. Circulation. 2010;122:1578–1587.CrossRefGoogle Scholar
  20. 20.
    Kim H, Britton GL, Peng T, et al. Nitric oxide-loaded echogenic liposomes for treatment of vasospasm following subarachnoid hemorrhage. Int J Nanomedicine 2014;9:155–165.Google Scholar
  21. 21.
    Huang SL, Kee PH, Kim H, et al. Nitric oxide-loaded echogenic liposomes for nitric oxide delivery and inhibition of intimal hyperplasia. J Am Coll Cardiol 2009;54:652–659.CrossRefGoogle Scholar
  22. 22.
    Dandekar MP, Peng T, McPherson DD, et al. Intravenous infusion of xenon-containing liposomes generates rapid antidepressant-like effects. Prog Neuro-Psychopharmacol Biol Psychiatry 2018;86:140–149.CrossRefGoogle Scholar
  23. 23.
    Huang SL, McPherson DD, Macdonald RC. A method to co-encapsulate gas and drugs in liposomes for ultrasound-controlled drug delivery. Ultrasound Med Biol 2008;34:1272–1280.CrossRefGoogle Scholar
  24. 24.
    Kwan JJ, Kaya M, Borden MA, Dayton PA. Theranostic oxygen delivery using ultrasound and microbubbles. Theranostics. 2012;2:1174–1184.CrossRefGoogle Scholar
  25. 25.
    Wang C, Yang F, Xu ZH, et al. Intravenous release of NO from lipidic microbubbles accelerates deep vein thrombosis resolution in a rat model. Thromb Res 2013;131:E31-E38.CrossRefGoogle Scholar
  26. 26.
    Fix SM, Borden MA, Dayton PA. Therapeutic gas delivery via microbubbles and liposomes. J Control Release 2015;209:139–149.CrossRefGoogle Scholar
  27. 27.
    Chen G, Yang L, Zhong L, et al. Delivery of hydrogen sulfide by ultrasound targeted microbubble destruction attenuates myocardial ischemia-reperfusion injury. Sci Rep 2016;6:30643.CrossRefGoogle Scholar
  28. 28.
    Eisenbrey JR, Shraim R, Liu JB, et al. Sensitization of hypoxic tumors to radiation therapy using ultrasound-sensitive oxygen microbubbles. Int J Radiat Oncol 2018;101:88–96.CrossRefGoogle Scholar
  29. 29.
    Owen J, McEwan C, Nesbitt H, et al. Reducing tumour hypoxia via oral administration of oxygen nanobubbles. PLoS One 2016;11.Google Scholar
  30. 30.
    Yang CJ, Xiao H, Sun Y, et al. Lipid microbubbles as ultrasound-stimulated oxygen carriers for controllable oxygen release for tumor reoxygenation. Ultrasound Med Biol 2018;44:416–425.CrossRefGoogle Scholar
  31. 31.
    Szijjarto C, Rossi S, Waton G, Krafft MP. Effects of perfluorocarbon gases on the size and stability characteristics of phospholipid-coated microbubbles: osmotic effect versus interfacial film stabilization. Langmuir. 2012;28:1182–1189.CrossRefGoogle Scholar
  32. 32.
    Kabalnov A, Bradley J, Flaim S, et al. Dissolution of multicomponent microbubbles in the bloodstream: 2. Experiment. Ultrasound Med Biol 1998;24:751–760.CrossRefGoogle Scholar
  33. 33.
    Goertz DE, de Jong N, van der Steen AFW. Attenuation and size distribution measurements of definity (TM) and manipulated definity (TM) populations. Ultrasound Med Biol 2007;33:1376–1388.CrossRefGoogle Scholar
  34. 34.
    Raymond JL, Haworth KJ, Bader KB, et al. Broadband attenuation measurements of phospholipid-shelled ultrasound contrast agents. Ultrasound Med Biol 2014;40:410–421.CrossRefGoogle Scholar
  35. 35.
    Altman PL. Handbook of respiration. Philadelphia: W. B. Saunders; 1959.Google Scholar
  36. 36.
    Mulvana H, Stride E, Tang MX, Hajnal JV, Eckersley RJ. The influence of gas saturation on microbubble stability. Ultrasound Med Biol 2012;38:1097–1100.CrossRefGoogle Scholar
  37. 37.
    Shekhar H, Smith NJ, Raymond JL, Holland CK. Effect of temperature on the size distribution, shell properties, and stability of definity (R). Ultrasound Med Biol 2018;44:434–446.CrossRefGoogle Scholar
  38. 38.
    Klegerman ME, Moody MR, Hurling JR, et al. Gas chromatography/mass spectrometry measurement of xenon in gas-loaded liposomes for neuroprotective applications. Rapid Commun Mass Spectrom 2017;31:1–8.CrossRefGoogle Scholar
  39. 39.
    Yeh SY, Peterson RE. Solubility of carbon dioxide, krypton, and xenon in lipids. J Pharm Sci 1963;52:453–458.CrossRefGoogle Scholar
  40. 40.
    Yeh SY, Peterson RE. Solubility of carbon dioxide, krypton, and xenon in aqueous solution. J Pharm Sci 1964;53:822–824.CrossRefGoogle Scholar
  41. 41.
    Tetreau C, Blouquit Y, Novikov E, Quiniou E, Lavalette D. Competition with xenon elicits ligand migration and escape pathways in myoglobin. Biophys J 2004;86:435–447.CrossRefGoogle Scholar
  42. 42.
    King AD. The solubility of gases in aqueous solutions of poly(propylene glycol). J Colloid Interface Sci 2001;243:457–462.CrossRefGoogle Scholar
  43. 43.
    Goto T, Suwa K, Uezono S, et al. The blood-gas partition coefficient of xenon may be lower than generally accepted. Br J Anaesth 1998;80:255–256.CrossRefGoogle Scholar
  44. 44.
    Kenwright DA, Thomson AJ, Hadoke PW, et al. A protocol for improved measurement of arterial flow rate in preclinical ultrasound. Ultrasound Int Open 2015;1:E46–52.CrossRefGoogle Scholar
  45. 45.
    Kubo-Inoue M, Egashira K, Usui M, et al. Long-term inhibition of nitric oxide synthesis increases arterial thrombogenecity in rat carotid artery. Am J Physiol-Heart C 2002;282:H1478-H1484.CrossRefGoogle Scholar
  46. 46.
    McDannold N, Arvanitis CD, Vykhodtseva N, Livingstone MS. Temporary disruption of the blood-brain barrier by use of ultrasound and microbubbles: safety and efficacy evaluation in rhesus macaques. Cancer Res 2012;72:3652–3663.CrossRefGoogle Scholar
  47. 47.
    Verbree J, Bronzwaer ASGT, Ghariq E, et al. Assessment of middle cerebral artery diameter during hypocapnia and hypercapnia in humans using ultra-high-field MRI. J Appl Physiol 2014;117:1084–1089.CrossRefGoogle Scholar
  48. 48.
    Alexandrov AV, Tsivgoulis G, Rubiera M, et al. End-diastolic velocity increase predicts recanalization and neurological improvement in patients with ischemic stroke with proximal arterial occlusions receiving reperfusion therapies. Stroke. 2010;41:948–952.CrossRefGoogle Scholar
  49. 49.
    Debbage PL, Griebel J, Ried M, et al. Lectin intravital perfusion studies in tumor-bearing mice: micrometer-resolution, wide-area mapping of microvascular labeling, distinguishing efficiently and inefficiently perfused microregions in the tumor. J Histochem Cytochem 1998;46:627–639.CrossRefGoogle Scholar
  50. 50.
    Widmaier EP, Raff H, Strang KT, Vander AJ. Vander’s human physiology: the mechanisms of body function. Fourteenth edition. ed. New York, NY: McGraw-Hill; 2016. 1 volume (various pagings) p.Google Scholar
  51. 51.
    Borden MA, Longo ML. Dissolution behavior of lipid monolayer-coated, air-filled microbubbles: effect of lipid hydrophobic chain length. Langmuir. 2002;18:9225–9233.CrossRefGoogle Scholar
  52. 52.
    Garg S, Thomas AA, Borden MA. The effect of lipid monolayer in-plane rigidity on in vivo microbubble circulation persistence. Biomaterials. 2013;34:6862–6870.CrossRefGoogle Scholar
  53. 53.
    Kim DH, Costello MJ, Duncan PB, Needham D. Mechanical properties and microstructure of polycrystalline phospholipid monolayer shells: novel solid microparticles. Langmuir. 2003;19:8455–8466.CrossRefGoogle Scholar
  54. 54.
    Borden MA, Longo ML. Oxygen permeability of fully condensed lipid monolayers. J Phys Chem B 2004;108:6009–6016.CrossRefGoogle Scholar
  55. 55.
    Chen CC, Borden MA. Ligand conjugation to bimodal poly(ethylene glycol) brush layers on microbubbles. Langmuir. 2010;26:13183–13194.CrossRefGoogle Scholar
  56. 56.
    Sarkar K, Katiyar A, Jain P. Growth and dissolution of an encapsulated contrast microbubble: effects of encapsulation permeability. Ultrasound Med Biol 2009;35:1385–1396.CrossRefGoogle Scholar
  57. 57.
    Abou-Saleh RH, Peyman SA, Johnson BRG, et al. The influence of intercalating perfluorohexane into lipid shells on nano and microbubble stability. Soft Matter 2016;12:7223–7230.CrossRefGoogle Scholar
  58. 58.
    Sutton JT, Raymond JL, Verleye MC, Pyne-Geithman GJ, Holland CK. Pulsed ultrasound enhances the delivery of nitric oxide from bubble liposomes to ex vivo porcine carotid tissue. Int J Nanomedicine 2014;9:4671–4683.CrossRefGoogle Scholar
  59. 59.
    Cloft HJ, Rabinstein A, Lanzino G, Kallmes DF. Intra-arterial stroke therapy: an assessment of demand and available work force. Am J Neuroradiol 2009;30:453–458.CrossRefGoogle Scholar
  60. 60.
    Abou-Chebl A. Intra-arterial therapy for acute ischemic stroke. Neurotherapeutics. 2011;8:400–413.CrossRefGoogle Scholar
  61. 61.
    Widmaier EP, Raff H, Strang KT. Vander’s human physiology: the mechanisms of body function. 2006.Google Scholar
  62. 62.
    Eisenbrey JR, Albala L, Kramer MR, et al. Development of an ultrasound sensitive oxygen carrier for oxygen delivery to hypoxic tissue. Int J Pharm 2015;478:361–367.CrossRefGoogle Scholar
  63. 63.
    Cavalli R, Bisazza A, Giustetto P, et al. Preparation and characterization of dextran nanobubbles for oxygen delivery. Int J Pharm 2009;381:160–165.CrossRefGoogle Scholar
  64. 64.
    McEwan C, Fowley C, Nomikou N, et al. Polymeric microbubbles as delivery vehicles for sensitizers in sonodynamic therapy. Langmuir. 2014;30:14926–14930.CrossRefGoogle Scholar
  65. 65.
    Cavalli R, Bisazza A, Rolfo A, et al. Ultrasound-mediated oxygen delivery from chitosan nanobubbles. Int J Pharm 2009;378:215–217.CrossRefGoogle Scholar
  66. 66.
    Fix SM, Nyankima AG, McSweeney MD, et al. Accelerated clearance of ultrasound contrast agents containing polyethylene glycol is associated with the generation of anti-polyethylene glycol antibodies. Ultrasound Med Biol 2018;44:1266–1280.CrossRefGoogle Scholar
  67. 67.
    Mulvana H, Stride E, Tang M, Hajnal JV, Eckersley R. Temperature-dependent differences in the nonlinear acoustic behavior of ultrasound contrast agents revealed by high-speed imaging and bulk acoustics. Ultrasound Med Biol 2011;37:1509–1517.CrossRefGoogle Scholar

Copyright information

© The American Society for Experimental NeuroTherapeutics, Inc. 2019

Authors and Affiliations

  • Himanshu Shekhar
    • 1
    Email author
  • Arunkumar Palaniappan
    • 1
  • Tao Peng
    • 2
  • Maxime Lafond
    • 1
  • Melanie R. Moody
    • 2
  • Kevin J. Haworth
    • 1
    • 3
  • Shaoling Huang
    • 2
  • David D. McPherson
    • 2
  • Christy K. Holland
    • 1
    • 3
  1. 1.Division of Cardiovascular Health and Disease, Department of Internal MedicineUniversity of CincinnatiCincinnatiUSA
  2. 2.Division of Cardiovascular Medicine, Department of Internal MedicineUniversity of Texas Health Science Center at HoustonHoustonUSA
  3. 3.Department of Biomedical EngineeringUniversity of CincinnatiCincinnatiUSA

Personalised recommendations