血腦屏障是哺乳類動物腦部具有的特異化結構，其存在能使腦組織免於有害物質侵入，但同時也增加了腦部疾病治療的困難，近年來已經發現多種方法能提升血腦屏障的通透性，其中聚焦式超音波配合微氣泡的方法，在藉由傳統磷脂質殼層微氣泡於超音波聲場下產生振動及破裂的現象，已被證實能夠局部且非侵入性地開啟血腦屏障，提升血管通透性及藥物輸送效率；然而磷脂質微氣泡破裂時所伴隨產生的慣性穴蝕效應在活體腦組織內可能造成不欲見之出血和傷害。因此，本研究發展出一種新型微氣泡(磷脂質包覆之聚合微氣泡)，藉由在磷脂質單層膜內之疏水單體及交聯劑的自由基聚合反應，於磷脂質殼層內部形成二維網狀聚合物。
製備上，先將磷脂質、聚合單體及交聯劑等材料經由強烈震盪形成微氣泡，再加入起始劑及加速劑在室溫環境下聚合3小時完成。接著我們測試磷脂質包覆之聚合微氣泡的物化及聲學性質，包括共振頻率、非線性共振能力、慣性穴蝕效應強度及擊破閥值。此外，為了避免高頻超音波(10-MHz)在組織內的衰減效應，我們另外製作了大粒徑的磷脂質包覆之聚合微氣泡並配合低頻超音波(2-MHz)來開啟血腦屏障，同時評估其開啟效率及出血情形。
低溫穿透式電子顯微鏡清楚驗證了此二維網狀聚合結構確實形成於磷脂質單層膜內的疏水相。小粒徑微氣泡方面，在低聲壓時磷脂質包覆之聚合微氣泡與未聚合微氣泡除了擁有相同的共振頻率(12-MHz)外，也在5-MHz處表現出相似的次諧波訊號強度；然而，在高聲壓時磷脂質包覆之聚合微氣泡的慣性穴蝕效應強度卻低了未聚合微氣泡1.3倍，且磷脂質包覆之聚合微氣泡的擊破閥值也低了未聚合微氣泡約800 KPa，此結果顯示網狀聚合物殼層不但不會影響磷脂質殼層的震動能力，還能降低其慣性穴蝕效應。另一方面，大粒徑磷脂質包覆之聚合微氣泡的聲學性質大部分皆與小粒徑的相似，除了其慣性穴蝕效應強度只有在超音波脈衝重複頻率高於20 Hz時才會較未聚合微氣泡來的低。最後，我們於活體實驗中使用此磷脂質包覆之聚合微氣泡配合2-MHz的超音波來開啟血腦屏障，結果顯示磷脂質包覆之聚合微氣泡造成了比未聚合微氣泡更多的依文氏藍染劑滲漏，亦即能較有效率地提升血腦屏障通透性，但卻能產生較少的出血傷害。
此研究首先發展了一種新型的微氣泡，其能夠在配合聚焦式超音波開啟血腦屏障時提供更安全的應用；然而，仍然有許多參數及材料可以更進一步調整和嘗試，例如優化磷脂質、聚合單體及交聯劑的材料組成比例，或是使用硬性材料像是熱固性高分子以形成更堅固的聚合物殼層而達到更好的應用效果。

Blood-brain barrier (BBB) is an unique structure in mammals’ brain. Although BBB can prevent harmful substances from entering brain tissues, it makes treatments of brain diseases difficult. Recently, a lot of methods have been discovered to enhance the permeability of BBB, among which traditional phospholipid-shelled microbubbles (MBs) combined with focused ultrasound (FUS) have been approved to induce non-invasive and local BBB disruption by the oscillation and destruction of MBs under insonification of acoustic field, facilitating vascular permeability and drug delivery efficiency. However, the destruction effects of phospholipid MBs may also accompany inertial cavitation (IC) that cause unwanted hemorrhage and damage in vivo. Therefore, in this study, a novel MBs (phospholipid-coated polymer MBs, PP-MBs) was developed by polymerization of hydrophobic monomer and cross-linker inside the phospholipid monolayer of MBs using a radical mechanism, forming a two-dimensional (2D) polymer network inside phospholipid shell.
During the fabrication of PP-MBs, phospholipids, monomer and cross-linker were intensively shook, followed by polymerization via adding initiator and accelerator for 3 hours at room temperature. Then, physicochemical and acoustic properties of PP-MBs, including resonance frequency, nonlinear resonance capacity, inertial cavitation (IC) dose and destruction threshold were measured. Beside, in order to avoid the attenuation effect of high frequency ultrasound (10-MHz) in tissues, PP-MBs with larger size were fabricated for BBB disruption with low frequency ultrasound (2-MHz), and the BBB disruption efficiency and hemorrhage levels were assessed.
Cryo-transmission electron microscopy provided clear evidence that the 2D polymer network structure was indeed incorporated inside the hydrophobic phase of phospholipid monolayer. In the aspects of PP-MBs with smaller size, PP-MBs and unpolymeried MBs behaved not only the same ultrasound resonance frequencies (measured as 12-MHz), but also similar subharmonic intensity characterized at 5-MHz under low acoustic pressure. However, under high acoustic pressure, the IC dose of PP-MBs was 1.3-fold lower than unpolymerized MBs. In addition, the destruction threshold of PP-MBs was about 800 KPa lower than unpolymerized MBs. These results suggested that the polymer network shell of PP-MBs could reduce the inertial cavitation with no influence on oscillation properties of phospholipid shell. On the other hand, PP-MBs with larger size showed similar acoustic properties with the smaller-sized PP-MBs, except for their lower IC dose under ultrasound pulse repetition frequency of >20 Hz than that of smaller-sized PP-MBs. Finally, PP-MBs were applied to disrupt BBB in vivo with 2-MHz FUS. PP-MBs were demonstrated to exhibit more Evans blue leakage (i.e. enhance the permeability of BBB more effectively) but less hemorrhage damage than unpolymerized MBs did.
In this study, we first developed a novel MBs which had great potential to provide safer application for BBB disruption in the combination with FUS. However, there were still some parameters and different materials to adjust and attempt. For example, optimizing the composition of phospholipid, monomer and cross-linker, or using stiff materials such as thermosetting polymers to form more rigid polymer shell to achieve better effectiveness.

[1] S. Mitragotri, "Innovation - Healing sound: the use of ultrasound in drug delivery and other therapeutic applications," Nature Reviews Drug Discovery, vol. 4, pp. 255-260, Mar 2005.
[2] R. Kunstfeld, G. Wickenhauser, U. Michaelis, M. Teifel, W. Umek, K. Naujoks, et al., "Paclitaxel encapsulated in cationic liposomes diminishes tumor angiogenesis and melanoma growth in a "humanized" SCID mouse model," Journal of Investigative Dermatology, vol. 120, pp. 476-482, Mar 2003.
[3] J. E. Kennedy, "High-intensity focused ultrasound in the treatment of solid tumours," Nature Reviews Cancer, vol. 5, pp. 321-327, Apr 2005.
[4] H. L. Liu, M. Y. Hua, P. Y. Chen, P. C. Chu, C. H. Pan, H. W. Yang, et al., "Blood-Brain Barrier Disruption with Focused Ultrasound Enhances Delivery of Chemotherapeutic Drugs for Glioblastoma Treatment," Radiology, vol. 255, pp. 415-425, May 2010.
[5] S. Tinkov, G. Winter, C. Coester, and R. Bekeredjian, "New doxorubicin-loaded phospholipid microbubbles for targeted tumor therapy: Part I - Formulation development and in-vitro characterization," Journal of Controlled Release, vol. 143, pp. 143-150, Apr 2 2010.
[6] J. A. Kang, X. L. Wu, Z. G. Wang, H. T. Ran, C. S. Xu, J. F. Wu, et al., "Antitumor Effect of Docetaxel-Loaded Lipid Microbubbles Combined With Ultrasound-Targeted Microbubble Activation on VX2 Rabbit Liver Tumors," Journal of Ultrasound in Medicine, vol. 29, pp. 61-70, Jan 2010.
[7] W. Xing, W. Z. Gang, Z. Yong, Z. Y. Yi, X. C. Shan, and R. H. Tao, "Treatment of Xenografted Ovarian Carcinoma Using Paclitaxel-loaded Ultrasound Microbubbles," Academic Radiology, vol. 15, pp. 1574-1579, Dec 2008.
[8] W. Lauterborn, T. Kurz, R. Geisler, D. Schanz, and O. Lindau, "Acoustic cavitation, bubble dynamics and sonoluminescence," Ultrasonics Sonochemistry, vol. 14, pp. 484-491, Apr 2007.
[9] I. Lentacker, S. C. De Smedt, and N. N. Sanders, "Drug loaded microbubble design for ultrasound triggered delivery," Soft Matter, vol. 5, pp. 2161-2170, 2009.
[10] V. Sboros, "Response of contrast agents to ultrasound," Advanced Drug Delivery Reviews, vol. 60, pp. 1117-1136, Jun 30 2008.
[11] N. Rapoport, D. A. Christensen, A. M. Kennedy, and K. H. Nam, "Cavitation Properties of Block Copolymer Stabilized Phase-Shift Nanoemulsions Used as Drug Carriers," Ultrasound in Medicine and Biology, vol. 36, pp. 419-429, Mar 2010.
[12] K. K. Shung, R. A. Sigelmann, and J. M. Reid, "Scattering of Ultrasound by Blood," Ieee Transactions on Biomedical Engineering, vol. 23, pp. 460-467, 1976.
[13] N. de Jong, A. Bouakaz, and P. Frinking, "Basic acoustic properties of microbubbles," Echocardiography-a Journal of Cardiovascular Ultrasound and Allied Techniques, vol. 19, pp. 229-240, Apr 2002.
[14] N. C. Nanda, R. Gramiak, and P. M. Shah, "Diagnosis of Aortic Root Dissection by Echocardiography," Circulation, vol. 48, pp. 506-513, 1973.
[15] M. C. Ziskin, Bonakdar.A, Weinstei.Dp, and P. R. Lynch, "Contrast Agents for Diagnostic Ultrasound," Investigative Radiology, vol. 7, pp. 500-505, 1972.
[16] M. A. Wheatley, B. Schrope, and P. Shen, "Contrast Agents for Diagnostic Ultrasound - Development and Evaluation of Polymer-Coated Microbubbles," Biomaterials, vol. 11, pp. 713-717, Nov 1990.
[17] J. Ophir and K. J. Parker, "Contrast Agents in Diagnostic Ultrasound," Ultrasound in Medicine and Biology, vol. 15, pp. 319-333, 1989.
[18] A. Kabalnov, J. Bradley, S. Flaim, D. Klein, T. Pelura, B. Peters, et al., "Dissolution of multicomponent microbubbles in the bloodstream: 2. Experiment," Ultrasound in Medicine and Biology, vol. 24, pp. 751-760, Jun 1998.
[19] A. Kabalnov, D. Klein, T. Pelura, E. Schutt, and J. Weers, "Dissolution of multicomponent microbubbles in the bloodstream: 1. Theory," Ultrasound in Medicine and Biology, vol. 24, pp. 739-749, Jun 1998.
[20] P. J. A. Frinking, A. Bouakaz, J. Kirkhorn, F. J. Ten Cate, and N. de Jong, "Ultrasound contrast imaging: Current and new potential methods," Ultrasound in Medicine and Biology, vol. 26, pp. 965-975, Jul 2000.
[21] S. Tinkov, R. Bekeredjian, G. Winter, and C. Coester, "Microbubbles as Ultrasound Triggered Drug Carriers," Journal of Pharmaceutical Sciences, vol. 98, pp. 1935-1961, Jun 2009.
[22] J. L. Cohen, J. Cheirif, D. S. Segar, L. D. Gillam, J. S. Gottdiener, E. Hausnerova, et al., "Improved left ventricular endocardial border delineation and opacification with OPTISON (FS069), a new echocardiographic contrast agent - Results of a phase III multicenter trial," Journal of the American College of Cardiology, vol. 32, pp. 746-752, Sep 1998.
[23] E. C. Unger, T. Porter, W. Culp, R. Labell, T. Matsunaga, and R. Zutshi, "Therapeutic applications of lipid-coated microbubbles," Advanced Drug Delivery Reviews, vol. 56, pp. 1291-1314, May 7 2004.
[24] P. B. Duncan and D. Needham, "Test of the Epstein-Plesset model for gas microparticle dissolution in aqueous media: Effect of surface tension and gas undersaturation in solution," Langmuir, vol. 20, pp. 2567-2578, Mar 30 2004.
[25] S. Lee, D. H. Kim, and D. Needham, "Equilibrium and dynamic interfacial tension measurements at microscopic interfaces using a micropipet technique. 1. A new method for determination of interfacial tension," Langmuir, vol. 17, pp. 5537-5543, Sep 4 2001.
[26] S. Lee, D. H. Kim, and D. Needham, "Equilibrium and dynamic interfacial tension measurements at microscopic interfaces using a micropipet technique. 2. Dynamics of phospholipid monolayer formation and equilibrium tensions at water-air interface," Langmuir, vol. 17, pp. 5544-5550, Sep 4 2001.
[27] V. D. Awasthi, D. Garcia, B. A. Goins, and W. T. Phillips, "Circulation and biodistribution profiles of long-circulating PEG-liposomes of various sizes in rabbits," International Journal of Pharmaceutics, vol. 253, pp. 121-132, Mar 6 2003.
[28] M. A. Borden, D. E. Kruse, C. F. Caskey, S. K. Zhao, P. A. Dayton, and K. W. Ferrara, "Influence of lipid shell physicochemical properties on ultrasound-induced microbubble destruction," Ieee Transactions on Ultrasonics Ferroelectrics and Frequency Control, vol. 52, pp. 1992-2002, Nov 2005.
[29] B. B. Goldberg and J. B. Liu, "Contrast agents in abdominal ultrasound," Current Trends in Digestive Ultrasonography, vol. 24, pp. 323-341, 1997.
[30] F. Cavalieri, A. El Hamassi, E. Chiessi, and G. Paradossi, "Stable polymeric microballoons as multifunctional device for biomedical uses: Synthesis and characterization," Langmuir, vol. 21, pp. 8758-8764, Sep 13 2005.
[31] K. Bjerknes, J. U. Braenden, G. Smistad, and L. Agerkvist, "Evaluation of different formulation studies on air-filled polymeric microcapsules by multivariate analysis," International Journal of Pharmaceutics, vol. 257, pp. 1-14, May 12 2003.
[32] W. J. Cui, J. Z. Bei, S. G. Wang, G. Zhi, Y. Y. Zhao, X. S. Zhou, et al., "Preparation and evaluation of poly(L-lactide-co-glycolide) (PLGA) microbubbles as a contrast agent for myocardial contrast echocardiography," Journal of Biomedical Materials Research Part B-Applied Biomaterials, vol. 73B, pp. 171-178, Apr 2005.
[33] J. D. Lathia, L. Leodore, and M. A. Wheatley, "Polymeric contrast agent with targeting potential," Ultrasonics, vol. 42, pp. 763-768, Apr 2004.
[34] M. A. Wheatley, F. Forsberg, K. Oum, R. Ro, and D. El-Sherif, "Comparison of in vitro and in vivo acoustic response of a novel 50 : 50 PLGA contrast agent," Ultrasonics, vol. 44, pp. 360-367, Nov 2006.
[35] M. Schneider, P. Bussat, M. B. Barrau, M. Arditi, F. Yan, and E. Hybl, "Polymeric Microballoons as Ultrasound Contrast Agents - Physical and Ultrasonic Properties Compared with Sonicated Albumin," Investigative Radiology, vol. 27, pp. 134-139, Feb 1992.
[36] H. Leong-Poi, J. Song, S. J. Rim, J. Christiansen, S. Kaul, and J. R. Lindner, "Influence of microbubble shell properties on ultrasound signal: Implications for low-power perfusion imaging," Journal of the American Society of Echocardiography, vol. 15, pp. 1269-1276, Oct 2002.
[37] S. H. Bloch, M. Wan, P. A. Dayton, and K. W. Ferrara, "Optical observation of lipid- and polymer-shelled ultrasound microbubble contrast agents," Applied Physics Letters, vol. 84, pp. 631-633, Jan 26 2004.
[38] F. Y. Yang, W. M. Fu, R. S. Yang, H. C. Lim, K. H. Kang, and W. L. Lin, "Quantitative evaluation of focused ultrasound with a contrast agent on blood-brain barrier disruption," Ultrasound in Medicine and Biology, vol. 33, pp. 1421-1427, Sep 2007.
[39] F. Wang, Y. Cheng, J. Mei, Y. Song, Y. Q. Yang, Y. J. Liu, et al., "Focused Ultrasound Microbubble Destruction-Mediated Changes in Blood-Brain Barrier Permeability Assessed by Contrast-Enhanced Magnetic Resonance Imaging," Journal of Ultrasound in Medicine, vol. 28, pp. 1501-1509, Nov 2009.
[40] F. Xie, M. D. Boska, J. Lof, M. G. Uberti, J. M. Tsutsui, and T. R. Porter, "Effects of Transcranial Ultrasound and Intravenous Microbubbles on Blood Brain Barrier Permeability in a Large Animal Model," Ultrasound in Medicine and Biology, vol. 34, pp. 2028-2034, Dec 2008.
[41] N. Vykhodtseva, N. McDannold, and K. Hynynen, "Progress and problems in the application of focused ultrasound for blood-brain barrier disruption," Ultrasonics, vol. 48, pp. 279-296, Aug 2008.
[42] W. M. Pardridge, "BBB-genomics: creating new openings for brain-drug targeting," Drug Discovery Today, vol. 6, pp. 381-383, Apr 15 2001.
[43] L. L. Muldoon, C. Soussain, K. Jahnke, C. Johanson, T. Siegal, Q. R. Smith, et al., "Chemotherapy delivery issues in central nervous system malignancy: a reality check," J Clin Oncol, vol. 25, pp. 2295-305, Jun 1 2007.
[44] L. Juillerat-Jeanneret, "The targeted delivery of cancer drugs across the blood-brain barrier: chemical modifications of drugs or drug-nanoparticles?," Drug Discovery Today, vol. 13, pp. 1099-1106, Dec 2008.
[45] L. H. Treat, N. McDannold, N. Vykhodtseva, Y. Z. Zhang, K. Tam, and K. Hynynen, "Targeted delivery of doxorubicin to the rat brain at therapeutic levels using MRI-guided focused ultrasound," International Journal of Cancer, vol. 121, pp. 901-907, Aug 15 2007.
[46] K. Hynynen, N. McDannold, N. A. Sheikov, F. A. Jolesz, and N. Vykhodtseva, "Local and reversible blood-brain barrier disruption by noninvasive focused ultrasound at frequencies suitable for trans-skull sonications," Neuroimage, vol. 24, pp. 12-20, Jan 1 2005.
[47] M. S. Khil, A. Kolozsvary, M. Apple, and J. H. Kim, "Increased tumor cures using combined radiosurgery and BCNU in the treatment of 9L glioma in the rat brain," International Journal of Radiation Oncology Biology Physics, vol. 47, pp. 511-516, May 1 2000.
[48] X. L. Xu, X. S. Chen, X. Y. Xu, T. C. Lu, X. Wang, L. X. Yang, et al., "BCNU-loaded PEG-PLLA ultrafine fibers and their in vitro antitumor activity against glioma C6 cells," Journal of Controlled Release, vol. 114, pp. 307-316, Sep 12 2006.
[49] H. L. Liu, Y. Y. Wai, P. H. Hsu, L. A. Lyu, J. S. Wu, C. R. Shen, et al., "In vivo assessment of macrophage CNS infiltration during disruption of the blood-brain barrier with focused ultrasound: a magnetic resonance imaging study (vol 30, pg 168, 2009)," Journal of Cerebral Blood Flow and Metabolism, vol. 30, pp. 674-674, Mar 2010.
[50] S. P. Qin and K. W. Ferrara, "Acoustic response of compliable microvessels containing ultrasound contrast agents," Physics in Medicine and Biology, vol. 51, pp. 5065-5088, Oct 21 2006.
[51] P. Dayton, A. Klibanov, G. Brandenburger, and K. Ferrara, "Acoustic radiation force in vivo: A mechanism to assist targeting of microbubbles," Ultrasound in Medicine and Biology, vol. 25, pp. 1195-1201, Oct 1999.
[52] J. Collis, R. Manasseh, P. Liovic, P. Tho, A. Ooi, K. Petkovic-Duran, et al., "Cavitation microstreaming and stress fields created by microbubbles," Ultrasonics, vol. 50, pp. 273-279, Feb 2010.
[53] H. L. Liu, C. H. Pan, C. Y. Ting, and M. J. Hsiao, "Opening of the Blood-Brain Barrier by Low-Frequency (28-Khz) Ultrasound: A Novel Pinhole-Assisted Mechanical Scanning Device," Ultrasound in Medicine and Biology, vol. 36, pp. 325-335, Feb 2010.
[54] J. Hotz and W. Meier, "Vesicle-templated polymer hollow spheres," Langmuir, vol. 14, pp. 1031-1036, Mar 3 1998.
[55] A. Graff, M. Winterhalter, and W. Meier, "Nanoreactors from polymer-stabilized liposomes," Langmuir, vol. 17, pp. 919-923, Feb 6 2001.
[56] J. F. P. D. Gomes, A. F. P. Sonnen, A. Kronenberger, J. Fritz, M. A. N. Coelho, D. Fournier, et al., "Stable polymethacrylate nanocapsules from ultraviolet light-induced template radical polymerization of unilamellar liposomes," Langmuir, vol. 22, pp. 7755-7759, Aug 29 2006.
[57] J. Y. Im, D. B. Kim, S. H. Lee, and Y. S. Lee, "Porous vesicles dispersible in organic solvents," Langmuir, vol. 19, pp. 6392-6396, Aug 5 2003.
[58] D. E. Goertz, N. de Jong, and A. F. van der Steen, "Attenuation and size distribution measurements of Definity and manipulated Definity populations," Ultrasound Med Biol, vol. 33, pp. 1376-88, Sep 2007.
[59] J. L. Raymond, K. J. Haworth, K. B. Bader, K. Radhakrishnan, J. K. Griffin, S. L. Huang, et al., "Broadband attenuation measurements of phospholipid-shelled ultrasound contrast agents," Ultrasound Med Biol, vol. 40, pp. 410-21, Feb 2014.
[60] J. A. Ketterling, J. Mamou, J. S. Allen, O. Aristizabal, R. G. Williamson, and D. H. Turnbull, "Excitation of polymer-shelled contrast agents with high-frequency ultrasound," Journal of the Acoustical Society of America, vol. 121, pp. El48-El53, Jan 2007.
[61] Y. S. Tung, F. Vlachos, J. A. Feshitan, M. A. Borden, and E. E. Konofagou, "The mechanism of interaction between focused ultrasound and microbubbles in blood-brain barrier opening in mice," Journal of the Acoustical Society of America, vol. 130, pp. 3059-3067, Nov 2011.
[62] J. Bauer-Marschallinger, T. Berer, H. Grun, H. Roitner, B. Reitinger, and P. Burgholzer, "Broadband high-frequency measurement of ultrasonic attenuation of tissues and liquids," IEEE Trans Ultrason Ferroelectr Freq Control, vol. 59, pp. 2631-45, Dec 2012.
[63] W. L. Nyborg, "Biological effects of ultrasound: Development of safety guidelines - Part I: Personal histories," Ultrasound in Medicine and Biology, vol. 26, pp. 911-964, Jul 2000.
[64] W. L. Nyborg, "Biological effects of ultrasound: Development of safety guidelines. Part II: General review," Ultrasound in Medicine and Biology, vol. 27, pp. 301-333, Mar 2001.
[65] W. D. OBrien and J. F. Zachary, "Lung damage assessment from exposure to pulsed-wave ultrasound in the rabbit, mouse, and pig," Ieee Transactions on Ultrasonics Ferroelectrics and Frequency Control, vol. 44, pp. 473-485, Mar 1997.
[66] P. J. Quinn, "A lipid-phase separation model of low-temperature damage to biological membranes," Cryobiology, vol. 22, pp. 128-46, Apr 1985.
[67] M. L. Kraft, P. K. Weber, M. L. Longo, I. D. Hutcheon, and S. G. Boxer, "Phase separation of lipid membranes analyzed with high-resolution secondary ion mass spectrometry," Science, vol. 313, pp. 1948-51, Sep 29 2006.
[68] R. Parthasarathy, C. H. Yu, and J. T. Groves, "Curvature-modulated phase separation in lipid bilayer membranes," Langmuir, vol. 22, pp. 5095-9, May 23 2006.
[69] J. J. Kwan and M. A. Borden, "Lipid monolayer collapse and microbubble stability," Adv Colloid Interface Sci, vol. 183-184, pp. 82-99, Nov 15 2012.
[70] M. E. Loveless, X. Li, J. Huamani, A. Lyshchik, B. Dawant, D. Hallahan, et al., "A Method for Assessing the Microvasculature in a Murine Tumor Model Using Contrast-Enhanced Ultrasonography," Journal of Ultrasound in Medicine, vol. 27, pp. 1699-1709, Dec 2008.
[71] D. E. Goertz, C. Wright, and K. Hynynen, "Contrast agent kinetics in the rabbit brain during exposure to therapeutic ultrasound," Ultrasound Med Biol, vol. 36, pp. 916-24, Jun 2010.
[72] T. Hirokawa, R. Karshafian, C. J. Pavlin, and P. N. Burns, "Insonation of the eye in the presence of microbubbles: preliminary study of the duration and degree of vascular bioeffects--work in progress," J Ultrasound Med, vol. 26, pp. 731-8, Jun 2007.
[73] S. B. Raymond, J. Skoch, K. Hynynen, and B. J. Bacskai, "Multiphoton imaging of ultrasound/Optison mediated cerebrovascular effects in vivo," J Cereb Blood Flow Metab, vol. 27, pp. 393-403, Feb 2007.