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1 :Graduate School of Biomedical Engineering Tohoku University 2 :Tohoku University Hospital

Drug delivery system with acoustic liposomes via the lymphatic vessels S higeki Kato 1 , Sachiko Horie 1 , Maya Sakamoto 2 , Shiro Mori 2 and Tetsuya Kodama 1. 1 :Graduate School of Biomedical Engineering Tohoku University 2 :Tohoku University Hospital. Aug. 13 th , 2012.

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1 :Graduate School of Biomedical Engineering Tohoku University 2 :Tohoku University Hospital

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  1. Drug delivery system with acoustic liposomes via the lymphatic vesselsShigeki Kato1, Sachiko Horie1, Maya Sakamoto2, Shiro Mori2 and Tetsuya Kodama1 1:Graduate School of Biomedical Engineering Tohoku University 2:Tohoku University Hospital Aug. 13th, 2012

  2. Lymphatic metastases Ran, S., et al., Pathophysiology, 2010; 17(4): 229-251. CAV2012

  3. Cancer treatment of lymph node metastases • Systemic administration causes severe side effects due to cytotoxicity to nomal tissues • Only a small fraction of drugs reach the target tissues CAV2012 Surgical dissection Radiation Chemotherapy Gene therapy

  4. Breast cancer categories Primary tumor Sentinel lymph node LN 1 LN 2 LN 3 … distant organs CAV2012 • TNM staging system • primary tumor size • lymph node involvement • whether the cancer has metastasized LN: lymph node

  5. Drug delivery via the lymphatic vessel Proper axillary lymph node Subiliac lymph node Lymph node (same size as it of human) Visualizing lymph route Develop local administration via lymphatic vessels for Drug Delivery System at T0, T1, T2, N0 stages CAV2012 Mouse model: MXH-10/Mo/lpr/lpr(MXH-10)

  6. Sonoporation Impulsive pressure cavitation nuclei Collapse of NMB and generation of cavitation nuclei US probe 00 Advantages Liquid membrane NMB • easy operation • low toxicity • low invasiveness • high tissue selectivity • Real-time monitoring of bubbles using an ultrasound imaging system nucleus Entry of exogenous molecules Transient permeabilization of cell membrane Liquid Nano/Micro Bubble(NMB) CAV2012

  7. Aim Evaluation of sonoporation efficiency of fluorescent molecules into the proper axillary lymph node via lymphatics CAV2012 • Exp.1 • To detect the lymphatic vessels in the proper axillarylymph node • Exp.2 • To deliver thefluorescent molecules into the proper axillarylymph node via the lymphatic vessels by sonoporation

  8. Materials • ~ To detect the lymphatic vessels • in the axillary lymph node ~ AL ＊ Kodama, T., et al., J Electron Microsc (Tokyo), 2010; 59(3): 187-196. CAV2012 • Animal • MXH-10/Mo/lpr/lpr(MXH-10) • male (n = 3) • 16 - 17 weeks of age • Imaging modality • High-frequency ultrasound (US) imaging system • central frequency: 55 MHz, • axial resolution: 30 μm • US contrast agent＊ • Acoustic liposome (AL) • ζ – potential: -4.1 ± 0.74 mV • mean diameter: 200 nm

  9. Methods • ~ To detect the lymphatic vessels • in the axillary lymph node ~ Mouse under anesthesia To acquire reference image of the axillary lymph node AL injection into the inguinal lymph node To detect the ALin the axillary lymph node US probe high-frequency US imaging system Proper axillary lymph node Lymphatic vessel syringe Subiliac lymph node Volume 200µL butterfly needle syringe pump(50μL/min) CAV2012

  10. Materials ~ To deliver thefluorescent molecules into the axillary lymph node via the lymphatic vessels by sonoporation ~ AL ＊ Kodama, T., et al., J Electron Microsc (Tokyo), 2010; 59(3): 187-196. CAV2012 • Animal • MXH-10 • male (n = 10) and female (n = 7) • 16 – 18 weeks of age • Imaging modality • High-frequency ultrasound (US) imaging system • central frequency: 25 MHz • axial resolution: 70 μm • US contrast agent＊ • Acoustic liposome (AL) • ζ – potential: -4.1 ± 0.74 mV • mean diameter: 200nm

  11. Materials ~ To deliver thefluorescent molecules into the axillary lymph node via the lymphatic vessels by sonoporation ~ ＊＊.Kodama, T., et al., Ultrasound Med Biol, 2010; 36(7): 1196-1205. CAV2012 • US exposurecondition＊＊ • US transducer: HONDA electronics Φ-12 (1 MHz) • Delivered molecule • TOTO-3 iodide • molecular weight: 1355 • absorption wavelength: 642 nm • emission wavelength: 660 nm

  12. Methods ~ To deliver thefluorescent molecules into the axillary lymph node via the lymphatic vessels by sonoporation ~ Mouse under anesthesia point of a needle Proper axillary lymph node high-frequency US imaging system Lymphatic vessel syringe US probe • PBS alone • TOTO-3 alone • TOTO-3 + US • TOTO-3 + AL + US Subiliac lymph node butterfly needle syringe pump(50μL/min) CAV2012

  13. INPUT MONITOR 1MHz Trigger CH1 CH2 Methods ~ To deliver thefluorescent molecules into the axillary lymph node via the lymphatic vessels by sonoporation ~ Mouse under anesthesia high-frequency US imaging system amplifier US probe Proper axillary lymph node US transducer Lymphatic vessel 1mm function generator oscilloscope syringe • PBS alone • TOTO-3 alone • TOTO-3 + US • TOTO-3 + AL + US Subiliac lymph node butterfly needle syringe pump(50μL/min) CAV2012

  14. Methods ~ To deliver thefluorescent molecules into the axillary lymph node via the lymphatic vessels by sonoporation ~ CAV2012 • Immunohistochemical evaluation • Lymph node dissection after treatment • Frozen to liquid nitrogen • Sliced by cryostat (10 μm) • Immunofluorescent staining • Nuclei: DAPI • Lymphatic vessel: anti-LYVE-1 (primary antibody) Alexa-488 (secondary antibody) • Confocal laser scanning microscope • Tissue damage evaluation • Hematoxylin & Eosin (HE) staining

  15. Methods ~ To deliver thefluorescent molecules into the axillary lymph node via the lymphatic vessels by sonoporation ~ R image  Measure grayscale intensity Original image G image B image CAV2012 • Analysis of the TOTO-3 distribution of the axillary lymph node • TOTO-3 bind to DNA  strong red fluorescence • RGB split  select “R (red)” image • Measure grayscale value • MAX: 255 (white) • MIN: 0 (black)

  16. Results and dicussions １ｍｍ Before arrival of the AL After arrival of the AL １ｍｍ Lymphatic vessels Proper axillarylymph node region CAV2012

  17. Results and discussions ROI PBS alone TOTO-3 alone 30μm Lymph node 100μm http://understandingcancer.co.uk/ news/page/9/ TOTO-3 + AL + US TOTO-3 + US CAV2012

  18. Results and discussions y y x x PBS alone TOTO-3 alone z z-axis  Grayscale intensity TOTO-3 + AL + US TOTO-3 + US CAV2012

  19. Results and discussions DAPI LYVE-1 TOTO-3 + AL + US TOTO-3 Merge CAV2012

  20. Results and discussions 20μm 20μm PBS alone TOTO-3 alone 20μm 20μm TOTO-3 + US TOTO-3 + AL + US No tissue damage CAV2012

  21. Results and discussion US wave liquid jet shock wave Lymphatic endothelial cell Transient hole Fluorescent agent CAV2012 Vascular permeabilization by sonoporation

  22. Conclusions CAV2012 Detection of the lymphatic vessels in the proper axillarylymph node Delivery of exogenous molecules into the lymphocytes of the proper axillarylymph node via the lymphatic vessel with US exposure Combined use of AL and US strongly enhanced the delivery efficiency

  23. Questions? Thank you! CAV2012

  24. Results PBS alone TOTO-3 alone TOTO-3 + AL + US TOTO-3 + US CAV2012

  25. Methods Referenceimage Comparativeimage Frame number Frame number 1 1 2 2 3 3 … … … … N N compare time time 1 2 3 … … N CAV2012

  26. 腋窩リンパ節内のリンパ管 CAV2012

  27. Methods CAV2012 • Hotspot ROI • RGB split  select “R” image • To select three ROI every image • To measure mean grayscale value in the ROI • MAX: 255 (white) • MIN: 0 (black)

  28. Results CAV2012

  29. Lymphatic vessel detection background CAV2012

  30. Methods (Exp.2) CAV2012 Experimental group

  31. methods Measure US field Y Z X Hydrophone US transducer Degassed tap water Oscilloscope Multifunction Synthesizer PC Stage Controller Bipolar Amplifier

  32. transducer is driven at several input effective voltages • hydrophone receives acoustic pressure, which is converted into electrical signals and then recorded as peak to peak voltage by oscilloscope • Vpeak= (Vpeak-peak) / 2 • IA = (Vpeak)2 / Kf2 Veff Kf2(acoustic intensity reply factor):0.0216[V2W-1cm2] Meff(hydrophone effective sensitivity):1.201[μV/Pa] IA

  33. PA [MPa]= 0.1225 × (IA)0.5 (Kf2 [V2W-1cm2])0.5/ Meff[μV/Pa] ≒ 0.1225 [Mpa・cm/(W)0.5] CAV2012

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