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DRUG DELIVERY SYSTEM WITH ACOUSTIC LIPOSOMES VIA THE LYMPHATIC VESSELS SHIGEKI 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 1 :Graduate School of Biomedical Engineering Tohoku University 2 :Tohoku University Hospital Aug. 13 th, 2012

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Lymphatic metastases 8/13/2012CAV2012 1 Ran, S., et al., Pathophysiology, 2010; 17(4): 229-251.

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Cancer treatment of lymph node metastases 8/13/2012CAV2012 2 Surgical dissection Radiation Chemotherapy Gene therapy Systemic administration causes severe side effects due to cytotoxicity to nomal tissues Only a small fraction of drugs reach the target tissues

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Breast cancer categories 8/13/2012CAV2012 3 TNM staging system primary tumor size lymph node involvement whether the cancer has metastasized stagesize T0not found T1less than 2cm T22 - 5 cm T3, T4more than 5cm stagemetastatic node N00 N11-3 N24-9 N3more than 10 stagedistant metastasis M0negative M1positive Primary tumor LN 1 LN 2 LN 3 LN: lymph node Sentinel lymph node distant organs

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Drug delivery via the lymphatic vessel 8/13/2012CAV2012 4 Mouse model: MXH-10/Mo/lpr/lpr (MXH-10) Lymph node (same size as it of human) Lymph node (same size as it of human) Proper axillary lymph node Proper axillary lymph node Subiliac lymph node Subiliac lymph node Visualizing lymph route Develop local administration via lymphatic vessels for Drug Delivery System at T0, T1, T2, N0 stages Develop local administration via lymphatic vessels for Drug Delivery System at T0, T1, T2, N0 stages

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Sonoporation 8/13/2012CAV2012 5 Collapse of NMB and generation of cavitation nuclei Nano/Micro Bubble (NMB) Liquid 0 US probe NMB cavitation nuclei Transient permeabilization of cell membrane Entry of exogenous molecules Impulsive pressure Advantages easy operation low toxicity low invasiveness high tissue selectivity Real-time monitoring of bubbles using an ultrasound imaging system membrane nucleus

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Aim 8/13/2012CAV2012 6 Exp.1 To detect the lymphatic vessels in the proper axillary lymph node Exp.2 To deliver the fluorescent molecules into the proper axillary lymph node via the lymphatic vessels by sonoporation Evaluation of sonoporation efficiency of fluorescent molecules into the proper axillary lymph node via lymphatics Evaluation of sonoporation efficiency of fluorescent molecules into the proper axillary lymph node via lymphatics

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8/13/2012CAV2012 7 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 m US contrast agent Acoustic liposome (AL) potential: -4.1 0.74 mV mean diameter: 200 nm Materials AL Kodama, T., et al., J Electron Microsc (Tokyo), 2010; 59(3): 187-196. ~ To detect the lymphatic vessels in the axillary lymph node ~

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8/13/2012CAV2012 8 syringe pump (50L/min) syringe butterfly needle US probehigh-frequency US imaging system Mouse under anesthesia Subiliac lymph node Subiliac lymph node Lymphatic vessel Proper axillary lymph node Proper axillary lymph node Volume 200L 1.To acquire reference image of the axillary lymph node 2.AL injection into the inguinal lymph node 3.To detect the AL in the axillary lymph node FOV (mm)Frame rate (Hz) Cine loop size ContrastthresholdGating 10.0 x 10.09 1330040300ON Methods ~ To detect the lymphatic vessels in the axillary lymph node ~

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8/13/2012CAV2012 9 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 m US contrast agent Acoustic liposome (AL) potential: -4.1 0.74 mV mean diameter: 200 nm Materials AL Kodama, T., et al., J Electron Microsc (Tokyo), 2010; 59(3): 187-196. ~ To deliver the fluorescent molecules into the axillary lymph node via the lymphatic vessels by sonoporation ~

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8/13/2012CAV2012 10 US exposure condition US transducer: HONDA electronics -12 (1 MHz) Delivered molecule TOTO-3 iodide molecular weight: 1355 absorption wavelength: 642 nm emission wavelength: 660 nm Materials Pulse number Duty cyclepressureExposure time 20020%0.67 MPa60 sec . Kodama, T., et al., Ultrasound Med Biol, 2010; 36(7): 1196-1205. ~ To deliver the fluorescent molecules into the axillary lymph node via the lymphatic vessels by sonoporation ~

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8/13/2012CAV2012 11 syringe pump (50L/min) syringe butterfly needle US probe high-frequency US imaging system Mouse under anesthesia Subiliac lymph node Subiliac lymph node Lymphatic vessel Proper axillary lymph node Proper axillary lymph node point of a needle PBS alone TOTO-3 alone TOTO-3 + US TOTO-3 + AL + US Methods PBS (L) TOTO-3 (L) AL (L) Total (L) PBS alone (n=3)20000 TOTO-3 alone (n=4) 100 0200 TOTO-3 + US (n=5) 100 0200 TOTO-3 + AL + US (n=5) 0100 200 ~ To deliver the fluorescent molecules into the axillary lymph node via the lymphatic vessels by sonoporation ~

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8/13/2012CAV2012 12 US transducer 1mm 1 MHz CH1 CH2 Trigger function generator amplifier INPUTMONITOR oscilloscope syringe pump (50L/min) syringe butterfly needle US probe high-frequency US imaging system Mouse under anesthesia Subiliac lymph node Subiliac lymph node Lymphatic vessel Proper axillary lymph node Proper axillary lymph node PBS alone TOTO-3 alone TOTO-3 + US TOTO-3 + AL + US Methods ~ To deliver the fluorescent molecules into the axillary lymph node via the lymphatic vessels by sonoporation ~

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Methods 8/13/2012CAV2012 13 Immunohistochemical evaluation Lymph node dissection after treatment Frozen to liquid nitrogen Sliced by cryostat (10 m) 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 ~ To deliver the fluorescent molecules into the axillary lymph node via the lymphatic vessels by sonoporation ~

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Methods 8/13/2012CAV2012 14 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) Original image G image B image Measure grayscale intensity ~ To deliver the fluorescent molecules into the axillary lymph node via the lymphatic vessels by sonoporation ~

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Results and dicussions 8/13/2012CAV2012 15 Before arrival of the AL After arrival of the AL Proper axillary lymph node region Lymphatic vessels

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Results and discussions 8/13/2012CAV2012 16 PBS aloneTOTO-3 alone TOTO-3 + US TOTO-3 + AL + US 30m 100m http://understandingcancer.co.uk/ news/page/9/ Lymph node ROI

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Results and discussions 8/13/2012CAV2012 17 PBS aloneTOTO-3 alone TOTO-3 + US TOTO-3 + AL + US x y xy z z-axis Grayscale intensity

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Results and discussions 8/13/2012CAV2012 18 DAPILYVE-1 TOTO-3Merge TOTO-3 + AL + US

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Results and discussions 8/13/2012CAV2012 19 20m PBS aloneTOTO-3 alone TOTO-3 + USTOTO-3 + AL + US 20m No tissue damage

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Results and discussion 8/13/2012CAV2012 20 Vascular permeabilization by sonoporation shock wave Lymphatic endothelial cell US wave Fluorescent agent Transient hole liquid jet

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Conclusions 8/13/2012CAV2012 21 Detection of the lymphatic vessels in the proper axillary lymph node Delivery of exogenous molecules into the lymphocytes of the proper axillary lymph node via the lymphatic vessel with US exposure Combined use of AL and US strongly enhanced the delivery efficiency

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Thank you! Questions? 22 8/13/2012 CAV2012

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Results 8/13/2012CAV2012 23 PBS aloneTOTO-3 alone TOTO-3 + US TOTO-3 + AL + US

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Methods 8/13/2012CAV2012 24 1 2 3 N time 1 2 3 N compare Reference imageComparative image Frame number 1 2 3 N

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8/13/2012CAV2012 25

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Methods 8/13/2012CAV2012 26 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) Group 1 (PBS alone) Group 2 (TOTO-3 alone) Group 3 (TOTO-3 + US) Group 4 (TOTO-3 + AL + US) 9141537

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Results 8/13/2012CAV2012 27

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background Lymphatic vessel detection 28 8/13/2012 CAV2012

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Methods (Exp.2) 8/13/2012CAV2012 29 Experimental group PBS (L) TOTO-3 (L) AL (L) Total (L) US exposure PBS alone (n=3)20000 TOTO-3 alone (n=4)100 0200 TOTO-3 + US (n=5)100 0200 TOTO-3 + AL + US (n=5)0100 200

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

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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 V peak = (V peak-peak ) / 2 I A = (V peak ) 2 / Kf 2 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 V peak = (V peak-peak ) / 2 I A = (V peak ) 2 / Kf 2 Kf 2 (acoustic intensity reply factor):0.0216[V 2 W -1 cm 2 ] Meff(hydrophone effective sensitivity):1.201[V/Pa] V eff IAIA

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P A [MPa]= 0.1225 (I A ) 0.5 (Kf 2 [V 2 W -1 cm 2 ]) 0.5 / M eff [V/Pa] 0.1225 [Mpa cm/(W) 0.5 ] P A [MPa]= 0.1225 (I A ) 0.5 (Kf 2 [V 2 W -1 cm 2 ]) 0.5 / M eff [V/Pa] 0.1225 [Mpa cm/(W) 0.5 ] 8/13/2012 CAV2012 32