Download - 1 :Graduate School of Biomedical Engineering Tohoku University 2 :Tohoku University Hospital
DRUG DELIVERY SYSTEM WITH ACOUSTIC LIPOSOMES VIA THE
LYMPHATIC VESSELS
SHIGEKI KATO1, SACHIKO HORIE1, MAYA SAKAMOTO2, SHIRO MORI2
AND TETSUYA KODAMA11:Graduate School of Biomedical Engineering
Tohoku University2:Tohoku University Hospital
Aug. 13th, 2012
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Lymphatic metastases
8/13/2012Ran, S., et al., Pathophysiology, 2010; 17(4): 229-251.
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Cancer treatment of lymph node metastases
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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
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TNM staging system primary tumor size
lymph node involvement
whether the cancer has metastasized
stage size
T0 not found
T1 less than 2cm
T2 2 - 5 cm
T3, T4 more than 5cm
stage metastatic node
N0 0
N1 1-3
N2 4-9
N3 more than 10
stage distant metastasis
M0 negative
M1 positive
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
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Mouse model: MXH-10/Mo/lpr/lpr (MXH-10)
Lymph node(same size as it of human)
Proper axillary lymph node
Subiliac lymph node
Visualizing lymph route
Develop local administration via lymphatic vessels for Drug Delivery System at T0, T1, T2, N0 stages
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Sonoporation
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Collapse of NMB andgeneration of cavitation nuclei
Nano/Micro Bubble(NMB)
Liquid
00
Liquid
US probe
NMB
cavitation nuclei
Transient permeabilizationof 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
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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
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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
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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syringe pump (50μL/min)
syringe
butterfly needle
US probe high-frequency USimaging system
Mouse under anesthesia
Subiliaclymph node
Lymphatic vessel
Proper axillarylymph node
Volume 200µL
1. To acquire reference image of the axillary lymph node
2. AL injection into the inguinal lymph node
3. To detect the ALin the axillary lymph node
FOV (mm) Frame rate (Hz)
Cine loop size
Contrast threshold Gating
10.0 x 10.0 9 – 13 300 40 300 ON
Methods ~ To detect the lymphatic vessels in the axillary lymph node ~
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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: 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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US exposure condition **
US transducer: HONDA electronics Φ-12 (1 MHz)
Delivered molecule TOTO-3 iodide
molecular weight: 1355 absorption wavelength: 642 nm emission wavelength: 660 nm
Materials
Pulse number
Duty cycle pressure Exposure time
200 20% 0.67 MPa 60 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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8/13/2012
syringe pump (50μL/min)
syringe
butterfly needle
US probe
high-frequency USimaging system
Mouse under anesthesia
Subiliac lymph node
Lymphatic vessel
Proper axillarylymph node
point of a needle
•PBS alone•TOTO-3 alone•TOTO-3 + US•TOTO-3 + AL + US
Methods
PBS (μL)
TOTO-3 (μL)
AL (μL)
Total(μL)
PBS alone (n=3) 200 0 0 200
TOTO-3 alone (n=4)
100 100 0 200
TOTO-3 + US (n=5)
100 100 0 200
TOTO-3 + AL + US (n=5)
0 100 100 200
~ To deliver the fluorescent molecules into the axillary lymph node via the lymphatic vessels by sonoporation ~
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US transducer
1mm
1 MHz
CH1CH2Trigger
function generator
amplifier
INPUT MONITOR
oscilloscope
syringe pump (50μL/min)
syringe
butterfly needle
US probe
high-frequency USimaging system
Mouse under anesthesia
Subiliac lymph node
Lymphatic vessel
Proper axillarylymph 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
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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
~ To deliver the fluorescent molecules into the axillary lymph node via the lymphatic vessels by sonoporation ~
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Methods
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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
R image
Gimage
Bimage
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
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Before arrival of the AL
1mm 1mm
After arrival of the AL
Proper axillary lymph node regionLymphatic vessels
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Results and discussions
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PBS alone TOTO-3 alone
TOTO-3 + US TOTO-3 + AL + US
30μm
100μm
http://understandingcancer.co.uk/news/page/9/
Lymph node
ROI
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Results and discussions
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PBS alone TOTO-3 alone
TOTO-3 + US TOTO-3 + AL + US
x
y
xy
z
z-axis Grayscale intensity
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Results and discussions
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DAPI LYVE-1
TOTO-3 Merge
TOTO-3 + AL + US
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Results and discussions
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20μm
PBS alone TOTO-3 alone
TOTO-3 + US TOTO-3 + AL + US
20μm
20μm 20μm
No tissue damage
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Results and discussion
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Vascular permeabilization by sonoporation
shock wave
Lymphatic endothelial cell
US wave
Fluorescent agent
Transient hole
liquid jet
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Conclusions
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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?23
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Results
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PBS alone TOTO-3 alone
TOTO-3 + US TOTO-3 + AL + US
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Methods
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…
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N …
time
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N …
time
compare
Reference image Comparative imageFrame number Frame number
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N …
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腋窩リンパ節内のリンパ管
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Methods
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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)
9 14 15 37
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Results
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group1 group2 group3 group4
mea
n gr
aysc
ale
inte
nsit
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backgroundLymphatic
vessel detection
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Methods (Exp.2)
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Experimental group
PBS (μL)
TOTO-3 (μL)
AL (μL)
Total(μL)
US exposure
PBS alone (n=3) 200 0 0 200 -TOTO-3 alone (n=4) 100 100 0 200 -TOTO-3 + US (n=5) 100 100 0 200 +TOTO-3 + AL + US (n=5)
0 100 100 200 +
Measure US fieldmethods
Oscilloscope
Stage Controller
Degassed tap water
Bipolar Amplifier
Multifunction Synthesizer
PC
US transducer
Hydrophone
X
Y
Z
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
Kf2(acoustic intensity reply factor):0.0216[V2W-1cm2]Meff(hydrophone effective sensitivity):1.201[μV/Pa]
Ve
ff
IA
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PA [MPa]= 0.1225 × (IA)0.5
(Kf2 [V2W-1cm2])0.5 / Meff [μV/Pa]
≒ 0.1225 [Mpa ・ cm/(W)0.5]
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