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.


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syringe

butterfly needle

US probe

high-frequency USimaging system


Subiliac lymph node

Lymphatic vessel


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


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US transducer

1mm

1 MHz

CH1CH2Trigger

function generator

amplifier

INPUT MONITOR

oscilloscope


syringe

butterfly needle

US probe

high-frequency USimaging system


Subiliac lymph node

Lymphatic vessel


•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


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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


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Results and dicussions

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Before arrival of the AL

１ｍｍ１ｍｍ

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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x

y

xy

z

z-axis Grayscale intensity

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DAPI LYVE-1

TOTO-3 Merge

TOTO-3 + AL + US

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20μm



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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Methods

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…

12

3

N …

time

…

12

3

N …

time

compare

Reference image Comparative imageFrame number Frame number

…

12

3

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

y

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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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1 :graduate school of biomedical engineering tohoku university 2 :tohoku university hospital

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