Sonodynamic Therapy
Literature Seminar2018. 12. 13
Kazuki Takahashi (M1)1Index
1. Introduction• About ultrasound• Various medical uses of ultrasound• Safety range of frequency and intensity

2. Sonodynamic therapy for cancer cells• Photodynamic therapy & sonodynamic therapy• Example of drugs used for sonodynamic therapy• Mechanism & Drug design2Index
1. Introduction• About ultrasound• Various medical uses of ultrasound• Safety range of frequency and intensity

2. Sonodynamic therapy for cancer cells• Photodynamic therapy & sonodynamic therapy• Example of drugs used for sonodynamic therapy• Mechanism & Drug design3About ultrasound• Ultrasound• a mechanical wave through the displacements of themolecules constituting the medium in which the wave istravelling.
• similar in character to audible sound, but at frequencies
greater than 20 kHz.

• a fundamentally different wave phenomenon fromelectromagnetic waves such as radio waves, infrared radiation
and X-rays.

4

Various medical uses of ultrasound• Diagnosis

https://www.philips.co.jp/healthcare/product/HC79
5200/epiq-7-ultrasound-machine#galleryTab=CLI• Lithotripsy

https://www.practo.com/health-wiki/lithotripsy-meaningprocedure-and-cost/134/article• Fracture healing

A. Harrison, et al., Ultrasonics, 2016, 70, 45• HIFU (High-intensity focused ultrasound)

James E. K, Nat. Rev. Cancer, 2005, 5, 321
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Practical range of frequency• In the frequency range between 1 MHz and 20 MHz• practical use in clinical medicine for diagnostic, therapeuticand destructive purposes.

• The attenuation of the ultrasonic energy depends onthe frequency of the wave.
• the average attenuation coefficient in soft tissue=0.5 dB cm⁻1 MHz⁻1
Gail ter Haar, The Safe Use of Ultrasound in Medical Diagnosis 3rd Edition, 2012, p9• 5 MHz: 80% loss within the first 2.8 cm of tissue.
• 3 MHz: 80% loss within the first 4.7 cm of tissue.
• 2 MHz: 80% loss within the first 7.0 cm of tissue.
• 1 MHz: 80% loss within the first 14 cm of tissue.

The treatments by US are available within the first 10 cm of tissue.
X. Qian, et al., Adv. Mater., 2016, 28, 80976

US intensity• High intensity of US makes burn and cell death.
• Low intensity is not effective.
• US intensity is recommended
between 0.1~5 MHzfor treatment.
W/cm
Atsumi Ohota, 理学療法の歩み, 2006, 17, 14

7Hydroxyl radical produced by cavitation• Ultrasonic irradiation of liquids causes acoustic cavitations, i.e., theformation, growth and implosive collapse of bubbles.
• Such cavitation generates local sites of high temperature andpressure for short periods of time.
• The extreme temperature conditions generated by a collapsing
bubble can also lead to the formation of radical chemical species.
Örjan J., et al., Sustainable and energy efficient leaching of tungsten (W) by ultrasoundcontrolled cavitation, 2018, p8

Sabina Z., et al., Proceedings, 2018,8 2, 188

Sonoluminescence produced by cavitation• The high temperatures generated in thecollapsing bubbles by the increasing pressure
would make the heated gases in the bubbles
emit light.
• The spectrum of MBSL in water shows peaks
that correspond to excited state OH* (mainlyat 310 nm)
Örjan J., et al., Sustainable and energy efficient leaching of tungsten (W) by ultrasoundcontrolled cavitation, 2018, p8

9Franc¸ois Hibou, Homeopathy, 2017, 106, 181

Short summary• US is used for Diagnosis, Fracture healing,
Lithotripsy, HIFU, and so on.
• US is able to penetrate into deep tissues (tencentimeter).
• Around 1 MHz of US frequency is recommended forpractical use into deep tissues .
• 0.1~5 W/cm2 of US intensity is also recommended.
• Cavitation in water generates hydroxy radical andother radical species, and makes sonoluminescence.
10Index1. Introduction• About ultrasound• Various medical uses of ultrasound• Safety range of frequency and intensity

2. Sonodynamic therapy for cancer cells• Photodynamic therapy & sonodynamic therapy• Example of drugs used for sonodynamic therapy• Mechanism & Drug design

11

Photodynamic therapy (PDT)• Side-effects are more suppressed.
• PDT is not used for tumors in the
deep place of the body.
Stanley B. B., et al.,Lancet Oncol., 2004, 5, 497

Bogdan, et al., Nanoscale Res. Lett., 2017, 12, 225

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Photodynamic Therapy (PDT)

Tianhong D., et al., Front. Microbiol., 2012, 3, 120

ROS produced by photosensitizer & light has cytotoxicity.
13Basic chemical structures for PDT

Ankit, S. et al. Drug Discovery Today. 2018, 23, 5

Sonodynamic therapy (SDT)

Bogdan, et al., Nanoscale Res. Lett., 2017, 12, 225

15

Sonodynamic therapy (SDT)• Deeper penetration• Non-invasive treatment• Good repeatability

Bogdan, et al., Nanoscale Res. Lett., 2017, 12, 225

16

First example of drugs used for SDT

Yumita, N et al., Jpn. J. Cancer Res., 1989, 80, 219

17

Unstained fraction

First example of drugs used for SDT

15304560

Exposure duration (s)△: 50 mg/mL Hp alone〇: US alone■: 50 mg/mL Hp + US

Yumita, N et al., Jpn. J. Cancer Res., 1989, 80, 219

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Other examples of drugs used for SDT
Porphyrin compound sensitizers

Xanthone compound sensitizers

non-steroid anti-inflammatory agent sensitizers

Other sound-sensitizers

Liu R., et al., Photodiagnosis and Photodynamic Therapy, 2017, 19, 159

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Mechanism of SDT (in vitro)• Investigating the US-responsiveness of a variety ofmetal-porphyrin complexes, freebase porphyrin andFe(III), Zn(II) and Pd(II) porphyrin.
• Analyzing their ROS generation under US exposure andrelated bio-eﬀects.
F. Giuntini, et al., Free Radical Biology and Medicine, 2018, 121, 190

20

Sono-induced ROS detected by EPR• Porphyrin 3 has the highest hydroxyl radical generationeﬃciency.
• Porphyrin 4 generated most singlet oxygens.
F. Giuntini, et al., Free Radical Biology and Medicine, 2018, 121, 190

21

Sonoluminescence• Sonoluminescence emission spectra• SL emitted both in water (blue) and in the presence ofporphyrins 3 (black) and 4 (red).
• The hydroxyl radical emission observed in the presenceof porphyrin 3.
F. Giuntini, et al., Free Radical Biology and Medicine, 2018, 121, 190

22

Effects of SDT on HT-29 cells• Porphyrin 1 and 2 did not cause a signiﬁcant reductionin HT-29 cell growth under US exposure.
• porphyrin 3 and 4 lead to a signiﬁcant HT-29 cell growthdecrease under US exposure.
F. Giuntini, et al., Free Radical Biology and Medicine, 2018, 121, 190

23

Cell death analysis

Detected by Dead Cell Apoptosis Kit, with Annexin V-Alexa Fluor® and propidium iodide.

• 4 + US induced both necrotic and apoptotic cell death.
• Light exposure was responsible for necrotic cell deathbecause the higher singlet oxygen production.
F. Giuntini, et al., Free Radical Biology and Medicine, 2018, 121, 190

24

Cell death by autophagy• HT-29 cells after SDT with porphyrin 4• Autophagic cell death was observed in this TEM image.
F. Giuntini, et al., Free Radical Biology and Medicine, 2018, 121, 190

25

Disscussion• Metal ions aﬀect the ability of porphyrins toundergo intersystem crossing (ISC) and can also
inﬂuence the lifetimes of the resulting excitedtriplets.
• Porphyrin activation by US can be mediated byphoto-activation via sonoluminescence.
• SDT of porphyrin 4 is not effective than PDT.

26

SDT vs PDT

US: 1.0 MHz, 1.0 W/cm2 output intensity, and 10% duty cycle for 1–2 min
Light: a halogen lamp (MHAA-100W; Kenis Ltd., Osaka, Japan) at 6000 lx for 5 min in a dark room at rt.

• Exposure to light in the presence of DEG was notcytotoxic for MKN-74 cells, whereas strong
phototoxicity was observed with ATX-70.
• MKN-74 cells were significantly damaged byultrasound irradiation in the presence of DEG,
whereas sonotoxicity with ATX-70 was less effective.
H. Tsuru, et al., Free Radical Biology and Medicine, 2012, 53, 464

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Tumor growth analysis
10 min at 1 MHz, with a 2.0 W/cm2• SDT with DEG three times a week for 2 weeks potently
inhibited tumor growth compared to SDT alone or notreatment.
H. Tsuru, et al., Free Radical Biology and Medicine, 2012, 53, 464

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SDT in vivo

• At the end of the experiment, no apparent adverse
side effects, including skin sensitivity, wereobserved in the mice injected with DEG.
H. Tsuru, et al., Free Radical Biology and Medicine, 2012, 53, 464

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Comparative histochemical analysis• spontaneous damage was observed on histochemical analysis in
the cancer tissue even without treatment, a large cavity wasobserved in tumor only after SDT with DEG.
H. Tsuru, et al., Free Radical Biology and Medicine, 2012, 53, 464

30

Antibody-drug conjugate (ADC)• Antibody can carry Drug to the target cells selectively.
• ADC is expected suppressing the side-effect tonormal cells.
31

ADC for Sonodynamic therapy• How to synthesize ADC (DAR is no data.)

Abe H., et al., Anticancer Research, 2002, 22, 1575 32

Example of ADC for SDT• SDT in vivo

1 MHz, 2 W/cm2, 10 min
F39/ATX-70 with US inhibited tumor
growth compared to US alone or ATX70 alone.

Abe H., et al., Anticancer Research, 2002, 22, 157533

Summary• US frequency is recommended for use around 1 MHz,US intensity is recommended at 1~5 W/cm2.
• SDT is a non-invasive treatment for tumors located in
the deep places of the body.
• Sonosensitizers are activated by sonoluminescence.
• ADC for SDT is few reported, but reported to beeffective to tumor.
• SDT and sonochemistry is a developing study, so it will
be developed more rapidly in the future.

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Appendix

35

US used at some friquency

X. Qian, et al., Adv. Mater., 2016, 28, 8097

36

HIFU
• HIFU (High-intensity focused ultrasound)•Thermal toxicity occurs if tissue
temperatures are raised above athreshold of 56°C for at least 1 second,leading to irreversible cell death throughcoagulative necrosis.
James E. K, Nat. Rev. Cancer, 2005, 5, 321

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Example of ADC for SDT• SDT in vivo

Abe H., et al., Anticancer Research, 2002, 22, 1575
38

SDT in vivo

• Visual inspection also showed increased deadcells after ultrasound irradiation with DEG
compared to no treatment or the control.
H. Tsuru, et al., Free Radical Biology and Medicine, 2012, 53, 464

39

Cell death analysis• No apoptotic signals increased after ultrasound irradiation with or without DEG,
according to Western blot analysis up to 24 h.

H. Tsuru, et al., Free Radical Biology and Medicine, 2012, 53, 464

40

Cell death analysis

H. Tsuru, et al., Free Radical Biology and Medicine, 2012, 53, 464

41

Photo-induced ROS detected by EPR• All porphyrins showed comparable singlet oxygengeneration eﬃciency.
• Light irradiation is more efficient than US exposure forexcitation.
F. Giuntini, et al., Free Radical Biology and Medicine, 2018, 121, 190

42

Oxidation of DMPO

Andrew L., et al., JBC, 2003, 278, 29410

43Effects of PDT on HT-29 cells• All porphyrins induced a signiﬁcant reduction in cellproliferation under light irradiation.
F. Giuntini, et al., Free Radical Biology and Medicine, 2018, 121, 190

44

ROS production after SDT• Porphyrin 3 and 4 showed ROS emission after USirradiation.
• Porphyrin 4 showed the highest US-responsiveness.
F. Giuntini, et al., Free Radical Biology and Medicine, 2018, 121, 190

45

ROS production after PDT• Every porphyrin produced more ROS after lightirradiation than after US irradiation.
• Porphyrin 4 showed the most ROS emission.
F. Giuntini, et al., Free Radical Biology and Medicine, 2018, 121, 190

46

Cell death analysis

47

Antibody-drug conjugate (ADC)• 2 types of Drug moiety• For PDT, SDT• Don’t have torelease theDrug moieties.

••

Common case
Need to release
the Drug moieties.
48

Enzyme & Photo cleavable linker• Enzyme-cleavable linker
Jessica R. M., et al.,The AAPS Journal, 2015, 17, 339• Photo-cleavable linker
Roger R. N., et al.,ACS Cent. Sci., 2017, 3, 329

49

Sonolysis linker• Sonolysis• Sonolysis linker will realize non-invasive treatmentof tumors in the deeper place of the body.

50

Example of sonolysis reaction• Sonolysis

etc…
Kenneth S. S., et al., Annu. Rev. Phys. Chem., 2008, 59, 659
Zineb B., et al., Turk. J. Chem., 2017, 41, 99
N.E. Chadi, et al., Sep. Purif. Technol., 2018, 200, 68

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Degradation of NBB by US in water• The NBB concentration decreased exponentially with time.
• In absence of substrate, the concentration of H2O2
increased linearly with sonication time.
H. Ferkous, et al., Ultrasonics Sonochemistry, 2015, 26, 40

52

Sonolysis by safe ultrasoundH. Ferkous, et al., Ultrasonics Sonochemistry, 2015, 26, 4053

Sonolysis by safe ultrasound

H. Ferkous, et al., Ultrasonics Sonochemistry, 2015, 26, 40

54

N2 inhibits sonolysis reaction competitively.
• N2 is reacted in water by US

J. Yao et al., Ultrason. Sonochem., 2018, 42, 42
Supeno, et al., Ultrason. Sonochem., 2000, 7, 109• N-N bond is cleavable in water under US exposure.55

H2O2 evolution (Method)

1 mL of potassium iodide (0.1 M) and 20 mL of ammonium heptamolybdate (0.01 M). Theiodide ion (I-) reacts with H2O2 to form the triiodide ion (I3-). The mixed solutions wereallowed to stand for 5 min before absorbance was measured. The absorbance wasrecorded with a UV–visible spectrophotometer (Lightwave II) at the maximum wavelengthof the formed triiodide (I3-) (352 nm; the molar absorptivity = 26,300 L mol-1 cm-1 ).
H. Ferkous, et al., Ultrasonics Sonochemistry, 2015, 26, 4056

COD abatement (Method)

COD: Chemical oxygen demand.
Test solution (2 mL) was transferred into the dichromate reagent and digested at 150 °Cfor 2 h. The optical density for the color change of dichromate solution was determinedwith a spectrophotometer at 440 nm.

H. Ferkous, et al., Ultrasonics Sonochemistry, 2015, 26, 4057