Nanoparticle-Augmented
Sonodynamic Therapy
Literature Seminar
2020/6/1
Wataru Atsumi (B4)

Contents• Introduction
About sonodynamic therapy
Benefits of using nanoparticles• Examples of Nanoparticle-assisted SDT
Self-assembled organic NPs
NPs-assisted SDT for glioma
NPs-assisted SDT for Alzheimer disease• Summary2

Photodynamic therapy (PDT) & Sonodynamic therapy (SDT)PDT• Limited penetration depth

SDT• High penetration depth• Less invasive

Chemical Engineering Journal, 2018, 340, 155–172

3

Possible mechanisms of SDT
Schematic diagram of the possible mechanism of SDT
Ultrasound (US)

Thermal effectSonoluminescence
Cavitation

Pyrolysis

ROS generation

Cell damage

Mechanical damage

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Possible mechanisms of SDT
1. Cavitation
Cavitation has three stages.
1. Nucleation
2. Bubble growth
3. Implosive collapse
When it collapses, the local
temperature and pressure canreach >10,000 K and 800 atm.

Walter de Gruyter, Berlin, Boston, Nanotechnology Reviews, 2012, 1, 173–182

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Possible mechanisms of SDT
2. Sonoluminescence• A concentration of energy caused by cavitation can generates light.
• Sonosensitizers are excited and generates ROS.
spectraChemical Engineering Journal, 2018, 340, 155–172
Thomas J Matula et al. Phy. Rev. Lett., 1995, 75, 2602–2605

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Possible mechanisms of SDT
3. Cellular toxicity by reactive oxygen species (ROS)

Generated ROS induces apoptosis→ Therapeutic effectChemical Engineering Journal, 2018, 340, 155–172

7Possible mechanisms of SDT
4. Other Effects• Pyrolysis of H2O
Cavitation also induces pyrolysis of H2O and generates ROS.
• Mechanical damage
The strong shock waves are generated by cavitation, resulting
in mechanical damage to the cell.
• Thermal Effect
The transformation and absorption of US energy cause
hyperthermia and induce necrosis.
Chemical Engineering Journal, 2018, 340, 155–172

8

Sonosensitizer

Porphyrins

Xanthenes

New Quinolones

Drug Discovery Today,
2014, 19, 504–506
Antitumor drugs

Other types

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Example of sonosensitizer
sonosensitizer: hematoporphyrin monomethyl ether (HMME)ROS generation by HMME+US→ Mitochondrial damage→ Induction of apoptosis→Therapeutic effect for C6 glioma cells
Condition
Frequency: 1MHzIntensity: 1W/cm2
Time: 60s
[HMME] = 10μg/mL

SDT can be a valid way to treat cancer.

S. Dai, S. Hu, C. Wu, Acta Neurochir.,2009, 151, 1655 –1661.

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Problems of traditional sonosensitizers
Because most of sonosensitizers are strongly hydrophobic,
there are some problems as below.
• Low bioavailability• Fast elimination out of the body
• Poor accumulation into targeted tissue
These problems hinder their clinical translation.

Xiaoqin Qian et al. Adv. Mater., 2016, 28, 8097–8129

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SDT augmented by nanoparticles (NPs)
Meritsü Large surface area suitable for chemical modification→ Improvement of biocompatibility, biodistribution and selectivityü Combination of SDT with other therapeutic modalities
ü Decrease of cavitation threshold by NPs→ Stabilization of nanobubbles on their surface and inside cavities→ Enhancement of the therapeutic efficiency
Xiaoqin Qian et al. Adv. Mater., 2016, 28, 8097–8129

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Contents• Introduction
About sonodynamic therapy
Benefits of using nanoparticles• Examples of Nanoparticle-assisted SDT
Self-assembled organic NPs
NPs-assisted SDT for gliomaNPs-assisted SDT for Alzheimer disease• Summary13

Self-assembled organic NPs
HA-PLGA@ART/ALA NPs• Poly(lactic-co-glycolic acid) (PLGA):
great biocompatibility and biodegradability• Hyaluronic acid (HA):
tumor-targeting• 4-hydrazinobenzoic acid(HBA):
pH-sensitive linkage• Artemisinin (ART):ROS generation (as a sonosensitizer)• Amino levulinic acid (ALA):
precursor of Heme and activate ART (also
as a sonosensitizer)

Lei Wang, et al. J. Controlled Release, 2018, 286, 74–84.

14Structure of HA-PLGA@ART/ALA NPsArtemisinin

NP were prepared by self-assembled method.
Hydrophobic ART was encapsulated in the inner core.

Lei Wang, et al. J. Controlled Release,15
2018, 286, 74–84.

In vitro pH-sensitive drug release assay
a. free ALA
b. free ART
c. ALA from NPs
d. ART from NPs• ALA were released under the acidic condition.
• At pH 5.5, ALA was released faster than ART, especially at theearly time points.
Lei Wang, et al. J. Controlled Release, 2018, 286, 74–84.

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Cytotoxicity and ROS production* More ROS generation by USConditionIntensity: 2 W/cm2
Time: 60 s
[ART] = 88 μM[ALA] = 0.5 mM** Combination of ALA & ART

a. Control

e. ART+ALA

b. US

f. ART+ALA+USc. ART

g. HA-PLGA@ART/ALA NPs

d. ART+US h. HA-PLGA@ART/ALANPs NPs + US
Lei Wang, et al. J. Controlled Release, 2018, 286, 74–84.

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Animal experiments
NPs + US group was the most effective.
ü More drug accumulation by HA
ü ART & ALA combination
ü More ROS production by US

a. Control

e. ART+ALA

b. US

f. ART+ALA+USc. ART

g. HA-PLGA@ART/ALA NPsd. ART+US h. HA-PLGA@ART/ALANPs NPs + US
Lei Wang, et al. J. Controlled Release, 2018, 286, 74–84.

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Animal experiments: Tumor siteü Tumor targeting by HA
ü Long circulation characteristic
Lei Wang, et al. J. Controlled Release, 2018, 286, 74–84.19

Contents• Introduction
About sonodynamic therapy
Benefits of using nanoparticles• Examples of Nanoparticle-assisted SDT
Self-assembled organic NPs
NPs-assisted SDT for glioma
NPs-assisted SDT for Alzheimer disease• Summary20

BBB opening by microbubbles and US

BBB (blood-brain barrier) is themain obstacle to the drug delivery.

BBB can be opened up locally andreversibly by Ultrasound-Targeted
Microbubble Destruction (UTMD).

Yue Sun et al. Biomater. Sci., 2019, 7, 985–994.
21
Xiaobing Wang et al. Ultrason. Sonochem., 2017, 37, 592–599

NPs-assisted SDT for glioma• Liposome
Drug carrier• DVDMS (Sinoporphyrin sodium)
Hydrophilic sonosensitizer• αvβ3 integrin
Specifically expressed in tumor cells• iRGDαvβ3 integrin-targeting peptide
DVDMS

Yue Sun et al. Biomater. Sci., 2019, 7, 985–994.

22BBB opening in vivo
Content of Evans blue in the brain

Condition
Frequency: 1.0 MHz
Power: 1 W
Exposure Time: 1 min
Interval Time: 1 s

BBB showed the highest
permeability 2h after UTMD.

DVDMS accumulation in orthotopic gliomas

The penetration of DVDMS with
UTMD was 7.2-fold higher thanthat without UTMD.
Yue Sun et al. Biomater. Sci., 2019, 7, 985–994.

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Sonodynamic effect in vivo
Condition(UTMD)
Frequency: 1.0 MHz
Power: 1 W
Exposure Time: 1 min
Interval Time: 1 s

Condition (SDT)
Frequency: 1.0 MHz
Power: 1 W
Exposure Time: 1 min

Survival time of mice treatedwith iRGD-Lipo-DVDMS-SDT
was longer than the others.
üBBB openingütumor targeting by iRGDüDVDMS delivery byliposome
Yue Sun et al. Biomater. Sci., 2019, 7, 985–994.

24

Contents• Introduction
About sonodynamic therapy
Benefits of using nanoparticles• Examples of Nanoparticle-assisted SDT
Self-assembled organic NPs
NPs-assisted SDT for glioma
NPs-assisted SDT for Alzheimer disease• Summary25

Alzheimer disease• Aggregation of Aβ is main cause ofAlzheimer disease (AD).
• Oxygenation of Aβ bysonosensitizer with US may be avalid way to treat AD.
• Tau hyperphosphorylation is also acause of AD.
AβGSK3βTau protein
PLiCl or PX

Tauphosphorylation• Inhibiting Glycoprotein synthasekinase-3 β (GSK3β) is a hopefultreatment.
Mengmeng Xu et al. ACS Appl. Mater. Interfaces,
2018, 10, 32965–32980

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IX-modified multifunctional NPs• Oxidized mesoporous carbon
nanosphere (OMCN): drug carrier• PpIX or PX: sonosensitizer andGSK3β inhibitor• Rabies Virus Glycoprotein (RVG):It improves BBB penetration andselectivity of brain cell

Sonosensitizer

Mengmeng Xu et al. ACS Appl. Mater. Interfaces,
2018, 10, 32965–32980

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Inhibition of Aβ aggregation
AFM image of Aβ

ThT fluorescence assay

Condition
Frequency: 1 MHz
Intensity: 3 W/cm2
Time: 3 min[NPs] = 20 μg/mLAβ immunostaining in the hippocampus

NPs + US remarkably
hindered Aβ aggregation.
Mengmeng Xu et al.
ACS Appl. Mater. Interfaces, 28
2018, 10, 32965–32980

Inhibition of tau hyperphosphorylation
Inhibition of tau phosphorylation assay

NPs effect in AD mice model

Treatment with NPs downregulated tau hyperphosphorylation.
Mengmeng Xu et al. ACS Appl. Mater. Interfaces,
2018, 10, 32965–32980

29Rescue of memory deficits
Morris Water Maze test

Nest construction

PX@OP@RVG group showed improved
learning ability.

Nest construction test showedsimilar results.

Mengmeng Xu et al. ACS Appl. Mater. Interfaces,
2018, 10, 32965–32980

30

Contents• Introduction
About sonodynamic therapy
Benefits of using nanoparticles• Examples of Nanoparticle-assisted SDT
Self-assembled organic NPs
NPs-assisted SDT for glioma
NPs-assisted SDT for Alzheimer disease• Summary31

Summary• SDT is a novel non-invasive therapeutic modality.
• NPs enhances therapeutic effect of SDT.
• BBB opening by UTMD can solve the main problem of drugdelivery system.
• NP-augmented SDT can be applied to not only cancer but also
Alzheimer disease.

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Appendix

33

Nanocup decreases cavitation threshold (P12)J. J. Kwan et al. Phys, Rev. E, 2015, 92,023019-1–023019-5.

Schematic of nanocup

Size measurement

Probability ofinertial cavitation
curve in each size34CD-44 targeting by hyaluronic acid (HA)

• HA recognize CD-44 receptors,
which are overexpressed in tumorcells.
• NPs are then internalized via
CD-44 mediated endocytosis.

Hae-Yong Seok et al. J. Controlled Release, 2018, 280, 20–30. 35

Characterization of NPs (P14–P19)
TEM imageSize distribution & Zeta potential

HA-PLGAHA-PLGA/ALA

HA-PLGA@ART/ALA

Lei Wang, et al. J. Controlled Release, 2018, 286, 74–84.

36

Cellular uptake and heme level (P14–19)
ALA is a precursor of PpIX, which shows
red-fluorescence.

1h2h

There was no remarkable difference
between ALA and NPs.

4h→NPs could enter cells efficiently.
Free ALANPs

Both ALA and NPs increased heme level.

Lei Wang, et al. J. Controlled Release, 2018, 286, 74–84.

37

Tumor targeting by iRGD (P22)
1. iRGD sequence binds to αvβ3/5integrin, which are specificallyexpressed in tumor vessels ortumor cells.
2. iRGD is hydrolyzed by hostprotease to expose CendR motif.
3. CendR interacts with neuropilin toinitiate internalization.
Yue Sun et al. Biomater. Sci., 2019, 7, 985–994.
38
Xiangsheng Liu et al. Mol. Syst. Des. Eng., 2017, 2, 370–379.

Rabies Virus Glycoprotein (RVG) drug delivery (P27)
The scheme of drug delivery by RVG peptide= n-acetylcholine
receptor

Yang Liu et al. Biomaterials, 2016, 80, 33–45.

Receptor mediated transcytosis

Zhui Zhang, Changyou Zhan,
Brain Targeted Drug Delivery system,
2019, 105–128.

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NP biodistribution (P26–30)
Quantitative analysis for the fluorescence in vivo

RVG-modified NPs had better circulation
and retention efficiency.

NPs remarkably accumulated in the brain.
→RVG active targeting and improved
penetration of BBB

Mengmeng Xu et al. ACS Appl. Mater. Interfaces,
2018, 10, 32965–32980.

40

NP biodistribution (P26–30)

Mengmeng Xu et al. ACS Appl. Mater. Interfaces,
2018, 10, 32965–32980.

41

Inhibition of Aβ aggregation (P28)

Anaerobic condition → None ROS production
ROS generation contributes to the inhibitory effect of US excited
PX@OP@RVGs on Aβ aggregation.

Mengmeng Xu et al. ACS Appl. Mater. Interfaces,
42
2018, 10, 32965–32980.

Inhibition of tau hyperphosphorylation (P29)

Mengmeng Xu et al.
ACS Appl. Mater. Interfaces,
2018, 10, 32965–32980.
43

Inorganic sonosensitizer

Not only organic molecules but also inorganic NPs
such as TiO2, ZnO, etc. act as a sonosensitizer.
Chemical Engineering Journal, 2018, 340, 155–172

44

Mesoporous titanium nanoparticle loading DTX
MTN@DTX-CD NP• Mesoporous TiO2 NPs (MTN):
Carrier and sonosensitizer• Docetaxel (DTX):
Anti-cancer drug, loaded in pores• β-cyclodextrin (β-CD):A bulky gatekeeper to block
mesopores
US-triggered drug release byROS sensitive linker

Jinjin Shi et al. ACS Appl. Mater. Interfaces, 2015, 7, 28554–28565.

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Synthesis of MTN@DTX-CD NPs

Jinjin Shi et al. ACS Appl. Mater. Interfaces, 2015, 7, 28554–28565.

46

1O generation ability
21O

2 production was more efficient withincreased concentration of MTN-CD.

US power density and irradiation time
were also important.

Jinjin Shi et al. ACS Appl. Mater. Interfaces, 2015, 7, 28554–28565.

47

US-triggered drug releaseβ-CD disperse showed the cleavage ofROS-sensitive linker was occurred.
β-CD

Rapid increase of DTX after USirradiation.
• 51.9% increase (4~7.5 h)• 28.4% increase (14~17 h)
DTXJinjin Shi et al. ACS Appl. Mater. Interfaces, 2015, 7, 28554–28565.

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Cell apoptosis assay
Average percentages← SDT only
← chemotherapy only
← SDT + chemotherapy

MTN@DTX-CD with US showed higher effect than the others.
→The combination of SDT & chemotherapy led to more apoptosis.
Jinjin Shi et al. ACS Appl. Mater. Interfaces, 2015, 7, 28554–28565.

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In vivo studies• Tumor targeting and accumulation• Long circulation ability
Jinjin Shi et al. ACS Appl. Mater. Interfaces, 2015, 7, 28554–28565.

50

In vivo studies

MTN-CD+US: SDT
DTX: chemotherapy
MTN/DTX-CD+US: the combination ofSDT & chemotherapy→More effective and less toxic side effectJinjin Shi et al. ACS Appl. Mater. Interfaces, 2015, 7, 28554–28565.

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