Improvement of Sonodynamic
Cancer Therapy by PorphyrinBased Sonosensitizer
Literature Seminar
2022/1/28
Keiichi Kawabata (M1)

Contents• Introduction• Improvement of ROS Generation Ability• Energy gap• Water solubility• Combination of SDT and PTT• Summary2

Contents• Introduction• Improvement of ROS Generation Ability• Energy gap• Water solubility• Combination of SDT and PTT• Summary3

Photodynamic Therapy (PDT) and
Sonodynamic Therapy (SDT)

Merits of SDT

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

Non-invasiveMore penetrative than PDT

4

Cavitation

J, Ö., et al., Sustainable and energy efficient leaching of
5tungsten (W) by ultrasound controlled cavitation, 2017, p8

Mechanism of SDT

Xing, X. et al., Coordination Chemistry
Reviews, 2021, 445, 214087
6

Generation of 1O2

McHale, A. P. et al., Therapeutic
Ultrasound, 2016, 880, 429–450
7Organic Sonosensitizers

Figure 2. (A–D) Chemical structures ofrepresentative organic sonosensitizers.

Yan, P., Liu, L. and Wang, P., ACS Appl.
Bio Mater. 2020, 3, 6, 3456–3475
8Porphyrins• Most widely used sonosensitizer• Large π-electron conjugated system• Various metals at center

N
HN

NH
N

9

Improvement points• Demerits of organic sonosensitizers• Phototoxicity• Low water solubility• Deficiency of targeting ability• Fast metabolism• A good sonosensitizer should• be of high sonosensitivity• be non-toxic in the absence of ultrasound• specifically accumulate in the target• be excreted from the body within a proper period
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Contents• Introduction• Improvement of ROS Generation Ability• Energy gap• Water solubility• Combination of SDT and PTT• Summary11

MTTP-HSA Nanocomplex• Metal-porphyrin
complexes wereencapsulated by
HSA (humanserum albumin)→solve, reduce
toxicity, prolong
circulation time,enhance effect• Red shift ofSoret band

Figure1
Schematic illustration and characterization of MTTP complexes and MTTP-HSA nanocomplexes. a) A scheme
illustrating the synthesis of MTTP complexes and the corresponding nanocomplexes of HSA. b) UV–vis absorption
spectra of TTP and the three metal-porphyrin coordination complexes in DMF; the inset is the Q-band absorption of
TTP and its metal complexes. c) UV–vis spectra of the three MTTP-HSA nanocomplexes in water; the absorption peakat 280 nm belongs to HSA. d) The size distribution of MTTP-HSA nanocomplexes, with the average diameter of about
60 nm. e–g) TEM images of MTTP-HSA nanocomplexes, showing spherical structures; the insets show the colors of thethree nanocomplex solutions (5.5  10−6 m in water), influenced by their metal centers.

Ma, A. et al., Small, 2019, 15, e1804028

12

1O Generation by MTTP-HSA
2• MTTP-HSA
generated notradicals but 1O2• MTTP-HSA
generated 1O2 up
to 11 cm awayfrom the US probe• MnTTP had thebest activity of thethree

Figure2
1O generation and the SDT effects of MTTP-HSAs irradiated by US treatment (1 MHz, 2 W cm−2, 50% duty cycle).
2a) ESR spectra for detecting 1O2 generation from these nanocomplexes after US treatment; 2,2,6,6tetramethylpiperidine (TEMP) without US irradiation was used as a control for comparison. Singlet oxygen was
significantly observed. b) ESR spectra for detecting radical generation from these nanocomplexes after US
treatment; 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) without US irradiation was used as a control forcomparison. No obvious radical species were clearly generated. c) The FL imaging for the detection for depthactivated 1O2 generation under US irradiation in mimic tissue using SOSG as 1O2 probe. Measurements weremade at depth up to 11 cm. d) 1O2 quantitative measurement of depth-dependent FL intensity of thesenanocomplexes after US irradiation. e) Photographs of the US treatment method and in vivo ROS fluorescentimages after 5 min of US treatment with the injection of MnTTP-HSA nanoparticles. f) The diagram illustrating
the possible US-excitation transfer between a metalloporphyrin complexes and oxygen molecule

Ma, A. et al., Small, 2019, 15, e1804028

13

HOMO-LUMO plots of MTTP

Figure2g) HOMO-LUMO plots of these simulated complexes by density functional theory (the orbital energies presented in eV).

• HOMO/LUMO of MnTTP are strongly localized on Mn2+
• HOMO/LUMO of TiOTTP/ZnTTP are mainly delocalizedon the p AOs of C/N atoms on the porphyrin• Energy gap of MnTTP was lower than that of
TiOTTP/ZnTTP→can be irradiated by low intensity→stronger ability of 1O2 generation

Ma, A. et al., Small,2019, 15, e1804028

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In Vivo SDT Efficiency of MnTTP-HSA• MnTTP-HSA + US
inhibited tumor
growth of both side
• Good biosafety andbiocompatibility

Figure5
In vivo SDT treatment efficiency of the MnTTP-HSA nanocomplex in nude mice bearing MCF-7 bilateral tumors.
The mice were divided into control (untreated) group, US only group, MnTTP-HSA group, and MnTTP-HSA + US
group. a) In vivo therapeutic protocol of SDT on MCF-7 tumor xenograft. The US probe was on the right tumor,
and the ultrasound wave penetrated from right to left (1.0 MHz, 2 W cm−2, 50% duty cycle, 5 min). The US
irradiation was conducted twice at 3 and 24 h, respectively. b,c) MCF-7 tumor growth curves of left side andright side, respectively, with various treatments (**p < 0.01). d,e) The corresponding excised tumor weights ofleft and right side, respectively, acquired at the end of treatments (**p < 0.01). f) Body weights of the mice
during the 15 d study period under the different conditions.

Ma, A. et al., Small, 2019, 15, e1804028 15

Contents• Introduction• Improvement of ROS Generation Ability• Energy gap• Water solubility• Combination of SDT and PTT• Summary

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Water-soluble Ir Complex• Hydrophobic compounds• low saturation concentration• aggregate in the body fluid• Porphyrin sulfonates arewater-soluble• IrTMPPS showed high watersolubility• HOMO/LUMO energy gap was
1.147 eV, easily excited by US

Figure 1. (a,b) UV–vis absorption spectra of TMPPS and IrTMPPS in the PBS
solution. (c) HOMO–LUMO plots of IrTMPPS by DFT.

Xie, J. et al., ACS Appl. Mater. Interfaces, 2021, 13, 24, 27934–27944
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1O Generation by IrTMPPS
2• 1O2 increased
depending on US
irradiation time
• DPA was oxidized
constantly under US• IrTMPPS had goodsono-stability• IrTMPPS produced
1O but not OH
2after US irradiation
Xie, J. et al., ACS Appl. Mater.
Interfaces, 2021, 13, 24, 27934–27944

Figure 2. (a) Time-dependent fluorescence intensity of SOSG indicating 1O2generation by IrTMPPS under US
irradiation. (b) Rate constant for SOSG fluorescence intensity depending on US irradiation time in the presenceof IrTMPPS. (c) Time-dependent absorption of DPA suggesting 1O2 generation by IrTMPPS under US irradiation.
(d) Rate constant for DPA absorption depending on the US irradiation time in the presence of IrTMPPS.
18(e) 1O2 generation of IrTMPPS was demonstrated by ESR spectra. The power of US was 0.3 W cm–2, 3.0 MHz. (f)Schematic illustration of 1O2 generation by IrTMPPS under US irradiation.

NADH Sonocatalytic oxidation• NADH decreased and NAD+
increased by IrTMPPS + US
• NADH oxidation was also
monitored with ESR and NMR• IrTMPPS consume NADH by
US irradiation at the cellularlevel →destroyed redox
balance induces cancer cell
death

Figure 3. (a) Reaction of IrTMPPS and NADH in PBS solution under US irradiation detected by
UV–vis spectra. (b) ESR spectra of NAD• radicals trapped by CYPMPO demonstrating NADH
oxidized by IrTMPPS under US irradiation. (c) Sonocatalytic oxidation of NADH (3.5 mM) by
IrTMPPS (0.25 mM) without US or with US irradiation was monitored by 1H NMR
spectroscopy. Peaks classified with blue triangles represent NADH and red circles represent
NAD+. (d) Schematic illustration of IrTMPPS for sonocatalytic oxidation of NADH andgeneration of 1O2. (e) NADH concentrations in the treated 4T1 cells. US irradiation: 3.0 MHz,
0.3 W cm–2.

Xie, J. et al., ACS Appl. Mater. Interfaces, 2021, 13, 24, 27934–27944

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Sono-toxicity of IrTMPPS In Vitro• Appropriate US irradiation
power was 0.3 W/cm2 andirradiation time was 20 min.
• IC50 of IrTMPPS was 3.43
μM toward 4T1 cells• IC50 toward other cancer
cells were all below 10 μM• With IrTMPPS and US, lotsof cancer cells died

Figure 5. (a) Cell viabilities of 4T1 cells incubated with IrTMPPS (10 μM) under different US
irradiation time. (b) Viability of 4T1 cells incubated with various concentrations of IrTMPPS
under US irradiation for 20 min. (c) Cytotoxic effects of IrTMPPS on different types of cellswith or without US irradiation. (d) Sono-toxicity (IC50, μM) of IrTMPPS toward different typesof cells. (e) Images of the living 4T1 cells co-stained with calcein AM (4 μM, 0.5 h) andpropidium iodide (6 μM, 0.5 h) after different treatments. Calcein AM: λex = 460 nm, λem =540  30 nm; PI: λex = 540 nm, λem = 610  30 nm.

Xie, J. et al., ACS Appl. Mater. Interfaces, 2021, 13, 24, 27934–27944

20

In Vivo SDT with IrTMPPS• 1O2 generated in deep tissue(>10 cm)• Low selectivity→intratumoral injection• Growth of tumors in micetreated with IrTMPPS + US
was suppressed
Figure 6. (a) Fluorescence imaging for investigating 1O2 generation in the presence of
IrTMPPS and SOSG probe in tissue-mimicking gel model (>10 cm) under 20 min US
irradiation. SOSG and IrTMPPS mixing solution was injected in every hole. (b) Schematic of
the in vivo therapeutic protocol for SDT. Mice were irradiated by US (0.3 W cm–2, 3.0 MHz)for 20 min after i.t. injected with 25 μL of PBS containing 500 μM IrTMPPS. (c) Tumor growth
curves after different treatments. Error bars were standard errors based on five mice pergroup. (d) Tumor weights of mice at day 14 after various treatments. (e) Representative
images of mice at day 14 after different treatments. (f) Photos of tumors of mice at 14 day.
***p < 0.001.

Xie, J. et al., ACS Appl. Mater. Interfaces, 2021,
13, 24, 27934–27944

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Anti-metastasis to Lungs• Mice treated with IrTMPPS + US
exhibited almost no lung metastasis
sites• IrTMPPS significantly inhibited tumor
lung metastasis
Figure 7. (c) Photos of India ink-stained lungs from mice at
40 days post various treatments. Spontaneous pulmonary
breast cancer metastasis sites are pointed out by red
circles.

Xie, J. et al., ACS Appl. Mater. Interfaces, 2021, 13, 24, 27934–27944
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Short Summary• SDT could treat deep-seated tumors.
• MnTTP-HSA presented the best SDT performancedue to the lowest HOMO−LUMO gap energy• SDT with IrTMPPS can suppress tumor growth andproliferation by generating 1O2 and oxidizing NADH
to NAD+.

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Contents• Introduction• Improvement of ROS Generation Ability• Energy gap• Water solubility• Combination of SDT and PTT• Summary24

Pt-CuS Nanoparticles (PCPT)• Photothermal Therapy (PTT)• Kill cancer cells with hyperthermia
produced by light energy• 808 nm laser + US
→Hyperthermia + 1O2
Pt: H2O2→O2
TAPP: Sonosensitizer
Temperature-sensitive polymer
Increase biocompatibility
prevent premature release of TAPP
Liang, S. et al., Nano Lett. 2019, 19, 6, 4134–4145

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Photothermal Effect of Pt-CuS in
Solution• Photothermal conversion efficiencies• Pt-CuS: 34.5%• CuS NPs: 23%• Stable conversion ability

Figure2. (f) Temperature elevation curves ofaqueous CuS and Pt-CuS solution with the same
concentration of Cu (100 ppm) at 0.8 W cm–2 808 nm
laser irradiation for 420 s. (g) Temperature elevation
curves of the aqueous Pt-CuS solution at various
concentrations of Cu upon 0.8 W cm–2 808 nm laser
irradiation for 420 s. (h) Temperature elevation
curves of the aqueous Pt-CuS solution (theconcentration of Cu is 100 ppm) under 808 nm laser
irradiation with various power densities for 7 min. (i)The photothermal stability of Pt-CuS solution (theconcentration of Cu is 100 ppm) under 0.8 W cm–
2808 nm laser irradiation.

Liang, S. et al., Nano Lett. 2019, 19, 6, 4134–4145
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In Vitro ROS Generation by PCPT• Catalase-like activity of Pt-CuS-P(H2O2→O2)• US exceeds light in 1O2 generation

Figure 3. (a) The production of O2 by
Pt-CuS (100 ppm of Cu) in theconcentration of 250 μM H2O2 in a
N2 environment. (b) UV–vis
absorption spectrum of theremainder H2O2 after reaction with
Pt-CuS and Pt-CuS-P at 37 and 40 C.
(c) The fluorescence intensity of SOSG
in the presence of PCPT with theconcentration of 50 ppm upon US (1.0MHz, 1.0 W cm–2, 60% duty cycle)
irradiation for fixed intervals
durations. (d) The fluorescenceintensity of SOSG in the presence of
PCPT with the concentration of 50
ppm upon US irradiation at varied
power densities. (e) The productionof 1O2 in the presence of PCPT (50ppm) at different conditions. (f)Relative 1O2 production from PCPT
after 650 nm NIR light or US
irradiation, with or without the screenof pork.

Liang, S. et al., Nano Lett. 2019, 19, 6, 4134–4145

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In Vitro Cytotoxicity of PCPT
• PCPT entered thecytoplasmic vesicles• PCPT + laser + US
produced more ROS
and enhanced
anticancer effect• SDT + PTT induced
extensive apoptosis
Figure 4. (a) Cellular endocytosis of PCPT by CT26 murine colon cancer cells at varied time points. (b)The viability of CT26 cells with different treatments, including control, only US, only laser, only PCPT,
PCPT combined with US irradiation, PCPT combined with 808 nm laser irradiation, and PCPT combinedwith 808 nm laser and US irradiation. (c) Cellular production of 1O2 with different treatments, including
control, only US, only laser, only PCPT, PCPT combined with US irradiation, PCPT combined with 808 nm
laser irradiation, and PCPT combined with 808 nm laser and US irradiation. (d) The fluorescence imagingof CT26 cells stained by calcein AM (green) and PI (red) after different treatments.

Liang, S. et al., Nano Lett. 2019, 19, 6, 4134–4145

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In Vivo Tumor Inhibition Ability• PCPT + US or Laser
Inhibited tumor growth, but recurrence• PCPT + US + Laser
Eradicate tumor, no reoccurrence

Figure 6. (a) Biodistribution of Pt in
tumor and main organs after
injection with PCPT intravenously
for 4, 8, 12, and 24 h. (b) Body
weight of CT26 tumor-bearing micewith different treatments during
therapeutic duration. (c) Tumor
volume change curves during
therapeutic duration. (d) Tumorweights harvested by mice at theend of various treatments. (e)Representative digital photos oftumor-bearing mice after different
therapies and digital photos oftumors from euthanized mice after
different treatments for 13 days.

Liang, S. et al.,Nano Lett. 2019,19, 6, 4134–4145
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Contents• Introduction• Improvement of ROS Generation Ability• Energy gap• Water solubility• Combination of SDT and PTT• Summary30

Summary• SDT is a promising strategy for cancer treatment• 1O2 generation ability may be related to energy gap• Solubility of sonosensitizer is important• Combination of SDT and other methods canstrengthen therapeutic effect

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APPENDIX

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In vitro SDT efficacy of MTTP-HSA
Figure3.
In vitro SDT efficacy of MTTP-HSA nanoparticles against
MCF-7 cells. a) In vitro cytotoxicity of the threenanoparticles at identical concentrations (5.5 m) against
MCF-7 cells after US irradiation (1 MHz, 2 W cm−2, 3 min);the MnTTP-HSA showed the best inhibition to MCF-7 cells
among the three nanoparticles (**p < 0.01). b) FL images of
MCF-7 cells stained by calcein-AM and PI after various
treatments; Viable cells were stained green with calceinAM, and dead/later apoptosis cells were stained red with
PI (scale bar = 100 µm). The MnTTP-HSA + US treatment
clearly induced apoptosis/necrosis. c) CLSM images of ROS
in MCF-7 cells strained by DCFH-DA after different
treatments (scale bar = 25 µm); the MnTTP-HSA + US
treatment produced a large quantity of ROS. d) The
intracellular quantitation of ROS generation after different
treatments. The intensity of the ROS signal was
significantly higher in the MnTTP-HSA + US group than theothers (**p < 0.01).

Ma, A. et al., Small,2019, 15, e1804028
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MRI/PA Dual-Modal Imaging of
the MnTTP-HSA

Ma, A. et al.,Figure4. MR and PA imaging of the MnTTP-HSA nanocomplex. a) Longitudinal relaxation rates (1/T ) versus the concentration of MnTTP-HSA (0, 0.2, 0.4, Small, 2019,
0.6, 0.8, and 1.0  10 m); the inset was the T -weighted MR images of MnTTP-HSA aqueous solutions at varied concentrations. b) Time-dependent invivo T -weighted MR images of MCF-7 tumor-bearing nude mice after the intravenous injection of MnTTP-HSA. c) Corresponding T -weighted MR signal 15, e1804028
−3

1

1

1

1

intensity of MnTTP-HSA in tumor varying with time (**p < 0.01). d) Photoacoustic (PA) signal intensity versus the concentration of MnTTP-HSA (0.1, 0.2,
0.4, 0.6, 0.8, and 1.0  10−3 m); the inset was the PA images at various concentrations of MnTTP (λex = 680 nm). e) Time-dependent PA images of MCF-7
solid tumors after the intravenous injection of MnTTP-HSA. f) The time-dependent PA signal intensity of MnTTP-HSA in tumor (**p < 0.01).

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Intracellular Localization and ROS
Generation• IrTMPPS specifically targeted lysosomes of 4T1 cells.
Figure 4. (a) Confocal microscopy images ofthe living cells treated with IrTMPPS (10 μM,
4 h) and co-stained with Lyso-Tracker Green(LTG, 5 μM, 45 min) or Mito Tracker Green(MTG, 200 nM, 45 min). IrTMPPS: λex = 405
nm, λem = 695  30; nm. MTG: λex = 488 nm,
λem = 516  30 nm; LTG: λex = 458 nm, λem =505  30 nm. (b) Confocal microscopy
images of 4T1 cells treated with IrTMPPS(20 μM, 4 h) and co-stained with SOSG in
the presence or absence of NaN3 under
different conditions. SOSG probe: λex = 488
nm, λem = 525  30 nm.

Xie, J. et al., ACS Appl.
Mater. Interfaces, 2021,
13, 24, 27934–27944
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In Vivo PA Imaging and Upregulationof Tumor Oxygenation

Figure 5. (a) In vivo NIR thermal imaging of CT26 tumor-bearing
mice after intravenous injection of PCPT (20 mg kg–1) for 12 hand combined with 808 nm laser irradiation. (b) In vitro PA
imaging of different concentrations of Pt-CuS NPs. (c) In vivo PA
imaging of the tumor-bearing mice intravenous injection with
PCPT (20 mg kg–1) at different times. (d) The blood oxygen
saturation of tumors before and after intravenous injection
with PCPT (20 mg kg–1) and combined with 808 nm laser
irradiation. (e) The representative immunofluorescence imagesof tumor slices after different treatments for 24 h. The nuclei
were stained with DAPI (blue); hypoxia areas were stained with
HIF-1α (green), and blood vessels were stained with VEGF (red).

Liang, S. et al., Nano Lett. 2019, 19, 6, 4134–4145

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