Peer review of: Preparation of TiH1.924 nanodots by liquid-phase exfoliation for enhanced sonodynamic cancer t (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 

This paper describes a new 2D material for cancer therapy. It combines the properties of TiH1.924 
nanodots as sono-sensitizers and as photothermal agents. 
The results are very interesting and well presented. They cover several aspects spanning from the 
mechanisms of ROS production to the in vitro and in vivo anticancer activity. It deserves 
publication in Nature Communications, but I have some questions that the authors should address 
in their revised version. 
DCFH-DA oxidation can be enzymatically catalyzed by the pair peroxidase/H2O2. Due to the 
existence of several substances that interfere with the formation of DCFH, this probe, when used 
in cellular systems, has better use as a marker of the cellular oxidative stress. So other ROS 
probes should be tested (see for example: Gomes et al. J Biochem. Biophys. Methods 2005, 65, 
45-80). 
The authors should better specify the ESR probes they used. It is not clear when TEMP or DMPO 
are used. Generation of singlet oxygen normally is generated via energy transfer mechanism, 
while in this case a charge transfer mechanism is proposed. The authors should clarify how singlet 
oxygen is generated with the appropriate probes. 
The authors say that they quantify the ROS produced. However, no quantification data are 
reported. 
Most of the characterizations seem to be performed in the material exfoliated into the organic 
solvents. This is not really clear, and should be specified in the legend of the figures. The 
exfoliated material has been dispersed in water for the biological test. This is not described clearly. 
Which are the conditions used? Centrifugation speed and time? Concentration of the stock 
solution? 
Nanosized hydrides can be much more reactive than bulk material. The authors should discuss the 
stability of this material particularly in water. 
How was the surface energy of the solvents measured? Are these data present in the literature? Is 
it possible to measure the surface energy of mixed solvent? I have a concern about this. Not sure 
it is possible. 
How reliable is the XPS to measure the ratio of the different Ti valences? Deconvolution of the 
peak is generally not precise. The authors should at least provide averages performing the analysis 
a certain number of times on different nanodot samples. 
The amount of PVP on the nanodots should be quantified. 
The authors expressed the concentration of titanium hydride in molar concentration (mM). Without 
the molecular weight of the material how is this possible? It is more appropriate to express the 
amount as mass per volume (µg/ml). 
 
Minor: 
Scheme 1 is mentioned, but I think it is probably Figure 1; 
“Solvent with different molecular structures….” has no meaning. Please modify 
Please check the abbreviations as NMP is not abbreviated for example. 
The superoxide O2- lacks of the radical. 
Some of the figure panels are to small and it is difficult to see some text or the structures (see 
solvent structures, which are useless though). 
 
 
 
Reviewer #2: 
Remarks to the Author: 
In this study, ultra-small titanium hydride (TiH1.924) nanodots have been prepared as the 
potential sonosensitizer from its powder form via the liquid-phase exfoliation. The nanodots 
showed better sonocatalytic activity than reported TiO2 NPs. The near IR absorption of the 
nanodots were used to produce mild photothermal heating to enhance intra-tumoral blood flow 
and improve tumor oxygenation. Consequently, a remarkable synergistic therapeutic effect is 
achieved in the combined photothermal-sonodynamic therapy. This study is interesting, but I 
recommend to improve the quality of the manuscript by considering following comments. 
 
1. Storage stability of the nanodots, obtained by exfoliation of TiH1.924 powder after PVP 
hydrophilization, is not provided. Thus, it is not sure whether these nanodots have similar or faster 
aggregation tendency, compared to hyrophilized TiO2 nanoparticles. 
2. The higher degree of exfoliation showed better sonocatalytic property, which might be mostly 
due to the increase in coordinately unsaturated Ti surface in the nanodots. What is the fate of the 
nanodots surface in H2O2-rich cancer environments? H2O2 has the ability to convert titanium 
hydride to oxygen deficient black TiO2 at elevated temperature, which is a better sonocatalyst 
than TiO2 nanoparticles. In cancer microenvironments, it is wondering if there is any formation of 
titanium oxide or oxohydrides in the nanodots surface. If H2O2-nanodots interaction occurs, is 
that influencing the sonocatalytic augmentation? 
3. What is the mode of interaction between PVP and surface of Ti having variable oxidation state? 
how strong or stable the interaction is? 
4. TiO2 nanoparticles with highly reactive exposed (0 0 1) facets can be prepared from TiO2 
nanoparticles by similar exfoliation technique to enhance the photocatalytic activity. Does liquid-
phase exfoliation make titanium hydride nanodots only having variable oxidation state or any 
reactive facet exposure, contributing to the sonocatalytic augmentation?