Bifunctional Magnetite–Gold Nanoparticles for Magneto-Mechanical Actuation and Cancer Cell Destruction

Round 1

Reviewer 1 Report

1. The measured diameters of the particles, depicted in Figures 1. b and 1. c, do not correspond with the sizes observed in Figure 1. a, according to the indicated scale bar.

2. The XRD patterns are obtained with cobalt radiation. In the literature, most of the diffractograms for magnetite are obtained by using copper radiation. The reference to support the explanation of the quantification and the identification of the peaks is referred to a previous work of some authors. It is important to analyse and compare the diffractograms and ensure that the obtained pattern corresponds to magnetite and gold, and excludes other iron oxides.

3. After calculating the magneto-mechanical force, it is indicated that a force of 100 pN can be obtained. A minimum concentration to achieve this force can be addressed.

4. After all the experiments, it is difficult to see the motivation for the use of gold instead of neat magnetite NPs. The plasmonic effect is mentioned in the abstract, but there is not any evidence of the suitability of gold to enhance cell death activation.

5. There is a lack of comparison of the magneto-mechanical effect produced inside the cells by the different directions of the applied magnetic fields.

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 2 Report

The article is devoted to the preparation, study of the physicochemical properties of magnetite nanoparticles with gold, as well as the study of their behavior in a magnetic field and interaction with cells as antitumor drugs with a magneto-mechanical effect. The article is of great interest from the point of view of studying the mechanism of interaction of nanoparticles with cells and their behavior in a magnetic field. However, at the same time, the authors poorly represent the physicochemical characteristics of the samples, and the samples themselves exhibit rather weak effects in terms of the magneto-mechanical effect in relation to tumor cells. In addition, the authors do not evaluate the fate of nanoparticles inside tumor cells, since this medium has a different pH, different from those used in the preparation of materials. Generally I recommend accepting the article for publication with minor modifications.

1.       From the introduction, it is not clear why the need to modify magnetite nanoparticles with gold is especially emphasized.

2.       Line 127: «redispersed in 9 mL of 0.1M PBS (pH = 8.3)» What is the reason for the choice of just such a pH for PBS? The physiological pH is 7.4, the pH inside the tumor cells is about 4.5, the pH in the tumor microenvironment is 6.5

3.       Lines 127-130: “Then, MNPs were mixed with a solution of 127 Sulfo-Cyanine5 NHS ester dye in 1 mL DMSO according to the protocol of the manufac- 128 turer and mixed at room temperature overnight. Fluorescently labeled MNPs were puri- 129 fied by multiple centrifugations, and then the resulting MNPs solution was dialyzed 130 against deionized water (MWCO 12 kDa) for 24 h.” What’s the reason of this treatment? What’s the final pH?

4.       The synthesis of magnetic nanoparticles with gold was announced, but in the section «2.1 Synthesis of Magnetic Nanoparticles» I did not see a mention of the gold precursor and at what stage it was added. It would be nice to provide a schematic diagram of the synthesis and structure of the final nanoparticles

5.       Line 149: “PHAN database”. Unfortunately, this is the first time I've heard of this database for XRF data analysis. Could you clarify how many records it contains and how relevant it is to use it for this analysis instead of the classic ICDD or COD?

6.       Line 159: MNPs-Cy5. Before that, it was not specified what this abbreviation means. Do I understand correctly that magnetite (with or without APTES?) was simply mixed with gold (in the form of salt or nanoparticles)? What is the reason you used distilled water and not cell medium? So it's more correct

7.       Line 140: “2.3 Dynamic Light Scattering (DLS) Measurements” Specify in what medium and at what temperature measurements were taken

8.       Line 277: “3.1 Magnetite-Gold Dumbbell Nanoparticles Characterization and Magnetic Properties”. Why are call its dumbbell? Its not clear from paper

9.       Lines 293-296: “According to the DLS measurements, MNPs had a hydrodynamic diameter of 251±23 nm, which means that they are stabilized in solution as conglomerates 294 of ~ a hundred individual MNPs. The Zeta potential of MNPs was positive (the value of 295 15±4 mV) due to the presence of amino groups on the MNPs’ surface from the APTES coating” Specify the pH at which the measurements were taken. It is not entirely correct to talk about stabilization in the context of one-time measurements: stabilization can be in time. Plus, the value of the zeta potential just indicates the instability of nanoparticles, since it is small.

10.   Line 299: “. From the spectrum, we extracted the volume fractions of phases 299 to be 96.0% (Fe3O4) and 4.0% (Au) (Figure S1 and Table S1).’ Please indicate how exactly the percentage of magnetite and gold was calculated from XRD? Does it contain maghemite, what is the formula of stoichiometric magnetite? Was an elemental analysis done to check the iron and gold content?

11.   The authors should study the structure of magnetite-gold nanoparticles more precisely. Do bonds form between magnetite and gold? If so, please provide IR spectra. In addition, the authors should explain the effect of gold nanoparticles on the magnetic properties? Does it exist.

12.   Specify, the data for TEM and magnetic properties are given for magnetite in the APTES shell?

13.   Line 351: “inside the cell’ how can particles of such a large size (about 200 nm) penetrate the cell, while for this their size should be in the range of 10-100 nm? Give the size distribution

14.   Line 427: “After the rotation field (1 Hz, 7 mT) was applied, some of these con- glomerates began to move, similar to the previous experiment (in water), with the exact parameters of the magnetic field forming short chains or keeping the dispersed form predominantly (Figure S6, S7, Video S5-S7).” I didn’t see the chains on video

15.   Line 457: “Thus, investigated MNPs provide a 457 magneto-mechanical effect on cells resulting in their death induction, but this effect depends on NPs concentration and specific cell line.” Why are you sure that cell death is associated with the mechanical movement of particles? It can also be caused by the release of iron ions during exposure to a magnetic field.

Author Response

Please see the attachment.

Author Response File: Author Response.docx