Synthesis of Conjugates of PEG-RGD Derivatives with Fe₃O₄ Magnetic Nanoparticles for Cell Labelling

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The paper entitled: 'Synthesis of Conjugates of PEG-RGD Derivative with Fe3O4 Magnetic Nanoparticles for Cell Labelling' is a carefully prepared work, both in terms of content and editing. The results presented can make a significant contribution to the field. However, I have a few comments that I would like the authors to address.

1. The abstract should include a few more key results of the paper (resulting from materials characteristics).

2. The statement at the beginning of section 2.1 – 'All reagents are commercially available' – is quite brief and appears to lack context. Please consider expanding on this point or incorporating it into the descriptions of reagent usage.

3. According to current IUPAC guidelines, polymers with multi-segment names should include brackets in their names. So, instead of polyethylene glycol, it is more appropriate to use the poly(ethylene glycol) form.

4. What was the procedure for preparing the material for FTIR measurements in ATR mode? Please include this information in the description of the methodology - in the case of powder materials, a special approach is required.

5. How many measurements were performed for each sample using EDX and CHN analyses? What is the measurement error associated with the values given in Table 1? Based on these experiments, the authors later draw many conclusions, hence including such information would be advisable.

6. Some of the graphs require minor editorial corrections. In Figure 1, different font sizes were used to indicate wavelengths, this needs to be unified. In Figure 2a and 2b, the scale should be enlarged, because the current one is difficult to read.

7. How were core and shell thickness measurements made? What does it look like from a statistical perspective (what error, variability).

 

8. Fe₃O₄ MNPs reduce cell viability and exhibit cytotoxic effects. Is this phenomenon observed in other cell types as well? If so, Is it safe to use such particles in applications related to a living organism, will they not have a destructive effect on other living tissues?

Author Response

We thank the reviewer for his efforts in reviewing our manuscript and for his valuable comments and suggestions.

 

Reviewer 1. Comments and Suggestions for Authors

 

The paper entitled: 'Synthesis of Conjugates of PEG-RGD Derivative with Fe3O4 Magnetic Nanoparticles for Cell Labelling' is a carefully prepared work, both in terms of content and editing. The results presented can make a significant contribution to the field. However, I have a few comments that I would like the authors to address.

1. The abstract should include a few more key results of the paper (resulting from materials characteristics).

Response: We have corrected the abstract.

2. The statement at the beginning of section 2.1 – 'All reagents are commercially available' – is quite brief and appears to lack context. Please consider expanding on this point or incorporating it into the descriptions of reagent usage.

Response: We have added the data.

3. According to current IUPAC guidelines, polymers with multi-segment names should include brackets in their names. So, instead of polyethylene glycol, it is more appropriate to use the poly(ethylene glycol) form.

Response: We have change to " poly(ethylene glycol)".

4. What was the procedure for preparing the material for FTIR measurements in ATR mode? Please include this information in the description of the methodology - in the case of powder materials, a special approach is required.

Response: We have added the information in “2.2. Characterization of Synthesized Materials”.

5. How many measurements were performed for each sample using EDX and CHN analyses? What is the measurement error associated with the values given in Table 1? Based on these experiments, the authors later draw many conclusions, hence including such information would be advisable.

Response: We have added the information in “2.2. Characterization of Synthesized Materials” and Table1.

6. Some of the graphs require minor editorial corrections. In Figure 1, different font sizes were used to indicate wavelengths, this need to be unified. In Figure 2a and 2b, the scale should be enlarged, because the current one is difficult to read.

Response: We have corrected the Figure 1, 2a and 2b.

7. How were core and shell thickness measurements made? What does it look like from a statistical perspective (what error, variability). Figure

Response: We have added the statistical data and examples of nanoparticle size measurements (P. 8,9). Text has been corrected.

8. Fe₃O₄ MNPs reduce cell viability and exhibit cytotoxic effects. Is this phenomenon observed in other cell types as well? If so, is it safe to use such particles in applications related to a living organism, will they not have a destructive effect on other living tissues?

Response: Recently, we have published the information regarding MNPs’ biocompatibility in our previous papers (https://dx.doi.org/10.2147/IJN.S111880, https://doi.org/10.3390/ijms23169093. https://doi.org/10.1021/acsami.1c07748; https://doi.org/10.1016/j.colsurfb.2022.112981, https://doi.org/10.1016/j.nano.2020.102317). The MNPs, including those modified with SiO₂, PEG, and peptides, are non-toxic to cells of various tumor lines and primary human cell cultures. Considering the current results, it can be seen that MNPs toxicity depends on the cell type and, for human cell line MDA-MB231 toxicity does not exceed 70% even when MNPs are used in high concentrations (more than 50 mg/ml). Generally, considering the ISO 10993-5:2009 standard [Standardization, I. O. F. (1999). ISO-10993-5: Biological Evaluation of Medical Devices Part 5: Test for Cytotoxicity: In Vitro Methods. ANSI/AAMI: Arlington, VA, USA.], if cell viability is above 70%, the compound can be recognized as non-toxic.

The adverse effect on cell viability can be due to the ability of iron oxide nanoparticles to induce the production of reactive oxygen species in cells. Also the ability of RGD derivatives to exhibit cytotoxicity against tumor cell lines are well known (for example, Cilengitide (c(RGDf-N(Me)K), which is used to treat glioblastoma [https://doi.org/10.1021/jm970832g]). So, we assumed that the negative effect of MNPs on 4T1 cell viability may be realized due to the RGD peptide. However, to make a final conclusion about safety of the MNPs for a living organisms, of course, more extensive biological studies are required, which are not the goals of this study.

We have corrected the text on P. 10.

 

On behalf of all coauthors.

Sincerely yours,

Dr. Alexander M. Demin

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The authors of the manuscript presented the synthesis and characterization of fluorescent-labeled PEG-RGD-SiO2@Fe3O4 magnetic nanoparticles, designed for cell labeling in potential oncology diagnostics. The nanoparticle characterization and stability, peptide loading, cellular update efficiency, cytotoxicity and magnetic and imaging properties were studied, demonstrating the successful conjugation of RGD peptides with selectively targeting cancer cells and showing promising future in improving diagnostic in MRI and fluorescent imaging. The following should be considered to further improve the manuscript before acceptance.

How the experimental results demonstrate the successful covalent fixing of SiO2 on the surface of Fe₃O₄ MNPs?

Could the authors provide their perspective on how surface modifications, such as PMIDA stabilization, influence nanoparticle-cell interactions at the molecular level?

The use of RGD peptides for targeting and Fe3O4 with SiO2-coated nanoparticles for MRI imaging and drug delivery is already well-documented in existing literature (DOI: 10.1021/acsami.6b09772, ACS Appl. Mater. Interfaces 2016, 8, 24502−24508; Materials 2018, 11, 324; doi:10.3390/ma11020324). To enhance the impact of this study, it would be valuable if the authors more clearly demonstrate the novelty of their work.

Author Response

We thank the reviewer for his efforts in reviewing our manuscript and for his valuable comments and suggestions.

 

Reviewer 2. Comments and Suggestions for Authors

The authors of the manuscript presented the synthesis and characterization of fluorescent-labeled PEG-RGD-SiO₂@Fe₃O₄ magnetic nanoparticles, designed for cell labeling in potential oncology diagnostics. The nanoparticle characterization and stability, peptide loading, cellular update efficiency, cytotoxicity and magnetic and imaging properties were studied, demonstrating the successful conjugation of RGD peptides with selectively targeting cancer cells and showing promising future in improving diagnostic in MRI and fluorescent imaging. The following should be considered to further improve the manuscript before acceptance.

1. How the experimental results demonstrate the successful covalent fixing of SiO₂ on the surface of Fe₃O₄ MNPs?

Response: This can be confirmed by the presence of absorption bands in the IR spectra in the region of ~560–700 cm^-1, which are characteristic of Fe–O stretching of Si–O–Fe bonds [43-46]. In the spectra of the MNPs synthesized in this work, there are a shoulders at ~621 cm^-1, which may indicate the Fe–O–Si bond formation (Fig. 1).

We have corrected the text on P. 5.

2. Could the authors provide their perspective on how surface modifications, such as PMIDA stabilization, influence nanoparticle-cell interactions at the molecular level?

Response: At this stage of the work, we cannot suggest any way of PMIDA influence on the interactions of nanoparticles with cells at the molecular level. However, obtained data can be explained either by the effect of PMIDA on the formation of a protein corona around the particles and increased shielding of the RGD peptide, or by the size effect of MNPs.

In the case of PMIDA-stabilized MNPs 20, serum proteins are likely to be adsorbed onto the MNP surface and, accordingly, shielded the RGD peptides to a greater extent than in the case of MNPs 19. This led to significant reduction of MNPs interaction with cell surface receptors. On the other hand, the hydrodynamic size of MNPs 20 obtained using PMIDA is smaller (Dh 91 nm, PdI 0.14) than the diameter of MNPs 19 obtained using bare MNPs (Dh 144 nm, PdI 0.27). It is possible that cells endocytosed larger MNPs to a greater extent. Thus, in this case, the size effect plays a predominant role in the process of MNPs uptake by cells.

Thus, based on the results of the work, it can be concluded that the PMIDA stabilization led to decrease in MNPs uptake by cells. The mechanism of how PMIDA affects the binding of MNPs to cells requires additional studies. Here we can state the negative effect of PMIDA on binding.

We have corrected the text on P. 11.

3. The use of RGD peptides for targeting and Fe3O4 with SiO₂-coated nanoparticles for MRI imaging and drug delivery is already well-documented in existing literature (DOI: 10.1021/acsami.6b09772, ACS Appl. Mater. Interfaces 2016, 8, 24502−24508; Materials 2018, 11, 324; doi:10.3390/ma11020324). To enhance the impact of this study, it would be valuable if the authors more clearly demonstrate the novelty of their work.

The use of RGD peptides for the synthesis of MR contrast agents based on MNPs has been well described. However, our research focused on the chemical aspect - the development of a new approach to the regiospecific conjugation of RGD-PEG with the groups on the nanoparticles’ surface. This approach allows us to excluded the risk of participation of the carboxyl groups of Asp and the guanidine group of Arg in the conjugation, it is important while Asp and Arg take part in the specific binding of the peptide to integrins. This work also compares the efficiency of conjugation of the RGD peptide PEG-derivatives with nanoparticles obtained without any surfactant or using N-(phosphonomethyl)iminodiacetic acid (PMIDA). The effect of the surfactant used on the initial stage of the MNPs surface modification on the cytotoxicity of the resultant MNPs and on the efficiency of cell labeling with MNPs was also studied, which favorably distinguishes this work from Refs. [24-27, 33-37].

We have corrected the text of our manuscript. Also, we have included ACS Appl. Mater. Interfaces 2016, 8, 24502−24508 and Materials 2018, 11, 324 in the References.

 

 

On behalf of all coauthors.

Sincerely yours,

Dr. Alexander M. Demin

Author Response File: Author Response.pdf