Novel Spectroscopic Studies of the Interaction of Three Different Types of Iron Oxide Nanoparticles with Albumin
, Plamen Zagorchev 2,3, Boris Tenchov 1 and Bissera Pilicheva 3,4,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript nanomaterials-3295833 provides a spectroscopic Study of the Interaction of Three Different Types of Iron Oxide Nanoparticles with Albumin. Finally, the type of interaction between the iron oxide nanoparticles and albumin is established based on the quenching data from the fluorescence data.
Specific Comments
The document has good level of English and the scientific background is sound. However, even if the specific coatings of the iron oxide nanoparticles haven’t been studied before, there are previous reports in the literature regarding the interaction of iron oxide nanoparticles and Human serum albumin (HSA). So, the novelty of the document is relative. In order to male the study more innovative, the authors should have invested more in other complimentary spectroscopic techniques, such as Fourier Transformed Infrared spectroscopy (FTIR), which helps to identify functional groups and surface chemistry. Also, techniques like X-ray Diffraction (XRD), Raman Spectroscopy, Electron Paramagnetic Resonance (EPR) could have been employed to provide a more complete insight regarding the full characterization of the chemical interactions between the nanoparticles and HSA. Furthermore, the use of Dynamic Light Scattering (DLS) would have been important to measure the size and distribution of the nanoparticles, providing insight into their stability and aggregation behavior. In addition, the authors could have developed some kind of in vivo studies to assess the cytotoxicity and distribution of the nanoparticles.
In summary, I think that this document lacks some novelty in terms of conceptual idea and is very incomplete in terms of methodological approach, needing a substantial improvement in order to present the quality needed to be published in an international and peer reviewed journal.
Author Response
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted in the re-submitted file.
We fully agree with the reviewer that the nanoparticles characterisation is needed, and the study seems to be in a preliminary stage based on the presented data. In fact, the fabricated nanoparticles have been thoroughly characterised by numerous methods and the results have been intended in another manuscript with a focus on preparation and characterisation. The goal of the suggested manuscript was to focus on binding properties of NPs with albumin. However, we respect the recommendation of the reviewer and the paper was revised as suggested and results regarding particle size, surface charge, etc. were included. Also, we have performed in vitro cell culture studies to investigate the NPs cytotoxicity together with other ex vivo investigations. The latter results are part of another project and cannot be included in this paper.
Reviewer 2 Report
Comments and Suggestions for AuthorsIn my opinion, the study is still in a very preliminary stage and contains several flaws. Most important is that while it provides some important data on the interaction of iron oxide nanoparticles with various coatings and albumin, it lacks of characterization of the nanoparticles, not only in terms of size and morphology but also in terms of their surface charge and surface state, which are critical for interpreting the results presented. Additionally, only a limited number of parameters have been examined, and there is no data regarding the stability of the interaction between the iron oxide nanoparticles and albumin. All these aspects must be taken into consideration to provide a complete study on the interaction of the nanoparticles used with proteins.
Author Response
Thank you very much for taking the time to review this manuscript. Please find the detailed responses below and the corresponding revisions/corrections highlighted in the re-submitted file.
We fully agree with the reviewer that the nanoparticles characterisation is needed, and the study seems to be in a preliminary stage based on the presented data. In fact, the fabricated nanoparticles have been thoroughly characterised by numerous methods and the results have been intended in another manuscript with a focus on preparation and characterisation. The goal of the suggested manuscript was to focus on binding properties of NPs with albumin. However, we respect the recommendation of the reviewer and the paper was revised as suggested and results regarding particle size, surface charge, etc. were included.
As per the reviewer’s comment on the stability of the interaction between NPs and albumin, the evaluation of the stability is established by analysing the obtained Stern–Volmer plots and parameters (Ksv, Kq) from the obtained by fluorescence spectroscopy data, and the changing in the peak of absorption of the protein before and after the interaction by obtained UV-Vis spectra.
In our study Table 2 presents the binding parameters estimated for IONP@UT, IONP@CV, and IONP@GL fitted according to the Stern-Volmer equation. The parameters show an inverse relationship between temperature and the calculated KSV and Kq values. According to the literature, this indicates that the quenching mechanism observed in the IONPs-HSA binding reaction is likely initiated by ground state STABLE complex formation rather than dynamic collision. A slight hyperchromic effect in the obtained UV-Vis spectra was observed in the range 267÷300 nm, indicates the interaction of HSA and IONPs in the ground state (a static mechanism). So, we could assume that the fluorophores of HSA were quenched using IONPs by stable complex formation.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe revised manuscript nanomaterials-3295833 is a significant improvement in comparison with its previous version. New characterization data is presented, thus making the manuscript scientifically more relevant and with a greater degree of novelty. There are some aspects that need to better presented and/or discussed, as indicated in the specific comments, but overall, I think that the manuscript now presents the minimum level of quality to be published in this journal. My recommendation is that, after the suggested revisions are made, the manuscript can be accepted for publication.
Specific comments
- (line 158). Figure 1 should be revised. The numbers in the scales are very difficult to read and I suggest to identity the different graphs with different letters in order to identify more easily which type of IONP they refer to.
- line 189 – I don’t agree the only the IONP-CV has a lower stability. Also, the IONP – UT has a low zeta potential (-20.67 mV), when the reference zeta potential for a good stability is ± 30 mV. Only the uncoated IONP and the IONP- GL match this “specification”. The discussion of this section should be revised.
Line 194 – table 1. The particle size is not ideal. In order to have nanoparticles with good technological properties particle size should be under 200 nm. This feature should also be addressed and discussed by the authors.
Author Response
The authors are very grateful to the reviewer for the evaluation of this work. The responses to the comments are provided below:
Comment 1: (line 158). Figure 1 should be revised. The numbers in the scales are very difficult to read and I suggest to identity the different graphs with different letters in order to identify more easily which type of IONP they refer to.
Response 1: Figure 1 was revised as recommended.
Comment 2: line 189 – I don’t agree the only the IONP-CV has a lower stability. Also, the IONP – UT has a low zeta potential (-20.67 mV), when the reference zeta potential for a good stability is ± 30 mV. Only the uncoated IONP and the IONP- GL match this “specification”. The discussion of this section should be revised.
Response 2: The discussion of that par was revised according to the reviewer's recommendation.
Comment 3: Line 194 – table 1. The particle size is not ideal. In order to have nanoparticles with good technological properties particle size should be under 200 nm. This feature should also be addressed and discussed by the authors.
We agree with the reviewer that the measured particle size is not ideal. In fact, the data presented refer to the average hydrodynamic particle diameter, as measured by DLS. The actual particle size is much smaller, as observed with TEM (TEM micrograph is attached for the reviewer's information only). We have added the word "hydrodynamic" for clarification.
