Fullerenes’ Interactions with Plasma Membranes: Insight from the MD Simulations
and Jirasak Wong-ekkabut 1,2,3,*Round 1
Reviewer 1 Report
The manuscript entitled “Fullerenes’ interactions with plasma membranes: Insight from the MD simulations“ by N,. Nisoh et al. reports the C60 interaction with aymmetric plasma membranes. A special attention is given to the aggregation mechanims of C60. Simulations reported in this work demonstrate clearly that the C60 clusters are generated at low concentration, these being prefrentialy situated in the inner leaflet, where there are the DAPE lipids. The discussions are well structured and experimental results prove conclusions of this work.
I recommend this article to be published in the Biomolecules journal in the present form.
Author Response
Reviewer: The manuscript entitled “Fullerenes’ interactions with plasma membranes: Insight from the MD simulations“ by N,. Nisoh et al. reports the C60 interaction with aymmetric plasma membranes. A special attention is given to the aggregation mechanims of C60. Simulations reported in this work demonstrate clearly that the C60 clusters are generated at low concentration, these being prefrentialy situated in the inner leaflet, where there are the DAPE lipids. The discussions are well structured and experimental results prove conclusions of this work.
I recommend this article to be published in the Biomolecules journal in the present
Response: We thank the reviewer for his/her encouraging comments.
Reviewer 2 Report
The paper by Nisoh et al. deals with the coarse-grained molecular dynamic of multicomponent plasma membrane in presence of different concentration of fullerenes. The manuscript contains interesting information on interaction of C60 with the model of plasma membrane. The authors characterised the penetration of fullerenes in plasma membrane and the location of fullerenes in the region of membrane with the highly unsaturated lipids composition.
The manuscript is well written and should be published in Biomolecules after some minor revision.
- In line 72 is reported that the plasma membranes are asymmetric due to a different level of unsaturation of alkyl chains of lipids between the inner and outer leaflets of membrane. But also a different distribution of lipids (PC, PE, PS, SM etc) is responsible of the asymmetric nature of membrane. Also the asymmetric composition of lipids should be added, in the manuscript, as feature of plasma membrane.
- Line 96: In figure S2 is not reported the density distributions of fullerenes but the snapshot at the end of simulation of the simulated systems. Please change the text.
- In table 1 is reported the system at fullerenes concentration 19 % (biased simulation) and figure 2 shows the density distribution of fullerenes and PO4 beads for biased simulation of this molecular system. In the section “Biased Molecular Dynamics Simulations” of Methodology this system is not reported (line 244). The authors should add the concentration at 19 % at the line 244. Why this system is not simulated in unbiased conditions?
- In Line 114 is reported: “In biophysical systems, the inner leaflets have typically higher levels of unsaturation than the outer leaflets”. I suggest to replace “biophysical systems” with "plasma membrane".
- Are the shadow matrix calculated for inner and outer leaflets of plasma membrane? Is the MSE calculated considering both leaflets? Please clarify this part.
- Figure 5 is very similar to the figure 3 of the previous article of the authors (BBA - Biomembranes 1862 (2020) 183328). I suggest the authors to report in figure 5 only the PMF profile of DAPC 0% and 40%, data obtained in this work, and describe the comparison with the PMF profiles of other lipids (DLPC, DSPC, DFPC) in the text.
- I suggest to add a figure to show the lipid organization around fullerenes molecules and rationalize, from atomic point of view, the preference of fullerenes to localize close to DA tails.
- In line 238 replace “All simulations were simulated for 10 us and..” in “All systems were simulated…”
Author Response
Reviewer2
Reviewer: The paper by Nisoh et al. deals with the coarse-grained molecular dynamic of multicomponent plasma membrane in presence of different concentration of fullerenes. The manuscript contains interesting information on interaction of C60 with the model of plasma membrane. The authors characterised the penetration of fullerenes in plasma membrane and the location of fullerenes in the region of membrane with the highly unsaturated lipids composition.
The manuscript is well written and should be published in Biomolecules after some minor revision.
Response: We thank the reviewer for his/her careful reading and constructive comments. The manuscript has been revised following the reviewer’s comments.
Reviewer: In line 72 is reported that the plasma membranes are asymmetric due to a different level of unsaturation of alkyl chains of lipids between the inner and outer leaflets of membrane. But also a different distribution of lipids (PC, PE, PS, SM etc) is responsible of the asymmetric nature of membrane. Also the asymmetric composition of lipids should be added, in the manuscript, as feature of plasma membrane.
Response: We thank the reviewer for this comment and have added the information in the manuscript.
Reviewer: Line 96: In figure S2 is not reported the density distributions of fullerenes but the snapshot at the end of simulation of the simulated systems. Please change the text.
Response: We apologize for this error. The manuscript has been corrected accordingly.
Reviewer: In table 1 is reported the system at fullerenes concentration 19 % (biased simulation) and figure 2 shows the density distribution of fullerenes and PO4 beads for biased simulation of this molecular system. In the section “Biased Molecular Dynamics Simulations” of Methodology this system is not reported (line 244). The authors should add the concentration at 19 % at the line 244. Why this system is not simulated in unbiased conditions?
Response: Our work focused on systems at fullerene concentrations of 5%, 10%, 20% and 30%. Under unbiased conditions at high fullerene concentrations (20%, 30%), large fullerene clusters in water induced membrane damage before entering the membrane. Therefore, we performed additional simulations using biased systems by gradually pulling fullerene into the plasma membrane one-by-one until saturation was reached at the concentration of 19% . We discuss the system at 19% in the section Biased Molecular Dynamics Simulations.
Reviewer: In Line 114 is reported: “In biophysical systems, the inner leaflets have typically higher levels of unsaturation than the outer leaflets”. I suggest to replace “biophysical systems” with "plasma membrane".
Response: We have made the changes as suggested.
Reviewer: Are the shadow matrix calculated for inner and outer leaflets of plasma membrane? Is the MSE calculated considering both leaflets? Please clarify this part.
Response: Since the fullerenes are located about the center of the bilayer, MSE was calculated considering both leaflets together. We have added this sentence in the MSE section.
Reviewer: Figure 5 is very similar to the figure 3 of the previous article of the authors (BBA - Biomembranes 1862 (2020) 183328). I suggest the authors to report in figure 5 only the PMF profile of DAPC 0% and 40%, data obtained in this work, and describe the comparison with the PMF profiles of other lipids (DLPC, DSPC, DFPC) in the text.
Response: Figure 5 was edited following the reviewer’s suggestion. The PMF profiles of DLPC, DSPC, DFPC are shown in Figure S6. We compare data with the PMF profiles of other lipids (DLPC, DSPC, DFPC) in the main text.
Reviewer: I suggest to add a figure to show the lipid organization around fullerenes molecules and rationalize, from atomic point of view, the preference of fullerenes to localize close to DA tails.
Response: We thank the reviewer for this suggestion and have added such a figure in Supporting Information.
Reviewer: In line 238 replace “All simulations were simulated for 10 us and..” in “All systems were simulated…”
Response: Corrected.
Reviewer 3 Report
The article entitled “Fullerenes’ interactions with plasma membranes: Insight from the MD simulations” investigated the behaviors of [60]fullerene in the plasma membranes consisting of multiple lipid components via coarse-grained molecular dynamic simulations. The results indicated that [60]fullerene migrated through the outer leaflet of the plasma membrane and aggregated in the regions of DA-tail lipids which are highly unsaturated. This result sounds interesting but ignores that hydrophobic fullerenes cannot exist as single molecules in physiological aqueous solutions. To improve the biocompatibility of fullerenes, it is commonly required to coat them with hydrophilic polymers (e.g., PEG, PVP) or make chemical derivation. Therefore, it is illogical to simulate the interaction of hydrophobic molecular [60]fullerene with plasma membranes in an aqueous solution, which is almost impossible in reality.
Author Response
Reviewer3
Reviewer: The article entitled “Fullerenes’ interactions with plasma membranes: Insight from the MD simulations” investigated the behaviors of [60]fullerene in the plasma membranes consisting of multiple lipid components via coarse-grained molecular dynamic simulations. The results indicated that [60]fullerene migrated through the outer leaflet of the plasma membrane and aggregated in the regions of DA-tail lipids which are highly unsaturated. This result sounds interesting but ignores that hydrophobic fullerenes cannot exist as single molecules in physiological aqueous solutions. To improve the biocompatibility of fullerenes, it is commonly required to coat them with hydrophilic polymers (e.g., PEG, PVP) or make chemical derivation. Therefore, it is illogical to simulate the interaction of hydrophobic molecular [60]fullerene with plasma membranes in an aqueous solution, which is almost impossible in reality.
Response: The reviewer is correct, and we are also well aware, in that fullerenes cannot exist as single molecules in a physiological solution and that they need to be coated for any practical purposes. It is, however, very important to understand the detailed interactions between fullerenes and plasma membranes and computer simulations offer a good method to do that. This information is critical in order to understand the origins of the different behaviors that coated fullerenes display, that is, what are the characteristics arising from the plain uncoated fullerenes vs those from the different coatings. Such understanding is also needed for rational design of coatings and functionalization. In our on-going follow-up study, we are working along those lines and have developed fullerenes with functional groups to study the effects of hydrophobically coated fullerenes on plasma membranes.
Round 2
Reviewer 3 Report
This work indicates that [60]fullerene may penetrate the plasma membrane and aggregate in the regions of unsaturated DA-tail lipids. The results probably provide information for further investigation of the interaction mechanism of functional fullerenes with cell membranes.
