Hierarchical Two-Dimensional Layered Double Hydroxide Coated Polydopamine Nanocarriers for Combined Chemodynamic and Photothermal Tumor Therapy
, Ravindranadh Koutavarapu 2,*, Dong-Yeon Lee 2,*, Jaesool Shim 3,* and Yaswanth Kuthati 1,*Round 1
Reviewer 1 Report
Major:
1. Why A549 cells are chosen and not any other cancer cell line? I would recommend the authors to use at least one more cancer cell line and perform the important experiments in it..e.g., MDA-MB-231 is a good model of invasive breast cancer, which is often used in such studies of nanocarriers. OR, the authors can also pick other lung cancer cell lines if they want this study to focus on lung cancer. A study based on just one cell line is not very convincing.
2. How many independent biological repeats for each experiment are done? Experiments should be done at least two times
3. Figure 4: Zero hours Control is missing. Why did the authors choose 24 hr for their study? I would suggest a time course 2, 8, 12,24, 48 hr and picking up the timepoint accordingly. In Figure 4A, cells don't look healthy; outline the membrane with the dotted line.
4. Compare and contrast different aspects of your NC with others already published with respect to ease of creating NCs, efficiency, mode of action, and toxic effects. At the moment, a separate discussion section is missing. I would strongly recommend the authors to have a separate discussion section or with the conclusion section and discuss aspects like these which I mentioned. Limitations and future directions of the study are also important to mention.
Minor:
1. What do the authors want to comment on the non-specific/toxic effects of CDT and PTT in general, and CuAl-LDH@DOX@PDA in particular? How could the normal cells be kept protected?
2. Line 168: the t-test used is paired or unpaired?
3. Line 230- reference for the pH?
4. Line 255- What is the optimal temperature based on the previous reports for photothermal performance in the tumor cells?
Author Response
We appreciate the efforts of the reviewers for their detailed and insightful comments which have helped us improve the quality of our manuscript. A point-by-point response to the reviewer’s comments is appended below for your convenience.
Major comments:
Comment 1: Why A549 cells are chosen and not any other cancer cell line? I would recommend the authors to use at least one more cancer cell line and perform the important experiments in it..e.g., MDA-MB-231 is a good model of invasive breast cancer, which is often used in such studies of nanocarriers. OR, the authors can also pick other lung cancer cell lines if they want this study to focus on lung cancer. A study based on just one cell line is not very convincing.
Response: We acknowledge the reviewer’s opinion. Our manuscript focussed primarily on evaluation of basic biological study with the materials synthesized. As suggested, we plan to extend this work further in more specific cell-lines with animal studies in the near future as a separate work. Thank you for your valuable comment.
Comment 2: How many independent biological repeats for each experiment are done? Experiments should be done at least two times.
Response: We acknowledge the reviewer’s opinion. In the present study, we have performed all cellular experiments in triplicates. Thank you for your valuable comment.
Comment 3: Figure 4: Zero hours Control is missing. Why did the authors choose 24 hr for their study? I would suggest a time course 2, 8, 12,24, 48 hr and picking up the timepoint accordingly. In Figure 4A, cells don't look healthy; outline the membrane with the dotted line.
Response: We acknowledge the reviewer’s opinion. Previously there were several reports on cell uptake mechanisms of Layered double hydroxides which showed 24 hours as an optimal uptake time. So, 24 hours is chosen in our experiments (Line 280-285, References 29,37). The cells are mounted on a slide for capturing images. The mounting procedure affects the cell morphology, in DOX uptake experiments cells look more healthy. All cells are seeded at one time with same batch of culture cells. Thank you for your valuable comment.
Comment 4: Compare and contrast different aspects of your NC with others already published with respect to ease of creating NCs, efficiency, mode of action, and toxic effects. At the moment, a separate discussion section is missing. I would strongly recommend the authors to have a separate discussion section or with the conclusion section and discuss aspects like these which I mentioned. Limitations and future directions of the study are also important to mention.
Response: We acknowledge the reviewer’s opinion. As suggested, we have compared LDH with other nanocarriers in the discussion section (Lines 236-248) and also included and separate section in the conclusions mentioning the limitations and future perspectives (Lines 339-345). Thank you for your valuable suggestion for strengthening the quality of the manuscript.
Minor comments:
Comment 1: What do the authors want to comment on the non-specific/toxic effects of CDT and PTT in general, and CuAl-LDH@DOX@PDA in particular? How could the normal cells be kept protected?
Response: We acknowledge the reviewer’s opinion. It is well known that the tumor micro-environment displays selective characteristics that are different from normal tissue. The CDT agents are triggered by specific tumor microenvironment changes, such as moderate acidity, hydrogen peroxide overexpression, hypoxia, and low levels of catalase. The PPT that employs photoconversion to yield heat for eradication has been investigated as a non-invasive cancer treatment therapy for selective suppression of cancers in specific regions. Thank you for your valuable comment.
Comment 2: Line 168: the t-test used is paired or unpaired?
Response: We acknowledge the reviewer’s opinion. Paired t-test were used. Thank you for your valuable comment.
Comment 3: Line 230- reference for the pH?
Response: We acknowledge the reviewer’s opinion. As suggested, reference for the pH was included in the revised manuscript (Reference 25). Thank you for your valuable comment.
Comment 4: Line 255- What is the optimal temperature based on the previous reports for photothermal performance in the tumor cells?
Response: We acknowledge the reviewer’s opinion. The optimal temperature is 43-450C for PTT performance according to previous reports. (References 34-36 , Line number-268). Thank you for your valuable comment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The paper “Hierarchical Two-Dimensional Layered Double Hydroxide Coated Polydopamine Nanocarriers for Combined Chemodynamic and Photothermal Tumor Therapy” by Prabhakar Busa, Ravindranadh Koutavarapu, Dong-Yeon Lee, Jaesool Shim3, and Yaswanth Kuthati has a good degree of originality and is well written.
The results are well presented.
However, my remark is about Figure 4. I suggest to modify this image by adding the scalebar to all fluorescence microscopic images and add in the caption of this figure the magnification. Also, I suggest to introduce, if possible, SEM images of the layers.
Author Response
We appreciate the efforts of the reviewers for their detailed and insightful comments which have helped us improve the quality of our manuscript. A point-by-point response to the reviewer’s comments is appended below for your convenience.
Comment: However, my remark is about Figure 4. I suggest to modify this image by adding the scalebar to all fluorescence microscopic images and add in the caption of this figure the magnification. Also, I suggest to introduce, if possible, SEM images of the layers.
Response: We acknowledge the reviewer’s opinion. As suggested, we added scale bars to all microscope images and magnification. The SEM images we are planned to include in our future research, and we don’t have the figures at this time. Thank you for your valuable comment.
Reviewer 3 Report
In my opinion, the presented manuscript needs a careful revision to be considered for publication in Coatings.
- All sections (Introduction, Methods, Discussion, and Results) need to be carefully reviewed and rewritten. There are some typographical and structural errors.
- The Introduction should be rewritten to present this topic better. There is no information about the toxicity of nanoparticles, which is an important problem in transferring this research from in vitro to in vivo studies.
- In general, the results are very brief, and the findings are not well described. Also, the Discussion is not well-written; it seems that it almost does not exist.
- The release of doxorubicin from CuAl-LDH@DOX@PDA particles is pH-dependent. The authors could highlight these findings and provide more descriptions of these findings in the Discussion section.
- Could the authors clarify which solvent for tested nanoparticles was used for biological studies?
- Are the tested particles biodegradable?
- Point 2.9. MTT assay; the procedure related to MTT assay is not clear. Could the authors explain why they added 0.5 mM H2O2 to the cells incubated with CuAl-LDH@DOX and CuAl-LDH@DOX@PDA and established pH by adding HCl? How was the pH in the well plate measured? Why did the authors establish pH after seeding the cells?
- There is no information, which kit was used to measure GSH level in A549 cells. The authors should include the manufacture name and method used to measure GSH concentration (colorimetric, fluorescent, or luminescent assay).
- Which concentrations of tested nanoparticles were used for ROS generation and GSH level measurement? The information about tested doses is essential.
- Did the authors include in the PTT study also the non-irradiated control?
- The Authors should calculate the IC50 values to describe the cytotoxic activity properly.
- In Figure 7, the authors stated that "Different NC's inhibits GSH production in 305 A549 cells compared with 0 μM of the treatment group"; however, they do not present the data for control cells. Also, could the authors double-check the statistical significance for CuAl-LDH@DOX@PDA + laser? Because the standard deviation seems to be very high.
- The ROS generation experiments need positive control.
- The statement "The size of LDH NC's is beneficial for cell uptake and drug release at the desired site in cancer cells" need at least one citation.
- Could the authors discuss how the zeta potential is important for biological activity?
- The authors stated that their nanoparticles are formulation with the controlled release. It seems, looking at Figure 3, that the release of doxorubicin from CuAl-LDH@DOX@PDA is slower than other tested particles; however, the level of DOX is approximately three times lower, and even after longer incubation time did not reach the similar concentration as it was measured for other particles. The controlled release is not just a lower release of tested material compared to others; it is not just a simple process; the controlled release means when the constant amount of drug is released per unit time, but the rate is independent of the concentration drug. In general, the idea of a controlled release drug delivery system is based on zero-order release, in which a drug is released at a constant rate. Thus, to support their conclusion about the controlled release, zero-order kinetics should be presented.
- There is no information on how many experiments were performed to determine the cytotoxic effect, ROS generation, drug release, and GSH level.
- The conclusion section is very short. The authors could comment on the further research and follow-up studies.
Minor comments:
The sentence "A549 cells were used for all in-vitro experiments, which were supplemented with Dulbecco's modified Eagle's medium (DMEM)" is incorrect; cells are not supplemented in DMEM; the cells are cultured using this type of medium.
The sentence "(MTT) assay performed to detect the cell viability in various treatment groups." In this assay, the authors do not "detect" viability; viability could be evaluated, measured, etc.
Author Response
We appreciate the efforts of the reviewers for their detailed and insightful comments which have helped us improve the quality of our manuscript. A point-by-point response to the reviewer’s comments is appended below for your convenience.
Major comments:
Comment 1: All sections (Introduction, Methods, Discussion, and Results) need to be carefully reviewed and rewritten. There are some typographical and structural errors.
Response: We acknowledge the reviewer’s opinion. As suggested, introduction, methods, discussion and results sections are revised carefully. Moreover, the typographical and structural errors are also modified in the revised manuscript. Thank you for your valuable suggestion for strengthening the quality of the manuscript.
Comment 2: The Introduction should be rewritten to present this topic better. There is no information about the toxicity of nanoparticles, which is an important problem in transferring this research from in vitro to in vivo studies.
Response: We acknowledge the reviewer’s opinion. The in vivo biocompatibility studies of the LDH NC’s is mentioned with necessary citations in the revised manuscript. (Reference 11-14, Lines 50-53). Thank you for your valuable suggestion for strengthening the quality of the manuscript.
Comment 3: In general, the results are very brief, and the findings are not well described. Also, the Discussion is not well-written; it seems that it almost does not exist.
Response: We acknowledge the reviewer’s opinion. As suggested, the discussion is carefully checked and we ensured all our results are discussed properly in the results and discussion section. The revised content in the discussion section is marked in blue color with necerssary citations. Thank you for your valuable suggestion for strengthening the quality of the manuscript.
Comment 4: The release of doxorubicin from CuAl-LDH@DOX@PDA particles is pH-dependent. The authors could highlight these findings and provide more descriptions of these findings in the Discussion section.
Response: We acknowledge the reviewer’s opinion. As suggested, we have highlighted the findings and provided detailed description of drug release and cited necessary papers in the discussion section. Lines 242-247. Thank you for your valuable suggestion for strengthening the quality of the manuscript.
Comment 5: Could the authors clarify which solvent for tested nanoparticles was used for biological studies?
Response: We acknowledge the reviewer’s opinion. FBS free cell culture medium (DMEM) used as a solvent for all biological studies. Thank you for your valuable comment.
Comment 6: Are the tested particles biodegradable?
Response: We acknowledge the reviewer’s opinion. Layered double hydroxide nanoparticles are biocompatible and natural biodegradability in an acidic tumor mciro environment and there are several reports on their biodegradability. (Reference 11 and 14).
Comment 7: Point 2.9. MTT assay; the procedure related to MTT assay is not clear. Could the authors explain why they added 0.5 mM H2O2 to the cells incubated with CuAl-LDH@DOX and CuAl-LDH@DOX@PDA and established pH by adding HCl? How was the pH in the well plate measured? Why did the authors establish pH after seeding the cells?
Response: We acknowledge the reviewer’s opinion. As suggested, MTT assay section is revised in the materials section of the revised manuscript. Thank you for your valuable suggestion for strengthening the quality of the manuscript.
Comment 8: There is no information, which kit was used to measure GSH level in A549 cells. The authors should include the manufacture name and method used to measure GSH concentration (colorimetric, fluorescent, or luminescent assay).
Response: We acknowledge the reviewer’s opinion. GSH-Glo (Promega) kit was used to measure GSH levels in A548 cells by luminescent assay. The details are mentioned in the revised materials and methods section. Thank you for your valuable comment.
Comment 9: Which concentrations of tested nanoparticles were used for ROS generation and GSH level measurement? The information about tested doses is essential.
Response: We acknowledge the reviewer’s opinion. 100 μg/mL of CuAl@DOX and CuAl@DOX@PDA used for ROS and GSH measurement assay. The doses are mentioned in the revised manuscript. (Lines 156 and 166). Thank you for your valuable comment.
Comment 10: Did the authors include in the PTT study also the non-irradiated control?
Response: We acknowledge the reviewer’s opinion. Cells that were not exposed to light served as negative individual control denoted as (-L) in all the groups and compared with light irradiated group. Thank you for your valuable comment.
Comment 11: The Authors should calculate the IC50 values to describe the cytotoxic activity properly.
Response: We acknowledge the reviewer’s opinion. As suggested, IC50 values are mentioned in lines 290-291 in the revised manuscript. Thank you for your valuable suggestion for strengthening the quality of the manuscript.
Comment 12: In Figure 7, the authors stated that “Different NC’s inhibits GSH production in 305 A549 cells compared with 0 μM of the treatment group”; however, they do not present the data for control cells. Also, could the authors double-check the statistical significance for CuAl-LDH@DOX@PDA + laser? Because the standard deviation seems to be very high.
Response: We acknowledge the reviewer’s opinion. The control luminisence is subtracted from treatment groups for the GSH production assay. The significance difference is double checked in the results. Thank you for your valuable comment.
Comment 13: The ROS generation experiments need positive control.
Response: We acknowledge the reviewer’s opinion. H2O2 was used as a positive control in our studies and is already included in the results. Thank you for your valuable comment.
Comment 14: The statement “The size of LDH NC's is beneficial for cell uptake and drug release at the desired site in cancer cells” need at least one citation.
Response: We acknowledge the reviewer’s opinion. As suggested, the citations are provided in the revised manuscript. Citation 8 and 25 (Line 205-210). Thank you for your valuable comment.
Comment 15: Could the authors discuss how the zeta potential is important for biological activity?
Response: We acknowledge the reviewer’s opinion. Zeta potential affects cell uptake. Positively charged nanoparticles are uptaken more efficiently whereas negatively and neutral charged nanoparticles have longer circulation times with in the body with less uptake by the organs. Thank you for your valuable comment.
Comment 16: The authors stated that their nanoparticles are formulation with the controlled release. It seems, looking at Figure 3, that the release of doxorubicin from CuAl-LDH@DOX@PDA is slower than other tested particles; however, the level of DOX is approximately three times lower, and even after longer incubation time did not reach the similar concentration as it was measured for other particles. The controlled release is not just a lower release of tested material compared to others; it is not just a simple process; the controlled release means when the constant amount of drug is released per unit time, but the rate is independent of the concentration drug. In general, the idea of a controlled release drug delivery system is based on zero-order release, in which a drug is released at a constant rate. Thus, to support their conclusion about the controlled release, zero-order kinetics should be presented.
Response: We acknowledge the reviewer’s opinion. CuAl-LDH@DOX@PDA displayed a extended release profile of DOX compared to CuAl-LDH@DOX. We revised the text in the manuscript to extended release and removed the term controlled drug release. Thank you for your valuable suggestion for strengthening the quality of the manuscript.
Comment 17: There is no information on how many experiments were performed to determine the cytotoxic effect, ROS generation, drug release, and GSH level.
Response: We acknowledge the reviewer’s opinion. As suggested, all the experiments were done in triplicates and mentioned in the revised manuscript. (Lines 134 and 163-169). Thank you for your valuable comment.
Comment 18: The conclusion section is very short. The authors could comment on the further research and follow-up studies.
Response: We acknowledge the reviewer’s opinion. As suggested, limitations and future perspectives are included in the conclusions section of the revised manuscript. Thank you for your valuable suggestion for strengthening the quality of the manuscript.
Minor comments:
Comment 1: The sentence “A549 cells were used for all in-vitro experiments, which were supplemented with Dulbecco’s modified Eagle’s medium (DMEM)” is incorrect; cells are not supplemented in DMEM; the cells are cultured using this type of medium.
Response: We acknowledge the reviewer’s opinion. The suggested corrections are included in the revised manuscript. Thank you for your valuable suggestion for strengthening the quality of the manuscript.
Comment 2: The sentence “(MTT) assay performed to detect the cell viability in various treatment groups”. In this assay, the authors do not “detect” viability; viability could be evaluated, measured, etc.
Response: We acknowledge the reviewer’s opinion. The suggested change is corrected in the revised manuscript. Thank you for your valuable suggestion for strengthening the quality of the manuscript.
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The authors addressed most of the concerns and the manuscript can now be accepted for publication!
Author Response
Coatings
Ms. Ref. No.: coatings-1325260
Title: Hierarchical Two-Dimensional Layered Double Hydroxide Coated Polydopamine Nanocarriers for Combined Chemodynamic and Photothermal Tumor Therapy
Response to Reviewer-1 Comments
Comment: The authors addressed most of the concerns and the manuscript can now be accepted for publication.
Response: We thank the reviewer for taking their valuable time and giving us valuable comments that helped us to improve the quality of our manuscript.
The authors are very thankful to the reviewer for their valuable suggestions for the improvement of the manuscript.
With regards
Ravindranadh Koutavarapu, Ph.D
Author Response File: Author Response.pdf
Reviewer 3 Report
Thank the Authors for answering my questions; however, some aspects still are not clear.
- Which type of plates were used to measure the fluorescence and luminescence using the plate reader?
- Which solvent was used to dissolve the formazan crystals?
- The answer to point 6 is not satisfactory. My question sounds: “are the tested particles biodegradable?” The authors stated that layered double hydroxide is biocompatible and biodegradable; however, the tested particles also consist of aluminum and copper, which presence and overloaded the cells are not unconcerned?
- Please could the authors re-check the manuscript and correct some English mistakes, for example, “Dulbecco’s modified Eagle’s medium (DMEM) along with 10% FBS. 1 % penicillin strepitous”, what does strepitous mean?; “Invitro ROS generation estimation assay”; “desired sSite of action.”
- To determine the IC50, the authors treated cells with various concentrations of NC. Could the authors specify the used concentrations? How did the authors calculate IC50?
- Which software was used to calculate statistical significance for GSH level?
- Which assay was used to measure the protein contents? Because the GSH level unit is expressed as nmol/mg protein.
- The authors used the abbreviation NC to describe the nanocarrier and negative control. Would you please harmonize the use of this abbreviation?
- The title of axis Y in figure 6 should be corrected (it is not ROS generation fluorescence); more appropriate is Fluorescence Intensity [arbitraty unit].
Author Response
Coatings
Ms. Ref. No.: coatings-1325260
Title: Hierarchical Two-Dimensional Layered Double Hydroxide Coated Polydopamine Nanocarriers for Combined Chemodynamic and Photothermal Tumor Therapy
Response to Reviewer-3 Comments
We appreciate the efforts of the reviewers for their detailed and insightful comments which has helped us improve the quality of our manuscript. A point-by-point response to the reviewer’s comments is appended below for your convenience.
Comment 1: Which type of plates were used to measure the fluorescence and luminescence using the plate reader?
Response: We acknowledge the reviewer’s opinion. We used Corning® 96-well plates with a transparent bottom and black side walls for fluorescent assays and completely transparent plates for luminescent assay (White and black plates have different reflective properties. The black plates absorb light and lower the background. On the other hand, white plates reflect the light and will increase the light yield signal). Mentioned in lines 161-162 and lines 167-168 in the revised manuscript. Thank you for your valuable suggestion for strengthening the quality of the manuscript.
Comment 2: Which solvent was used to dissolve the formazan crystals?
Response: We acknowledge the reviewer’s opinion. We used dimethyl sulfoxide (DMSO) solvent to dissolve the formazan crystals. Mentioned in line 155 in the revised manuscript. Thank you for your valuable suggestion for strengthening the quality of the manuscript.
Comment 3: The answer to point 6 is not satisfactory. My question sounds: “are the tested particles biodegradable?” The authors stated that layered double hydroxide is biocompatible and biodegradable; however, the tested particles also consist of aluminum and copper, which presence and overloaded the cells are not unconcerned?
Response: We acknowledge the reviewer’s opinion. CuAl-LDH is biocompatible. We have mentioned in the manuscript text that LDH is biocompatible and did not mention their biodegradability. The metal ions are incorporated in the LDH frame as they can induce intracellular depletion of reduced glutathione (GSH) levels along with the reduction of Cu2+ to Cu+. The Cu+ ions in turn react with DOX leading to the generation of intracellular hydrogen peroxide (H2O2) molecules to produce the highly toxic hydroxyl radicals (.OH) through a Fenton-like reaction. Free LDH with metal in the interlayers are reported to be highly biocompatible in previous reports (Reference 20: Wang, Z.; Fu, L.; Zhu, Y.; Wang, S.; Shen, G.; Jin, L.; Liang, R. Chemodynamic/photothermal synergistic therapy based on Ce-doped Cu–Al layered double hydroxides). Thank you for your valuable suggestion for strengthening the quality of the manuscript.
Comment 4: Please could the authors re-check the manuscript and correct some English mistakes, for example, “Dulbecco’s modified Eagle’s medium (DMEM) along with 10% FBS. 1 % penicillin strepitous”, what does strepitous mean?; “Invitro ROS generation estimation assay”; “desired sSite of action.”
Response: We acknowledge the reviewer’s opinion. English mistakes are corrected according to the suggestion in the revised manuscript. Thank you for your valuable suggestion for strengthening the quality of the manuscript.
Comment 5: To determine the IC50, the authors treated cells with various concentrations of NC. Could the authors specify the used concentrations? How did the authors calculate IC50?
Response: We acknowledge the reviewer’s opinion. The 50 μg of DOX loaded and PDA coated NC was used as initial concentration and further diluted by performing the serial dilutions to respective specific concentrations to treat the cancer cells. The DOX concentration was estimated from the drug loading percentage calculations. The IC50 was calculated by GraphPad prism software following the previous reports. Thank you for your valuable comment.
Comment 6: Which software was used to calculate statistical significance for GSH level?
Response: We acknowledge the reviewer’s opinion. We used Graphpad prism 5 to calculate the statistical significances. Mentioned in line 178 in the revisd manuscript. Thank you for your valuable suggestion for strengthening the quality of the manuscript.
Comment 7: Which assay was used to measure the protein contents? Because the GSH level unit is expressed as nmol/mg protein.
Response: We acknowledge the reviewer’s opinion. Bicinchonic acid assay (BCA) was used to measure the protein contents. Mentioned in line 173 in the revised manuscript. Thank you for your valuable suggestion for strengthening the quality of the manuscript.
Comment 8: The authors used the abbreviation NC to describe the nanocarrier and negative control. Would you please harmonize the use of this abbreviation?
Response: We acknowledge the reviewer’s opinion. Negative control is used in the text to describe negative control and nanocarrier is described as NC in the revised manuscript. Thank you for your valuable comment.
Comment 9: The title of axis Y in figure 6 should be corrected (it is not ROS generation fluorescence); more appropriate is Fluorescence Intensity [arbitraty unit].
Response: We acknowledge the reviewer’s opinion. As suggested, it was corrected in the revised manuscript. Thank you for your valuable suggestion for strengthening the quality of the manuscript.
The authors are very thankful to the reviewer for their valuable suggestions for the improvement of the manuscript.
All the modifications are shown in blue color in the revised manuscript.
With regards
Ravindranadh Koutavarapu, Ph.D
Author Response File: Author Response.pdf
