Differences among Unique Nanoparticle Protein Corona Constructs: A Case Study Using Data Analytics and Multi-Variant Visualization to Describe Physicochemical Characteristics
Round 1
Reviewer 1 Report
The manuscript describes a very useful tool for representing data derived from the analysis of complex protein coronas found around AuNPs in serum media. Comparison of various representations derived from Excel, ending in the heat-map approach, demonstrates that the authors are well aware of the complexities of corona content and analysis.
However, the manuscript does appear to be an exercise in how to use Excel to plot and draw graphs, or to represent data in a table. Heat-maps are not a novel concept and have been used in 'omics' research for some considerable time. The novelty presented here is the use of heat-maps in analysis of data concerning the protein corona. The authors have focused upon corona composition but have not commented broadly on other methods of depicting complex multi-variate data.
Overall, the manuscript does present a novel method for presenting corona composition data. The Introduction leads the reader to believe that the manuscript will describe matters in more depth with substantial new data. However, the data obtained and presented is sufficient to demonstrate the concept. I trust that the authors will continue to investigate protein coronas and their role in nanomedicine delivery.
The manuscript does contain some minor typographical errors. I recommend that the manuscript be accepted for publication once these have been attended to.
Author Response
Thank you for sending our manuscript out for review. The reviewers' feedback was constructive and informative. I believe we have addressed the concerns raised by the reviewers and brought forth by you. I request the opportunity to re-submit our manuscript to your esteemed journal once again. The most notable changes include further justification of the use of engineered nanoparticles and its surrounding surface functionalization as payload delivery vehicles in nano-enabled drug products. Furthermore, we corrected all of the typographical and formatted errors mentioned by both reviewers.
In this letter, we have provided a point-by-point reply that indicates how the authors have revised the manuscript to address the concerns raised. We have reviewed and edited the manuscript text for fine/minor spelling and/or formatting errors.
In addition to the point-by-point reply included in our revised manuscript file, we also marked all changes using the Word feature “Track Changes” within the manuscript body.
Our manuscript has been reviewed for compliance in accordance with the INSTRUCTIONS TO AUTHORS document.
Thank you for the opportunity to resubmit our manuscript.
Sincerely,
Christie M. Sayes
Responses to Reviewer 1 Critiques:
The manuscript describes a very useful tool for representing data derived from the analysis of complex protein coronas found around AuNPs in serum media. Comparison of various representations derived from Excel, ending in the heat-map approach, demonstrates that the authors are well aware of the complexities of corona content and analysis.
Response: We thank the reviewer for commenting on our paper’s content. Our intention is two-fold: first, we want to demonstrate the utility of alternative data visualization types in the field of nanomaterial characterization; and second, we aimed to highlight the differences among and between protein corona composition among a suite of similarly produced engineered nanomaterials (of same chemical core composition).
However, the manuscript does appear to be an exercise in how to use Excel to plot and draw graphs, or to represent data in a table. Heat-maps are not a novel concept and have been used in 'omics' research for some considerable time. The novelty presented here is the use of heat-maps in analysis of data concerning the protein corona. The authors have focused upon corona composition but have not commented broadly on other methods of depicting complex multi-variate data.
Response: We thank the reviewer for his/her critique of the work. We certainly agree that the Excel methods used in this manuscript are not novel or unique. Our intention was not to convey that message. Instead, we aim to highlight the array of varying interpretations that could be gained after displaying the same data in multiple forms. We have edited the text on page 2 (line 75) to now read, “The results presented in this manuscript serve two purposes. First, the data sets expand upon the available data in the protein corona field and its exploitation in pharmaceutical drug development. Second, the data visualization highlights the array of varying interpretations that could be gained after displaying the same data in multiple forms. Therefore, the visualization and presentation of results is of relevance to toxicologists and pharmacologists alike.”
Overall, the manuscript does present a novel method for presenting corona composition data. The Introduction leads the reader to believe that the manuscript will describe matters in more depth with substantial new data. However, the data obtained and presented is sufficient to demonstrate the concept. I trust that the authors will continue to investigate protein coronas and their role in nanomedicine delivery.
Response: We thank the reviewer for this insight. On page 1 (line 39) we clarified the motivation of this work by editing the following text, “This creates a new interface between the pristine particle and the surrounding biological fluid. The formation of a protein corona has been shown to change the particle’s biological effects.” This change, along with the edits made on page 2 (line 75) provides a better description of the paper’s goals.
The manuscript does contain some minor typographical errors. I recommend that the manuscript be accepted for publication once these have been attended to.
Response: The manuscript has been thoroughly reviewed and edited for typos and formatting errors.
Reviewer 2 Report
I think it is an interesting article very nicely written. However in 'Materials and Methods' section more details need to be provided so that the experiments could be reproduced in another laboratory. Hence, I recommend publication of this article after minor corrections.
Introduction:
lines 43-45: could you provide 1-2 examples to support this statement?
lines 49-53: could you be more precise? Which nanoparticles (polymeric, metallic, ...)?
lines 72-73- what kind of payloads? to which tissues (1-2 examples)
Materials and Methods
Line 90: please, correct (30 2 nm?)
Regarding the size determination by TEM, how many particles did you analyse? how was the diameter determined (average?), any information about the size distribution? The size- is it radius or diameter?
Figure 2: please, define the scale bar.
The gold nanoparticles were obtained in a liquid or powder form? did you purchase the coated particles or did you perform coating in your lab?
Please, provide more information about the incubation with serum. What was the nanoparticle concentration? how were the nanoparticles (with adsorbed proteins) separated from the serum containing non-adsorbed proteins? Please, provide more detains how were the adsorbed proteins removed from particle surface? Incubation with/in which medium? Did you verify the removal of proteins from the nanoparticle (by TEM for example)?
Author Response
Thank you for sending our manuscript out for review. The reviewers' feedback was constructive and informative. I believe we have addressed the concerns raised by the reviewers and brought forth by you. I request the opportunity to re-submit our manuscript to your esteemed journal once again. The most notable changes include further justification of the use of engineered nanoparticles and its surrounding surface functionalization as payload delivery vehicles in nano-enabled drug products. Furthermore, we corrected all of the typographical and formatted errors mentioned by both reviewers.
In this letter, we have provided a point-by-point reply that indicates how the authors have revised the manuscript to address the concerns raised. We have reviewed and edited the manuscript text for fine/minor spelling and/or formatting errors.
In addition to the point-by-point reply included in our revised manuscript file, we also marked all changes using the Word feature “Track Changes” within the manuscript body.
Our manuscript has been reviewed for compliance in accordance with the INSTRUCTIONS TO AUTHORS document.
Thank you for the opportunity to resubmit our manuscript.
Sincerely,
Christie M. Sayes
Responses to Reviewer 2 Critiques:
I think it is an interesting article very nicely written. However, in 'Materials and Methods' section more details need to be provided so that the experiments could be reproduced in another laboratory. Hence, I recommend publication of this article after minor corrections.
Response: We thank Reviewer 2 for the summary and suggestions. We have edited the “protein corona sample preparation” paragraph, per the comments noted below as well as added a new paragraph that details the characterization performed on the nanoparticles used in the study.
On page 3 (line 94-109), the paragraph now reads, “Protein Corona Sample Preparation. In this study, three different nanoparticle coatings were subjected to two different serum-types and incubated for 24 hours. Gold nanoparticles of 30 nm were purchased from NanoHybrids, Austin, Texas, USA. Particles were purchased in aqueous dispersions with one of the following coatings: polyethylene glycol (PEG), carboxylic acid (COOH), or amine (NH3). The primary particle size of each nanoparticle was measured as 30 +/- 2 nm using transmission electron microscopy (TEM). The different coatings provided a variety of charges among the coatings, with the PEG producing a neutrally charged surface, the COOH producing a negatively charged surface, and the NH3producing a positively charged surface. This variance assisted in indicating the effects of surface charge on the formation of the protein corona. The serum-types used included fetal bovine, termed serum-type 1, and equine, termed serum-type 2 (Equitech-Bio Inc., Kerrville, Texas, USA). Particles were incubated with serum at a 1:1 ratio (at 37 deg. C) for 24 hours. Coronas were separated from particles in two steps. First, the particle-corona suspension was centrifuged to remove excess proteins not adhered to particle surfaces. Second, the proteins were removed from the particle surface using a lysis buffer of 8 M Urea at 95 deg. C for 5 minutes and then centrifuged to separate the proteins from the nanoparticles.
On page 3 (line 110), the paragraph now reads, “Characterization of the Nanoparticle System. Figure 2 shows the representative transmission electron micrographs (TEM) of the gold nanoparticles before serum incubation (A) versus the gold nanoparticle after 24-hour incubation with serum-type 1 (B). Briefly, samples were deposited on 200 mesh carbon coated copper grid (EMS, Hatfield, PA, USA), left to dry at room temperature for 5 minutes, and washed with PBS twice. TEM images were collected using a JEM-1010 (JEOL Inc., Peabody, MA, USA) microscope at 60 kV with a spot size of 2. [42, 43] To determine the average corona diameter, 100 nanoparticles imaged in TEM micrographs were measured using Cell Sens version 1.13 from Olympus Corp. The hydrodynamic diameter was determined through dynamic light scattering (DLS), along with the polydispersity index (PdI), where NPPC with ST1 was determined to be highly monodisperse in particle population (as indicated by a low PdI of 0.144), while NPPC with ST2 was determined to be moderately monodisperse in particle population (as indicated by a PdI of 0.483).
Introduction:
lines 43-45: could you provide 1-2 examples to support this statement?
lines 49-53: could you be more precise? Which nanoparticles (polymeric, metallic, ...)?
lines 72-73- what kind of payloads? to which tissues (1-2 examples)
Response: We thank the reviewer for their insight. We have added more details on the following pages:
Lines 43-45: On page 2 (lines 45-51): “The cloak of proteins around particles in biological fluids has been shown to enhance or diminish the particle’s specificity to a desired location, incite or inhibit its ability to permeate into a cell, and heighten or mitigate a biochemical response. When a protein corona forms it is more likely to distribute and become available due to the nano-bio interface that forms around the surface of the nanoparticle. Furthermore, this protein cloud can also change the biodistribution of the nanoparticle system depending on the biomolecular composition formed around the surface. Consequently, if the drug is reliant on specific proteins binding to the surface of the nanoparticle, then the particle could be translocated into an unintended location if non-targeted proteins form around the particle.”
Lines 49-53: On page 2, now lines 55-59 “Many different nanoparticles, such as anatase titania, alpha-quartz silica, and gold colloids, have distinct crystal structures (as opposed to amorphous materials of similar chemical composition). Notably, crystal structure has been associated with increased cytotoxicity in mammalian cell populations [14-17]. Nanoparticle size has been implicated as a main driver in systemic distribution [15, 18-20]. Surface charge is related to cellular uptake mechanisms [21-24].”
Lines 72-73: One page 2, now lines 78-80: “Gold nanoparticles are important because they offer the opportunity to advance pharmaceutical treatments by delivering direct payloads to target tissues. For example, in Ghosh et. al. hybrid AuNP-polymers transfected vectors into kidney cells [9, 32-37].”
Materials and Methods:
line 90: please, correct (30 2 nm?)
Response: Thank you for pointing this out. The text has been corrected on page 3 (line 112).
Regarding the size determination by TEM, how many particles did you analyze? how was the diameter determined (average?), any information about the size distribution? The size- is it radius or diameter?
Response: Thank you for your response. The text has been corrected to add in more details regarding the hydrodynamic diameter and particle diameter using DLS and TEM techniques. These details are described in lines 114 to 118.
Figure 2: please, define the scale bar.
Response: Thank you for highlighting this. The scale bar has been defined as 25 nm.
The gold nanoparticles were obtained in a liquid or powder form? did you purchase the coated particles, or did you perform coating in your lab?
Response: We thank reviewer 2 for their recommendation to add more details about the nanoparticles used in manuscript. We have adding in more information in lines (96-101).
Please, provide more information about the incubation with serum. What was the nanoparticle concentration? how were the nanoparticles (with adsorbed proteins) separated from the serum containing non-adsorbed proteins? Please, provide more details how were the adsorbed proteins removed from particle surface? Incubation with/in which medium? Did you verify the removal of proteins from the nanoparticle (by TEM for example)?
Response: We acknowledge reviewer 2’s comment concerning the level of specificity in the corona formation and removal. We have added in details to lines (104-109).
