5-Fluorouracil Loaded Biogenic and Albumin Capped Gold Nanoparticles Using Bacterial Enzyme—In Vitro-In Silico Gastroplus® Simulation and Prediction
Round 1
Reviewer 1 Report
The authors present in situ biosynthesis of 5-fluorouracil (5-FU) loaded gold nanoparticles that are capped with albumin, and are intended for treatment of cancer. Through in vitro assays on MCF-7 cells lines, the novel nanoparticles loaded with 5-FU are shown to have superior anticancer efficacy than free 5-FU. The nanoformulation also had improved PK profile than the free drug, and sensitivity analysis using PBPK modeling revealed the parameters responsible for improved kinetics.
Some points that need to be addressed:
-Define error bars in caption of Figures 2, 6.
-Include scale bar in Figure 7.
-Quantitative measurements need to be provided for cell inhibition and apoptosis assays. This should be followed by the appropriate statistical tests to check for statistical significance.
-Proofreading for typographical and grammatical errors needed.
Author Response
Response letter to reviewers Date: 17-11-2020
Title: 5-Fluorouracil loaded biogenic and albumin capped gold nanoparticles using bacterial enzyme: In vitro-in silico GastroPlus® simulation and prediction
Authors: Wael A. Mahdi, Afzal Hussain*, Mohammad Ramzan
Manuscript ID: processes-999175
Reviewer 1
The authors present in situ biosynthesis of 5-fluorouracil (5-FU) loaded gold nanoparticles that are capped with albumin, and are intended for treatment of cancer. Through in vitro assays on MCF-7 cells lines, the novel nanoparticles loaded with 5-FU are shown to have superior anticancer efficacy than free 5-FU. The nanoformulation also had improved PK profile than the free drug, and sensitivity analysis using PBPK modeling revealed the parameters responsible for improved kinetics.
Some points that need to be addressed:
Comment 1: Define error bars in caption of Figures 2, 6.
Response 1: I have revised the caption of figure 2 and 6 as per suggestion. The changes have been highlighted with red text in revised manuscript. Now, the figure number is 2 and 5.
Comment 2: Include scale bar in Figure 7.
Response 2: I have revised the caption of figure 7 and replaced figure 7 (now figure 6) as per suggestion.
Comment 3: Quantitative measurements need to be provided for cell inhibition and apoptosis assays. This should be followed by the appropriate statistical tests to check for statistical significance.
Response 3: The cell inhibition was compared against control group and statistically presented in caption of the figure 6. I have revised the caption of figure 6 according for statistical significance differences. Moreover, I have replaced the figure 6 after marking “*” for statistically significant formulations producing profound cytotoxicity or cells inhibition.
Comment 4: Proofreading for typographical and grammatical errors needed.
Response 4: I have revised the whole manuscript for typo and grammar errors and the changes have been highlighted with red text.
Reviewer 2 Report
This paper reports on the preparation and characterization of 5-fluorouracil loaded gold nanoparticles, manufactured through a bacterial extract ; the study evaluated the effect of bacterial extract and nanoparticle concentration on the cytotoxic effect, and applied Gastroplus for the prediction of pharmacokinetic parameters of the formulation.
The reported results are quite promising, but a few points, in my opinion, are to be addressed:
- I was wondering why the Authors used bovine serum albumin instead of human albumin.
- The Authors have not fully explored yet the potential of gold NP for imaging and photothermal therapy. An in vivo study including these aspects would make the research more complete.
- As apparently some molecules of the bacterial lysate remain adsorbed on the NP, did the Authors consider any immunogenicity issue of the particles?
- The Authors should study also the effect of other variables on particle size, such as temperature, pH, exc., ideally with an experimental design.
- Did the Authors verify the stability of the prepared particles in terms of dimensions and zeta potential?
Besides, quite a number of mistakes have to be amended:
- Some references have been written in superscript (i.e., lines 80, 139, 204, 206, 308, 430…).
- Sometimes words are missing, i.e. in lines 84, 151; the sentence in line 556 is not clear, it needs rephrasing; lines 581-588 are very confusing, with several mistakes (“vial cells”, “100 µ/ml”) and need to be carefully rewritten; in line 543 I guess the Authors intended mM, not Mm.
- In line 117 I think the acronym FGNP is confusing, as 5-FU loaded gold nanoparticles have been designated as 5-GP later on; in line 134 I guess blank formulation corresponds to capped placebo, already described in line 126; in line 419 I guess “capped” must be replaced with “uncapped”.
- In Paragraph 2.3.2. the Authors repeat that the samples were diluted with water twice; in line 305 “Rod-shaped” is written twice.
- In line 622 the reference to the EPR effect is not pertinent, in my opinion, as the NP have been tested in vitro only, so the EPR effect still has to be confirmed in vivo.
- I can’t understand the values of EE% and DL% illustrated in Figure 2: they do not correspond to the values reported in Table 2. In any case, as the values have been reported both in Table 2 and in lines 424-425, Figs. 2 and 3 are redundant.
- In Figure 5 I would suggest the Authors to include the results of drug release study up to 48 h.
-In Supplementary File S1 I guess cell viability is represented, not cell cytotoxicity; have the Authors any idea why F15 at 10 mcg/mL concentration is more cytotoxic than at 100 mcg/mL?
Moreover, English wording and phrasing have to be carefully checked throughout the paper (i.e. lines 148, 176, 191, 201-202, 305, 308 578, 617, 711..); mL requires capital letter on the L; in line 299 Students requires capital letter; also, the confidence intervals of the mean diameters should not report the decimal figures, in my opinion, as well as in line 568.
I appreciate the efforts of the Authors to fully characterize the formulation they prepared, but due to the many critical points, I would recommend a major revision of their work.
Author Response
Response letter to reviewers Date: 17-11-2020
Title: 5-Fluorouracil loaded biogenic and albumin capped gold nanoparticles using bacterial enzyme: In vitro-in silico GastroPlus® simulation and prediction
Authors: Wael A. Mahdi, Afzal Hussain*, Mohammad Ramzan
Manuscript ID: processes-999175
Reviewer 2
This paper reports on the preparation and characterization of 5-fluorouracil loaded gold nanoparticles, manufactured through a bacterial extract; the study evaluated the effect of bacterial extract and nanoparticle concentration on the cytotoxic effect, and applied Gastroplus for the prediction of pharmacokinetic parameters of the formulation.
The reported results are quite promising, but a few points, in my opinion, are to be addressed:
Comment 1: I was wondering why the Authors used bovine serum albumin instead of human albumin.
Response 1: Fundamentally, both HSA and BSA are proteins and they slightly differ due to tryptophan content and varied optical properties. HSA contains one tryptophan in peptide whereas BSA contains one as reported by authors (Steinhardt J, Krijn J, Leidy JG. Differences between Bovine and Human Serum Albumins: Binding Isotherms, Optical Rotatory Dispersion, Viscosity, Hydrogen Ion Titration, and Fluorescence Effects. Biochemistry, 1971, 10 (22): 4005- 4015). Despite these differences, we selected BSA due to a reasonable condition in this investigation. The purpose of BSA was to utilized as a coating agent over the gold nanoparticles for increased drug loading, entrapment efficiency, biocompatibility and maximum interaction with 5-FU. It was reported that 5-FU exhibits its maximum drug loading and entrapment with polymer at pH below 6 due to maximized protonation (cited in the manuscript). It was reported that BSA exists in two distinguishable unfolded states at pH range of 4.8-5.6 as compared to HSA (single unfold state) (Maghsoudi A, Shojaosadati SA, Farahani EV. 5-Fluorouracil-Loaded BSA Nanoparticles: Formulation Optimization and In Vitro Release Study. AAPS PharmSciTech, 2008, 9 (4): 1092-1096). Therefore, it was optionally good for me so that I can achieve maximum drug loading, entrapment and coating over gold nanoparticles. Moreover, BSA based nanoparticles revealed sustained release of 5-FU as reported before (doi: 10.1208/s12249-008-9146-5). That was why BSA was selected instead of HSA. This is a preliminary study and we would optimize and explore as a comparative study using HSA and BSA for pharmacokinetics and pharmacodynamics studies in next continued study. I have changes the result and discussion part in this concern with suitable references and changes have been highlighted with red text in revised manuscript.
Comment 2: The Authors have not fully explored yet the potential of gold NP for imaging and photothermal therapy. An in vivo study including these aspects would make the research more complete.
Response 2: It is absolutely reasonable comment. As I replied in response 1, this is a part of major project and remaining ex vivo (using rat model) and in vivo studies are ongoing which would be published in future publication (very soon). Moreover, I would explore the potential of gold NP for imaging and photothermal therapy which may be a sound section of the project. In this investigation, we mainly explored preparation, variables and in silico GastroPlus based prediction and simulation followed by cytotoxicity studies.
Comment 3: As apparently some molecules of the bacterial lysate remain adsorbed on the NP, did the Authors consider any immunogenicity issue of the particles?
Response 3: In this article, we have not performed ex vivo, in vivo and immunological assessment. We are covering these studied in next part of our publication. This is an important aspect which would be studied in acute and chronic toxicity studies in mice model.
Comment 4: The Authors should study also the effect of other variables on particle size, such as temperature, pH, exc., ideally with an experimental design.
Response 4: Thank you for your valuable suggestion. In this study, we optimized based on CLS concentration and gold salt. I shall use Design of Expert for optimization of nanoparticles based these findings. Therefore, these optimization, pre-optimization and post-optimization are part of this major project. We have to cover in our upcoming publication.
Comment 5: Did the Authors verify the stability of the prepared particles in terms of dimensions and zeta potential?
Response 5: Yes. We have kept the optimized formulation for long term stability (12 months) at four temperatures (2-8 °C, 25 °C, 40°C and 60 °C) as per ICH guidelines in term of particle size (d90 and d10), zeta potential, drug content and impurities (known and unknown impurities).It has been 3 months and the samples are loaded in stability chambers under regular monitoring (physical visualization and chemical analysis).
Comment 6: Besides, quite a number of mistakes have to be amended: Some references have been written in superscript (i.e., lines 80, 139, 204, 206, 308, 430…).
Response 6: I have revised the manuscript as per your suggestions. These changes were highlighted with red text in revised manuscript. Now, the number of references are changed due to addition of new references after 39.
Comment 7: Sometimes words are missing, i.e. in lines 84, 151; the sentence in line 556 is not clear, it needs rephrasing; lines 581-588 are very confusing, with several mistakes (“vial cells”, “100 µ/ml”) and need to be carefully rewritten; in line 543 I guess the Authors intended mM, not Mm.
Response 7: Dear reviewer, these suggested confusing sentences are rewritten to avoid confusion and to make meaningful. There were typo errors and these were corrected in revised manuscript with red coloured text.
Comment 8: In line 117 I think the acronym FGNP is confusing, as 5-FU loaded gold nanoparticles have been designated as 5-GP later on; in line 134 I guess blank formulation corresponds to capped placebo, already described in line 126; in line 419 I guess “capped” must be replaced with “uncapped”.
Response 8: I have removed “FGNP” to avoid confusion from line 117. Similarly, I deleted the sentence from the line 134 due to repetition. Yes, I replaced with “uncapped in line 419”. These changes were addressed by red text in revised manuscript.
Comment 9: In Paragraph 2.3.2. the Authors repeat that the samples were diluted with water twice; in line 305 “Rod-shaped” is written twice.
Response 9: I have revised the section 2.3.2 and rewritten the sentence to remove repetition. Similarly, I revised the sentence of line 305 (rod shaped).
Comment 10: In line 622 the reference to the EPR effect is not pertinent, in my opinion, as the NP have been tested in vitro only, so the EPR effect still has to be confirmed in vivo.
Response 10: I have replaced the reference 55 for EPR explanation. In the current study, only in vitro study was conducted and rationalized the findings based on literature support for improved cytotoxicity potential of formulation.
Comment 12: I can’t understand the values of EE% and DL% illustrated in Figure 2: they do not correspond to the values reported in Table 2. In any case, as the values have been reported both in Table 2 and in lines 424-425, Figs. 2 and 3 are redundant.
Response 12: I have revised the figure 2 and replaced with revised figure 2. Moreover, Figure 3 was removed as it was already presented in figure 2. The changes were highlighted with red text in revised file.
Comment 13: In Figure 5 I would suggest the Authors to include the results of drug release study up to 48 h.
Response 13: I have replaced figure 5 and it is now figure 4 after renumbering in revised file.
Comment 14: In Supplementary File S1 I guess cell viability is represented, not cell cytotoxicity; have the Authors any idea why F15 at 10 mcg/mL concentration is more cytotoxic than at 100 mcg/mL?
Response 14: Thank you for pointing out. The label for F15 was wrongly presented. I have revised the suppl S1 as per suggestion.
Comment 15: Moreover, English wording and phrasing have to be carefully checked throughout the paper (i.e. lines 148, 176, 191, 201-202, 305, 308 578, 617, 711..); mL requires capital letter on the L; in line 299 Students requires capital letter; also, the confidence intervals of the mean diameters should not report the decimal figures, in my opinion, as well as in line 568.
Response 15: Corrected as per suggestion.
I appreciate the efforts of the Authors to fully characterize the formulation they prepared, but due to the many critical points, I would recommend a major revision of their work.
…………………….Thank you for valuable comments and suggestions…………………………
Round 2
Reviewer 2 Report
The revised version of the manuscript "5-Fluorouracil loaded biogenic and albumin capped gold nanoparticles using bacterial enzyme: In vitro-in silico GastroPlus® simulation and prediction" has been extensively improved, and almost all the critical points have been addressed.
As stated by the Authors, a few of the missing data, related to in vivo application of this technology, will be presented in a following paper, which I am looking forward to reading.
Anyway, I suggest the Authors to re-read the paper carefully, as English wording and phrasing are still perfectible.
Author Response
Response letter to reviewers Date: 25-11-2020
Manuscript ID: processes-999175
Minor revision R2
Title: 5-Fluorouracil loaded biogenic and albumin capped gold nanoparticles using bacterial enzyme: In vitro-in silico GastroPlus® simulation and prediction
Authors: Wael A. Mahdi*, Afzal Hussain*, Mohd. Ramzan
Comments and Suggestions for Authors (Reviewer 2)
Comment 1: The revised version of the manuscript "5-Fluorouracil loaded biogenic and albumin capped gold nanoparticles using bacterial enzyme: In vitro-in silico GastroPlus® simulation and prediction" has been extensively improved, and almost all the critical points have been addressed.
As stated by the Authors, a few of the missing data, related to in vivo application of this technology, will be presented in a following paper, which I am looking forward to reading.
Anyway, I suggest the Authors to re-read the paper carefully, as English wording and phrasing are still perfectible.
Response 1: Dear reviewer, I would like to give a brief introduction of GastroPlus software used in this study. The software uses in vitro drug release data and predicted in vivo performance in human or other selected animal model. I have used in vitro release data as input parameter and physicochemical properties of the drug in “compound tab”. NO any in vivo data was used to simulate the in vitro release. Notably, IVIVC can be simulated based in vitro release data and in vivo kinetics (plasma drug concentration) of the drug. However, I had not used in vivo data in this study. That is why I have not included IVIVC simulation profile which needs in vivo data from selected animal model. Secondly, I predicted in vivo performance of the formulation as compared to free drug solution based on in vitro findings. In section, 3.3.5, I have revised the sentence as “The drug release kinetics is important for assessing behaviour of drug release from the tailored NPs as it can be easy to predict in vivo absorption pattern”.
Similarly, section 3.3.9, GastroPlusTM predicted the effect of various factor (related to compound and formulation) on PK parameters. Finally, there was a comparative regional absorption of both formulation from different segments of human GIT as shown in figure 11. All these findings were predicted based on in vitro data obtained in this study and values related to drug published in literature (table 3).
Finally, I have revised the whole manuscript for typo, punctuation, phrases, wording, and grammar mistakes. The changes have been highlighted in revised manuscript file.
………………………………..Thank you……………………………………………….
