Improvement of Drug-Loading Properties of Hydroxyapatite Particles Using Triethylamine as a Capping Agent: A Novel Approach
, Haotian Lin 1, Hao Huang 1, Keke Song 2, Ke Duan 3, Tailin Guo 2,* and Jie Weng 1,2,*Round 1
Reviewer 1 Report
Dear Authors, in your interesting manuscript, the following points should be added/changed to further improve it:
- Introduction: I note that a method of synthesis has already been developed that allows to obtain nano HAP with a practically identical crystal structure as in bone (doi:10.3762/bjnano.7.153). I think it is worth adding a comment with the information that the natural bone is made of, among others, nano HAP (results of XRD studies of bone samples, doi:10.3762/bjnano.7.153). The method I mentioned allows to obtain nano HAP with an controlled size.
I would like to point out that the drying method is the dominant factor causing aggregation of nanomaterials in wet syntheses. It is worth informing the reader about this. One way to reduce aggregation of nanomaterials is to use freeze drying.
- Preparation of porous n-HAPs: Please add a table with information about the composition and preparation of samples. This will make it easier to compare the differences between the samples.
- Characterization: Note to the sentence “Dynamic light scattering (DLS) was measured by Zeta potential analyzer (Zetasizer Nano instrument, Anton Paar, Austria).”, the Zetasizer Nano is a model manufactured by the Malvern company. How the average particle size was investigated using the "Zeta potential analyzer"? There is no description of how the samples are prepared for DLS measurement. There is no information about DLS measurement statistics. There is no information about the preparation of samples for the analysis of specific surface area.
- Materials and Methods: Sedimentation experiment was not described.
- Results - Preparation of n-HAPs by TEA capping: The DLS results should be presented in the table (average size, polydispersity index). Please give me the physical meaning of this statement “The particle size distribution of TEA+ET group was the most concentrated among the three groups". Please tell me and give examples of how to compare sample concentrations using DLS results? Where are the standard deviations for the DLS measurement results? Why the authors did not show complete plots of size distributions (appear to be clippings of the results)? The authors claim that the obtained samples have a potential application in drug delivery, so why did they not perform DLS tests in a fluids used in medicine, eg phosphate-buffered saline.
- Results - Preparation of n-HAPs by TEA capping: Why did the authors not measure the zeta potential?
- Results - The crystallization properties of n-HAPs could be increased by TEA capping: Why the authors did not specify the size of the crystallites?
- Results - The crystallization properties of n-HAPs could be increased by TEA capping: I have a comment on the sentence „By comparing the XRD patterns of three groups of HAP particles, it was found that the diffraction peak of TEA+ET group was stronger, sharp and symmetrical than the two other groups.” Please explain to me what the author meant by using the word “symmetrical”? Whether the size of the NPs has an effect on the XRD results (e.g. width = sharpness)?
- Results - The crystallization properties of n-HAPs could be increased by TEA capping: Why was particle size not determined from the TEM results?
- Results - Causes of phenotype of n-HAPs: Please correct the error regarding figure numbering.
Author Response
Please refer to the attached response.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
In their manuscript, Wen and colleagues provide a modified triethylamine (TEA) capped coprecipitation template method to prepare hydroxyapatite nanoparticles (n-HAPs). Specifically, after testing different conditions, the technique better results in crystallization and dispersion when using Ca(OH)2 as a template, TEA as a capping agent and ethanol as solvent. The manuscript reports few methods to characterize the prepared n-HAPs, which are overall sufficient. No data collected on cells are reported.
I have two major concerns regarding the prepared n-HAPs:
- The first concern is the scalability of the optimized preparation process, as it involves long times (e.g. “…aging 2 days…”). Please comment and eventually include this limitation in the conclusion section.
- The second concern regards the stability of the prepared n-HAPs. Authors must compare the stability of n-HAPs prepared by their method with one of the n-HAPs prepared with the conventional method.
Other major points:
- Line 137: please report details of the drying process.
- The authors should add the polydispersity and zeta potential of n-HAPs prepared with conventional method and n-HAPs prepared with the modified method.
- Figure 5 does not report the standard deviation. Authors should add it. Also, was a statistical analysis done to determine if the release profiles’ differences were significant? Please revise.
Below some minor points:
- Figure 2: add “A” and “B”.
- Section 3.5: check if the Figures cited are the correct ones.
Author Response
Reviewer: 2
We appreciate the comments and suggestions provided by the reviewers. According to the reviewers’ comments, we have tried our best to improve our manuscript to meet with the requirements of your journal. In this revised version, changes to our manuscript were all highlighted. A Point-by-point responses to the reviewers are listed below.
. I have two major concerns regarding the prepared n-HAPs:
- The first concern is the scalability of the optimized preparation process, as it involves long times (e.g. “…aging 2 days…”). Please comment and eventually include this limitation in the conclusion section.
Response: Thanks for your comment. We mentioned the aging process in the conclusion of line 348.
- The second concern regards the stability of the prepared n-HAPs. Authors must compare the stability of n-HAPs prepared by their method with one of the n-HAPs prepared with the conventional method.
Response: We design a comparative sample stability experiment. The experiment was performed by blending 1ml of undried sample stock solution with 10ml of PBS, shaking at 37°C, and taking 1ml of samples every other day to test DLS. The experimental data is shown in Figure 1E, and the result analysis is shown in line 184 of the article.
Other major points:
- Line 137: please report details of the drying process.
Response: We have added details of the drying process.
- The authors should add the polydispersity and zeta potential of n-HAPs prepared with conventional method and n-HAPs prepared with the modified method.
Response: We have added Table 2, Table 3 and Table 4 to supplement the missing information above.
- Figure 5 does not report the standard deviation. Authors should add it. Also, was a statistical analysis done to determine if the release profiles’ differences were significant? Please revise.
Response: We have added the standard deviation to figure 5
Below some minor points:
- Figure 2: add “A” and “B”.
Response: We have added “A” and “B” in Figure 2.
- Section 3.5: check if the Figures cited are the correct ones.
Response: Thank you for your reminder. The Figures cited have been corrected, 5.5 × 10-6 should be 5.02 × 10-6 (CRC Handbook of Chemistry and Physics, 84 th ed [M], CRC, Boca Raton, 2011) in line 303 of the article.
Round 2
Reviewer 1 Report
The authors addressed most of the comments and questions I formulated during the first round of revision. The revised version of the manuscript is more complete and readable. However, I have a few more comments:
- Materials and Methods - Preparation of porous n-HA: Please add definitions of "RO"(Table 1)
- Materials and Methods – Characterization: please add information about "ultrasonic vibration" (time, power, model, manufacturer) [142].
- Results - Preparation of n-HAPs by TEA capping: Please round to whole numbers the results of "Hydrodynamic diameter" (Table 2, Table 3).
- Results - Preparation of n-HAPs by TEA capping: The Polydispersity index value is a dimensionless value (Table 2, Table 3). Please see ISO 22412: 2017 Particle size analysis - Dynamic light scattering (DLS).
- Results - Preparation of n-HAPs by TEA capping: Please complete the description (name) in Figure 1. I also suggest in the name of the figure to provide an explanation of all abbreviations used in the figure. Please make it easier for the reader to read the results.
- Results - Causes of phenotype of n-HAPs: Please correct the error regarding figure numbering.
Response: We have corrected the number error.
Reply reviewer: The authors did not remove the Figure numbering error. I note that Figure 5 is “Figure 5. Drug release profile of IBU-loaded HAP in PBS.”
Author Response
Comments and Suggestions for Authors
The authors addressed most of the comments and questions I formulated during the first round of revision. The revised version of the manuscript is more complete and readable. However, I have a few more comments:
1. Materials and Methods - Preparation of porous n-HA: Please add definitions of "RO"(Table 1)
Response: We added an explanation of abbreviations to the comments in Table 1 in line 126.
2. Materials and Methods – Characterization: please add information about "ultrasonic vibration" (time, power, model, manufacturer) [142].
Response: We added the equipment information in line 136.
3. Results - Preparation of n-HAPs by TEA capping: Please round to whole numbers the results of "Hydrodynamic diameter" (Table 2, Table 3).
Response: The contents of the table have been modified.
4. Results - Preparation of n-HAPs by TEA capping: The Polydispersity index value is a dimensionless value (Table 2, Table 3). Please see ISO 22412: 2017 Particle size analysis - Dynamic light scattering (DLS).
Response: The contents of the table have been modified.
5. Results - Preparation of n-HAPs by TEA capping: Please complete the description (name) in Figure 1. I also suggest in the name of the figure to provide an explanation of all abbreviations used in the figure. Please make it easier for the reader to read the results.
Response: We added an explanation of abbreviations to the comments in Figure 1 in line 232.
6. Results - Causes of phenotype of n-HAPs: Please correct the error regarding figure numbering.
Response: We have carefully corrected the mistake regarding figure numbering Figure 5, Figure 6A, Figure 6B, Figure 6C, Figure 6D.
Response: We have corrected the number error.
Reply reviewer: The authors did not remove the Figure numbering error. I note that Figure 5 is “Figure 5. Drug release profile of IBU-loaded HAP in PBS.”
Response: We are very sorry for our negligence. We have carefully corrected the mistake regarding figure numbering Figure 5, Figure 6A, Figure 6B, Figure 6C, Figure 6D. The text and the figure have one-to-one correspondence.
All English grammar issues have been professionally edited.
Reviewer 2 Report
The revision only partially improved the manuscript. Please refer to the comments below.
- I am still concerned about the scalability of the optimized preparation process, as it involves long times (e.g. “…aging 2 days…”). The sentence added in the conclusion section did not adequately discuss this aspect.
- I am okay with the stability experiment performed, but the methodology must be included in the materials and methods section. Figure 1 should be better explained in the caption.
- The authors added the polydispersity and zeta potential of n-HAPs prepared with conventional method and n-HAPs prepared with the modified method. However, the size is too big to define the prepared samples as nanoparticles.
- The authors added standard deviation in Figure 5, but if a statistical analysis has been performed is not mentioned yet.
Author Response
Comments and Suggestions for Authors
The revision only partially improved the manuscript. Please refer to the comments below.
- I am still concerned about the scalability of the optimized preparation process, as it involves long times (e.g. “…aging 2 days…”). The sentence added in the conclusion section did not adequately discuss this aspect.
Response: We have fully emphasized and explained the two days of aging in line 178, 276 and 380. For this method, we refer to the work of Shao et al (Shao, C.; Jin, B.; Mu, Z.; Lu, H.; Tang, R., Repair of tooth enamel by a biomimetic mineralization frontier ensuring epitaxial growth. Science Advances 2019, 5 (8), eaaw9569.). This literature has fully proved and emphasized that it takes at least 2 days for the transformation of amorphous calcium phosphate (ACP) to HAP. In order to ensure that our products are HAP, we choose a 2-day aging time.
- I am okay with the stability experiment performed, but the methodology must be included in the materials and methods section. Figure 1 should be better explained in the caption.
Response: We have added stability experiment methodology in line 138. The description of the experimental results is added between lines 201 and 205. We added an explanation of abbreviations to the comments in Figure 1 and explained the detection time.
- The authors added the polydispersity and zeta potential of n-HAPs prepared with conventional method and n-HAPs prepared with the modified method. However, the size is too big to define the prepared samples as nanoparticles.
Response: The big particle size was due to the solvent effect in the hydrated state, and the particles showing a larger hydrodynamic volume, but DLS is only an auxiliary judgment of particle size, the main results should be based on SEM and TEM results. Thanks for the reviewer’s reminder. To avoid misunderstanding, we corrected the description, such as “which leads to the agglomeration of particles in PBS”.
- The authors added standard deviation in Figure 5, but if a statistical analysis has been performed is not mentioned yet.
Response: We added “2.5 Statistical Analysis” in line 171.
All English grammar issues have been professionally edited.
Round 3
Reviewer 2 Report
I still have the following severe observations:
- The authors state they have added stability experiment methodology in line 138. I still cannot see reported how stability studies were conducted in the materials and methods section.
- The size of the prepared sample still is a significant concern. I am firmly convinced that they cannot be considered nanoparticles but must be considered microparticles. Also, I’m afraid I disagree that the DLS is only an auxiliary judgment of particle size. The article should be refocused on proposing the preparation of microparticles instead of nanoparticles.
Author Response
We appreciate the comments and suggestions provided by the reviewers. According to the reviewers’comments, we have tried our best to improve our manuscript to meet with the requirements of your journal.
I still have the following severe observations:
- The authors state they have added stability experiment methodology in line 138. I still cannot see reported how stability studies were conducted in the materials and methods section.
Response: We rewrite the part of the experimental method, and write the Sedimentation experiment and material stability experiment separately on line 153 “2.4 Sedimentation experiment and material stability experiment”.
- The size of the prepared sample still is a significant concern. I am firmly convinced that they cannot be considered nanoparticles but must be considered microparticles. Also, I’m afraid I disagree that the DLS is only an auxiliary judgment of particle size. The article should be refocused on proposing the preparation of microparticles instead of nanoparticles.
Response: Thank the judges for their comments on our work. We have modified the relevant description to increase the accuracy of the language. If you have any new opinions and suggestions, we are willing to communicate and discuss with you further. We used the description of “nanocrystals” and “micro/nano-sized agglomerates of HAP nanocrystals”. And corrected the error of nano-hydroxyapatite particles.
We checked and corrected minor errors in Table 3 and Figure 1.
Round 4
Reviewer 2 Report
I appreciate the efforts the authors made in revising the manuscript. Although they could further improve it, I can recommend the publication of the article in this form.
Author Response
Thank you so much for your comments!
