Comparison of Analytical Methods for the Detection of Residual Crosslinker in Hyaluronic Acid Hydrogel Films

Round 1

Reviewer 1 Report

The manuscript entitled, ‘Comparison of analytical methods for the detection of residual crosslinker in hyaluronic acid hydrogel films’ reported residual crosslinked determination in hydrogel fabrication.. I am mentioning some points which should be justified before publication;

1.      The abstract is too big. Better to brief with some data.

2.      Tetra acrylic monomer is quite susceptible to form dendrimer. How that was justified?

3.      In equation 2, what the ‘*’ signifies?

4.      The conclusion is too short. The summary of the work should rewritten with the major findings.

5.      Some articles related to hydrogel should have importance here for your reference: https://0-doi-org.brum.beds.ac.uk/10.1021/acsabm.2c00664; https://0-doi-org.brum.beds.ac.uk/10.1016/j.bioactmat.2022.10.028; https://0-doi-org.brum.beds.ac.uk/10.1016/j.cej.2022.138494.  

Author Response

Thank you to the Reviewer for their comments & suggestions.

The manuscript entitled, ‘Comparison of analytical methods for the detection of residual crosslinker in hyaluronic acid hydrogel films’ reported residual crosslinked determination in hydrogel fabrication.. I am mentioning some points which should be justified before publication;

1. The abstract is too big. Better to brief with some data.

Please see the revised abstract which now includes data from the results.

2. Tetra acrylic monomer is quite susceptible to form dendrimer. How that was justified?

It is likely indeed that part of the tetra acrylic monomer could form dendrimers instead of enabling the crosslinking of the polymer. The molecular weight of the dendrimers would determine their safety. If these PT dendrimers are lipophilic (similarly to PT), they would be extracted during the process we used for the extraction of the PT monomer. From the GC spectra (Figure 2) it can be deduced that the residual PT was in monomer form, considering the predominant PT monomer peak (peak with highest signal intensity) and the absence of high-mass peaks with high signal intensity.

It is however more likely these dendrimers would have a hydrophilic surface consisting of hydroxyl groups; in that case the dendrimers would be removed together with the residual uncrosslinked HA when the crosslinked films are rinsed/washed with water.    

3. In equation 2, what the ‘*’ signifies?

It signifies the mathematical multiplication sign; it has now been replaced with the correct sign “×”

4. The conclusion is too short. The summary of the work should rewritten with the major findings.

Please see the re-written Conclusion on the revised manuscript which now contains clarifications of the key findings. 

5. Some articles related to hydrogel should have importance here for your reference: https://0-doi-org.brum.beds.ac.uk/10.1021/acsabm.2c00664; https://0-doi-org.brum.beds.ac.uk/10.1016/j.bioactmat.2022.10.028; https://0-doi-org.brum.beds.ac.uk/10.1016/j.cej.2022.138494.  

These references have now been added to the revised manuscript (References 19, 20, 21)

Reviewer 2 Report

Comments and suggestion for the manuscript Comparison of analytical methods for the detection of residual crosslinker in hyaluronic acid hydrogel films

Introduction: should be more focused on the aim of the study which is more analytical. 

line 131-133 : which was the viscosity of the HA solution? which type of stirring was used? why the bubbles were removed after the formation of the hydrogel? it would not be esier to remove from the starting solution before the mixing with the crosslinking agent? As reported, it seems the stirring was low intensity, meaning the creation of the bubbles should be minimal.

PT is immiscible in water, how it was supposed to form homogeneous solution with HA?

line 133-136: the author report that the hydrogel was puored in petri dish and let dry for 4-5 days and then the crosslinking reaction was performed in oven to obtain xerogels. It looks a bit confused, could you please refrease it.

line 266-268 : how the samples were washed? if with distilled water, could you specify the washing method.

line 271-274 : the authors report the effect of the freeze-drying in imporving the PT extraction. Well, it is quite obvious that if the extraction is performed in hydrated hydrogel, the ability of chloroform to diffuse, dissolve PT and favor the release is definitely lower than in dried hydrogel. This is not a question of porosity. However,  was the swelling  of the freeze dried hydrogel in chloroform evaluated?

Figure 3A : what do you mean with blank hydrogel without swelling and freeze drying? is it not swollen and not dried?

In several part the authors defined swollen hydrogels as washed. What is the difference between having an hydrated hydrogels ( swollen) and washed? Could you be more precise?

Figure 3 : the same scale should be used in the SEM micrographs

3.3 NMR analysis: provide a better discussion and calculation

Author Response

Reviewer 2

Comments and suggestion for the manuscript Comparison of analytical methods for the detection of residual crosslinker in hyaluronic acid hydrogel films

Introduction: should be more focused on the aim of the study which is more analytical.

In the Introduction we attempted to set the context of our study and the importance of analytical testing for these hydrogels. In the last paragraph of the Introduction we focus on the analytical techniques that have been used in literature and the reasons for choosing GC and NMR analysis.

line 131-133 : which was the viscosity of the HA solution? which type of stirring was used? why the bubbles were removed after the formation of the hydrogel? it would not be esier to remove from the starting solution before the mixing with the crosslinking agent? As reported, it seems the stirring was low intensity, meaning the creation of the bubbles should be minimal.

Please see added comment on the revised manuscript. In addition to this; we didn’t measure the viscosity of the HA solution but it was low enough to enable mixing of the HA using a stirrer (please see the type of stirrer on our manuscript) Indeed, we could have avoided the formation of bubbles at the initial stirring of the HA in distilled water i.e. before the addition of the crosslinker.

PT is immiscible in water, how it was supposed to form homogeneous solution with HA?

Please see added comment on the revised manuscript. In addition to this; the aim is not to form a homogeneous solution, but to enable the crosslinking reaction between PT monomers with the linear HA polymer. The first step is the generation of PT nucleophiles via its carbonyl group, under alkaline conditions and high temperature. The PT nucleophiles then abstract hydrogen atoms from the hydroxyl groups of HA, to generate HA radicals, which then bind with the PT radicals to form crosslinks (C-O-C).

We have explained the crosslinking reaction between HA and PT in our previous papers.

line 133-136: the author report that the hydrogel was puored in petri dish and let dry for 4-5 days and then the crosslinking reaction was performed in oven to obtain xerogels. It looks a bit confused, could you please refrease it.

We have now rephrased these sentences.

line 266-268 : how the samples were washed? if with distilled water, could you specify the washing method.

This clarification has now been added, please see lines 278-279 on the revised manuscript.

line 271-274 : the authors report the effect of the freeze-drying in imporving the PT extraction. Well, it is quite obvious that if the extraction is performed in hydrated hydrogel, the ability of chloroform to diffuse, dissolve PT and favor the release is definitely lower than in dried hydrogel. This is not a question of porosity. However, was the swelling of the freeze dried hydrogel in chloroform evaluated?

Yes, this was indeed one of the first parameters we evaluated in order to explain the mechanism of PT extraction; we found that the hydrogels do not swell at all in chloroform. Please find this clarification on the revised manuscript. 

Figure 3A : what do you mean with blank hydrogel without swelling and freeze drying? is it not swollen and not dried?

Please see the detailed revised caption on Figure 3. The “Blank” hydrogels were non-treated compared to hydrogels B & C; ie. They were not swollen in water or freeze-dried, ie they were xerogels.  

In several part the authors defined swollen hydrogels as washed. What is the difference between having an hydrated hydrogels ( swollen) and washed? Could you be more precise?

The term “washed” is used interchangeably to the term “swollen”. The term “washed” also hints to the removal of residual uncrosslinked polymer.

Figure 3 : the same scale should be used in the SEM micrographs

Please see the revised caption to Figure 3 detailing the scales.

3.3 NMR analysis: provide a better discussion and calculation

Please see revised section 3.3.

Reviewer 3 Report

The Authors presented an article entitled: “Comparison of analytical methods for the detection of residual crosslinker in hyaluronic acid hydrogel films.”

The manuscript is interesting, and it is pointed out the problem of skin sensitization caused by monomers residues derived after incomplete crosslinking when performing polymerizing reactions; furthermore, the safety of the ingredients used for formulating cosmetic products in crucial to assess before marketing, and the quality control is needed, as stabilized by the cosmetic regulation.

It appears quite organized.

However, the Authors should follow the suggestions listed below before the publication:

In the “Introduction”, please, check the English form of the following sentence, reported at line 45: “[..] it’s a science is focused and based [..]”.

Again, in the “Introduction”, as a suggestion, please, note that the “dermal/intra-dermal filler injections” referred at line 53, cannot be considered as cosmetic treatments.

Again, in the “Introduction”, in my opinion, it should be better to immediately report the definition of the expression “Type 1” when referring to the sentence at lines 57-58 (“Therefore, cross-linking of the HA polymer in hydrogel films (Type 1) [..]”), in order to clarify the expressed concepts.

In the “Materials and methods” section, sub-section “2.3.3. ¹HNMR Spectroscopy”, please, it should be better to explain the abbreviation TMS reported at line 165.

Again, in the “Materials and methods” section, sub-section “2.3.4 Gas Chromatography analysis”, please, check and correct the error typo “spitless”, reported at line 184.

In the “Results & Discussion” section, sub-section “3.1 PT description”, please, it should be better to explain the abbreviation “Peta” reported at line 242.

In the “Results & Discussion” section, sub-section “3.1 PT description”, please, check and correct the error typo “identefied”, reported at line 250.

In figure 2, panel B, please, it should be better to report the parameters referred to the x and y axes.

In figure 3, in my opinion, it should be better to report the scale bars directly on the collected images.

In the “Results & Discussion” section, sub-section “3.2 SEM scanning electron microscope”, when referring to figure 3 description, please, it should be better to specify that the panel A is related to blank untreated samples, differently from B and C panels.

Figure 4 appears poorly resolved; thus, It should be better to replace it.

In the “Results & Discussion” section, sub-section 3.2.4 Sensitivity, please, improve the following sentence reported at lines 475-477: “Table (5) illustrated the PT concentration representing to LOD was about 0.000032%m/m, while the 0.00013 %m/m was indicative to the LOQ value based on this finding, as the lowest LOD, LOQ values concluded the method was highly sensitive”, in order to better clarify the expressed concepts.

Please, check in the manuscript that the reference 36 is missing.

Author Response

Reviewer 3

The Authors presented an article entitled: “Comparison of analytical methods for the detection of residual crosslinker in hyaluronic acid hydrogel films.”

The manuscript is interesting, and it is pointed out the problem of skin sensitization caused by monomers residues derived after incomplete crosslinking when performing polymerizing reactions; furthermore, the safety of the ingredients used for formulating cosmetic products in crucial to assess before marketing, and the quality control is needed, as stabilized by the cosmetic regulation.

It appears quite organized.

However, the Authors should follow the suggestions listed below before the publication:

In the “Introduction”, please, check the English form of the following sentence, reported at line 45: “[..] it’s a science is focused and based [..]”.

Thank you, this has now been corrected on the revised manuscript.

Again, in the “Introduction”, as a suggestion, please, note that the “dermal/intra-dermal filler injections” referred at line 53, cannot be considered as cosmetic treatments.

Thank you. Please see the revised manuscript where the filler injections are now specified with the term “aesthetic skin rejuvenation treatments".

Again, in the “Introduction”, in my opinion, it should be better to immediately report the definition of the expression “Type 1” when referring to the sentence at lines 57-58 (“Therefore, cross-linking of the HA polymer in hydrogel films (Type 1) [..]”), in order to clarify the expressed concepts.

Done

In the “Materials and methods” section, sub-section “2.3.3. ¹HNMR Spectroscopy”, please, it should be better to explain the abbreviation TMS reported at line 165.

Done

Again, in the “Materials and methods” section, sub-section “2.3.4 Gas Chromatography analysis”, please, check and correct the error typo “spitless”, reported at line 184.

Done

In the “Results & Discussion” section, sub-section “3.1 PT description”, please, it should be better to explain the abbreviation “Peta” reported at line 242.

Done. It is now clarified on the revised manuscript that this is the tri-acrylate.

In the “Results & Discussion” section, sub-section “3.1 PT description”, please, check and correct the error typo “identefied”, reported at line 250.

Done

In figure 2, panel B, please, it should be better to report the parameters referred to the x and y axes.

Please see revised manuscript (Figure 2) with added explanations of the x & y axes.

In figure 3, in my opinion, it should be better to report the scale bars directly on the collected images.

Done, please see revised caption for Figure 3.

In the “Results & Discussion” section, sub-section “3.2 SEM scanning electron microscope”, when referring to figure 3 description, please, it should be better to specify that the panel A is related to blank untreated samples, differently from B and C panels.

Please see the revised caption for Figure 3 which provides better clarity.

Figure 4 appears poorly resolved; thus, It should be better to replace it.

Please see the revised Figure 4 and accompanying comment to it. This is not a chromatogram, and the resolution is therefore not relevant. The figure shows the characteristic 1H multiplets arising from the PT molecule.

In the “Results & Discussion” section, sub-section 3.2.4 Sensitivity, please, improve the following sentence reported at lines 475-477: “Table (5) illustrated the PT concentration representing to LOD was about 0.000032%m/m, while the 0.00013 %m/m was indicative to the LOQ value based on this finding, as the lowest LOD, LOQ values concluded the method was highly sensitive”, in order to better clarify the expressed concepts.

Thank you, the sentence has now been improved.

Please, check in the manuscript that the reference 36 is missing.

The missing reference has now been added. 

Round 2

Reviewer 1 Report

This article can be published in its present form. 

Reviewer 3 Report

The Authors have mostly complied with the requested changes into the manuscript for improving it before the publication.