Studies on Novel Methods for Formulating Novel Cross-Linked Hydrogel Films of Hyaluronic Acid

Round 1

Reviewer 1 Report

The authors have presented in this manuscript a synthesis of hydrogel films based on cross-linking reaction between HA and pentaerythritol tetra-acrylate. They applied different crosslinking methods of the film formulation.

Comments:

Introduction is too long

The fig 1 and the points of reaction process can be moved to Results and discussion

Part: Result and discussions

Discussion of advantages and disadvantages of novel cross -linked film must be included.

Pentaerythritol tetra-acrylate is considered for Body Contact or reactivity as  moderate hazardous substance according to O OSHA 29 CFR 1910.1200. However it must be taken under consideration that although Skin contact is not thought to have harmful health effects, however the material may still produce health damage following entry through wounds, lesions or abrasions. Therefore the authors of this work should consider the possible toxic effect of their novel hydrogel  films.

Application of strong base as NaOH raises some doubts whether such a film (with high pH) will not irritate the skin?!

Are the authors sure that such films are not photosensitizing, under the influence of UV, as far as it is known, free radicals are formed, which can cause harmful effects on health ?!

Information about the possible harmful effects of such hydrogel films and the references have to be included.

Figures: the spectra in the drawings (Fig. 6 7, 8, 10, 11, 12, 13, 16, 17) should be in bold and the inscriptions enlarged and clear. They are unreadable in the present forms.

In Conclusion the authors should mention also future toxicological studies of this novel cross-linked HA hydrogel films. Such studies are indispensable  before the films will be used for cosmetics.

Language

I recommend that the authors replace some words, e.g. disadvantageous instead of detrimental.

Major revision is necessary.

Author Response

The authors have presented in this manuscript a synthesis of hydrogel films based on cross-linking reaction between HA and pentaerythritol tetra-acrylate. They applied different crosslinking methods of the film formulation.

Comments:

Introduction is too long

We believe that the Introduction is concise, informative and up-to-date, setting the context of our work and explaining the scope of our study.

 

The fig 1 and the points of reaction process can be moved to Results and discussion

Please see Figures 3 & 4 showing the reaction process.

 

Part: Result and discussions

Discussion of advantages and disadvantages of novel cross -linked film must be included.

A paragraph has been added at the end of the “Results and Discussion”, explaining the advantages and potential limitations that need to be further investigated.  

 

Pentaerythritol tetra-acrylate is considered for Body Contact or reactivity as  moderate hazardous substance according to O OSHA 29 CFR 1910.1200. However it must be taken under consideration that although Skin contact is not thought to have harmful health effects, however the material may still produce health damage following entry through wounds, lesions or abrasions. Therefore the authors of this work should consider the possible toxic effect of their novel hydrogel  films.

Pentaerythritol tetra-acrylate (PT) may have harmful effects if it remains uncrosslinked because due to its low MW it would be likely to diffuse via the skin. During the cross-linking reaction PT is incorporated in the hydrogel’s structure and therefore is no longer harmful. It is therefore important to conduct residual content analysis for PT in the optimised hydrogel films. This is our next task and we will carry it out using the method we have previously developed for the quantification of residual PT in PEO hydrogel films (See Reference 17 on the revised paper)  

 

Application of strong base as NaOH raises some doubts whether such a film (with high pH) will not irritate the skin?!

The basic mixture (pH=11) was used at the initial stage of the patch’s manufacture. The pH of the swollen cross-linked films is no longer basic as the xerogels are swollen in distilled water.

 

Are the authors sure that such films are not photosensitizing, under the influence of UV, as far as it is known, free radicals are formed, which can cause harmful effects on health ?!

This could be a possibility if unreacted PT monomer was present in the cross-linked films. As we explain above, we will be conducting residual content analysis for PT and will adjust the PT amount in the formulation if necessary, so as to ensure that all PT gets used during the cross-linking reaction.  

 

Information about the possible harmful effects of such hydrogel films and the references have to be included.

A reference [18] has been added to the revised manuscript on the toxicity studies of PT monomer and how it favourably compares to other similar monomers.

Figures: the spectra in the drawings (Fig. 6 7, 8, 10, 11, 12, 13, 16, 17) should be in bold and the inscriptions enlarged and clear. They are unreadable in the present forms.

Done

 

In Conclusion the authors should mention also future toxicological studies of this novel cross-linked HA hydrogel films. Such studies are indispensable  before the films will be used for cosmetics.

The toxicological safety evaluation of the films has been added as a future study in the Conclusion. This will be done via the determination of the residual PT content in the cross-linked films.

 

Language

I recommend that the authors replace some words, e.g. disadvantageous instead of detrimental.

Done

 

Major revision is necessary.

All additions to the manuscript are shown in blue colour. The language throughout the manuscript has been revised extensively.

 

Reviewer 2 Report

This paper developed a novel method for the formulation optimization of hydrogel films of hyaluronic acid. The overall work can aid to development relevant formulations. In addition, the result and discussion seem well structured and might provide a significant information. However, the optimization method that might be main topic in this manuscript was not presented, the OFAT presented in this manuscript is not an optimization method. Therefore, the reviewer come to the decision that the advances presented in this manuscript were not sufficient enough to have immediate impact to the scope of this journal. The authors should present DoE with statistical relationships between response factors and control factors to optimize formulation or process method. 

Additionally, all figures should be improved to present better resolution.

Author Response

This paper developed a novel method for the formulation optimization of hydrogel films of hyaluronic acid. The overall work can aid to development relevant formulations. In addition, the result and discussion seem well structured and might provide a significant information. However, the optimization method that might be main topic in this manuscript was not presented, the OFAT presented in this manuscript is not an optimization method. Therefore, the reviewer come to the decision that the advances presented in this manuscript were not sufficient enough to have immediate impact to the scope of this journal. The authors should present DoE with statistical relationships between response factors and control factors to optimize formulation or process method. 

The aim of our work was to explore the synthesis of novel cross-linked HA-PT hydrogel films using PT as the cross-linking agent and to deduce an optimum formulation and reaction process by evaluating the effects of the following independent variables on cross-linking:

pH of the formulation mixture before cross-linking, PT concentration (10, 15, 20, 30 % w/w) in the formulation, type of cross-linking method (UV-irradiation, microwaving and oven-assisted crosslinking), and exposure time of the film to the cross-linking method

The efficacy of the cross-linking reaction was evaluated using swelling studies (dependent parameters) and Fourier transform infrared (FTIR) spectroscopy for the characterization of the xerogel HA-PT film formulations.

An OFAT (one factor at a time) approach was applied with regards to the aforementioned independent variables and statistical analysis was used to evaluate significant differences among the measured dependent parameters.

However, our intention was not to follow a DoE approach and therefore we have removed the word “optimisation” from the title of our manuscript.

 

Additionally, all figures should be improved to present better resolution.

The resolution of all Figures has been improved.

Reviewer 3 Report

The paper deals with an interesting subject which is the analysis and optimization of crosslinking method for the formulation of innovative hyaluronic acid (HA) hydrogel films using pentaerythritol tetra-acrylate as the crosslinking agent. In particular the authors studied the formulation over a range of pH values and using different crosslinking methods (UV radiation, microwaving, oven heating).

Unfortunately, in my opinion the paper cannot be accepted in the present form, the article should be significantly improved.

The introduction should be rewritten, including more updated references on the topic. HA has been widely applied in the biomedical field, specifically in the cosmetics. The authors should better highlight the novelty of their functionalization and what are the advantages of this chemical modification compared to the already reported functionalization strategies. The results are not well presented by the authors. They should reduce the number of images as well as the number of tables. I suggest to include some of the data into the supplementary information. Did the authors evaluate the stability of the HA-PT hydrogel under basic conditions? Did they evaluate the degradation profile and the effect of pH=11 on Mw of HA? Taking into consideration the cosmetics application, the authors should check the biocompatibity of HA-PT. The authors should improve the hydrogels’ nomenclature. Actually, it’s really difficult to identify the samples. Furthermore, some samples (i.e. HA2 at pH=2 and pH=11, are missing). The authors should check all the samples.

 

Minor revisions

In the abstract, the following sentence: “FTIR data suggests formation of ester bond between the carbonyl of the HA and hydroxyl group of the PT acrylate group.” Should be replaced by: “FTIR data suggest formation of ester bond between the carbonyl of the HA and hydroxyl group of the PT acrylate group.”. In paragraph 3.3.2, the following sentence: “This indicate that microwave cross-linking method was better than UV-method,..” should be replaced by: “This indicates that microwave cross-linking method was better than UV-method,…”.

Author Response

The paper deals with an interesting subject which is the analysis and optimization of crosslinking method for the formulation of innovative hyaluronic acid (HA) hydrogel films using pentaerythritol tetra-acrylate as the crosslinking agent. In particular the authors studied the formulation over a range of pH values and using different crosslinking methods (UV radiation, microwaving, oven heating).

Unfortunately, in my opinion the paper cannot be accepted in the present form, the article should be significantly improved.

The introduction should be rewritten, including more updated references on the topic. HA has been widely applied in the biomedical field, specifically in the cosmetics.

We have tried to keep our Introduction concise and up-to-date. Please see our References [2], [3] and [8] regarding biomedical applications. If the Reviewer could please specify any other references, we would be happy to include them in the Introduction.

 

The authors should better highlight the novelty of their functionalization and what are the advantages of this chemical modification compared to the already reported functionalization strategies.

We have added the following explanation at the end of the Introduction:

“In contrast to other reported methods, our approach is intended to avoid additional functionalisation of the HA prior or during the cross-linking reaction, thus avoiding the use of organic solvents and multiple reaction steps. As such, our overall aim is to provide a simple and robust one-step crosslinking method for high MW HA.”   

 

The results are not well presented by the authors. They should reduce the number of images as well as the number of tables. I suggest to include some of the data into the supplementary information.

Instead of reducing the number of Tables and Figures, we have re-written most of the Results & Discussions section to remove descriptive narratives of the data presented on the Tables and Figures. There is now a more coherent explanation and discussion of the results.

 

Did the authors evaluate the stability of the HA-PT hydrogel under basic conditions?

The crosslinked HA-PT hydrogels are not exposed to basic conditions, the swelling is carried out in distilled water.

 

Did they evaluate the degradation profile and the effect of pH=11 on Mw of HA?

pH=11 was found to be optimum during the initial formulation stages when mixing HA with PT. During this formulation stage, HA was exposed to basic conditions for about 7 days until complete drying of the casted uncrosslinked film.

We haven’t evaluated the degradation profile of HA throughout the above process but we can conduct this as a future study. However, the FTIR results and swelling behaviour of the cross-linked films, indicate the presence of high MW HA that was able to be cross-linked in the presence of PT to form 3D films that can reversibly turn from xerogel to hydrogel.

 

Taking into consideration the cosmetics application, the authors should check the biocompatibity of HA-PT.

The biocompatibility evaluation will involve future studies to quantify the residual PT monomer content in the hydrogel films considering that PT monomer is a potential skin sensitiser (see the additions we have made to the discussion and conclusion).

Based on the results of the residual content analysis, we will adjust the PT amount in the formulation if necessary, so as to ensure that all PT gets used during the cross-linking reaction.

 

The authors should improve the hydrogels’ nomenclature. Actually, it’s really difficult to identify the samples.

We have added a definition of the numbers used for naming each of our hydrogel formulations. Eg. HA2-PT20 means that HA was dissolved in a concentration of 2%w/v in the initial mixture and that the concentration of PT in the final product (xerogel) was 20%w/w.

 

Furthermore, some samples (i.e. HA2 at pH=2 and pH=11, are missing). The authors should check all the samples.

The HA2 samples, ie HA without PT, were formulated at both pH=2 and pH=11 to examine the possibility of auto-crosslinking of HA. However these films dissolved during the swelling testing indicating that the crosslinking agent is essential for the crosslinking of HA. We have now added these films on Tables 1 and 2.

 

Minor revisions

In the abstract, the following sentence: “FTIR data suggests formation of ester bond between the carbonyl of the HA and hydroxyl group of the PT acrylate group.” Should be replaced by: “FTIR data suggest formation of ester bond between the carbonyl of the HA and hydroxyl group of the PT acrylate group.”. In paragraph 3.3.2, the following sentence: “This indicate that microwave cross-linking method was better than UV-method,..” should be replaced by: “This indicates that microwave cross-linking method was better than UV-method,…”. 

Done. In addition we have improved the language throughout the manuscript. 

 

Round 2

Reviewer 1 Report

The manuscript in the present form can be published.

Reviewer 2 Report

The revision was sufficient to address my concern. It is recommended to be published.

Reviewer 3 Report

The paper can be accepted in the present form. The authors improved the quality of the paper.