Sol-Gel Synthesis of Caffeic Acid Entrapped in Silica/Polyethylene Glycol Based Organic-Inorganic Hybrids: Drug Delivery and Biological Properties

Round 1

Reviewer 1 Report

The reviewed manuscript presents results on studying properties of organic-inorganic system composed of silica, polyethylene glycol, and caffeic acid. The performed experiments are well described, most of the analyses were performed correctly and were presented in an approachable way. However, I find lack of some important results to confirm the statements and conclusions as well as lack of discussion on the investigated system in context of other research. Therefore, my suggestion to Editors would be to reject the manuscript and to the Authors to reconsider application of the material and redesign samples’ characterization to get full desired information about the material. The detailed remarks are as follow:

1. The system cannot be considered as novel material because similar materials have been already described in literature, including papers published by the Authors. Some of the presented results were also reported (for example in ref. 32)

2. The application of the material is not clear – would it be drug delivery or regenerative medicine, or something else? In any case, the cytotoxicity tests would be obligatory. The sol-gel material is not annealed thus remaining reagents or solvents might have toxic effect on cells. 

3. In the studies, there is missing a reference material, i.e. silica sample (S) and silica/PEG (SP). Would they have, for example, antibacterial effect?

4. Can this system be considered as “bioactive”? Is the material undergoing transformation in SBF or it is just the formation of HA on its surface?

5. Were the samples washed after tests in SBF? If not (as presented in the manuscript), they should contain also salts from the SBF on the surface and the EDS analysis would not show correctly the samples’ composition. What is the Ca:P ratio?

6. It would be needed to present tests confirming activity of oxidized caffeic acid in the investigated system (e.g. antioxidant properties).

7. What is the meaning of “Abs(%)” shown in figure 2? Why not Absorbance? Why “zero point” starts at (0, 18.15) not (0,0)? The calibration curve together wit absorption spectra could be presented in the supplementary file.

Other remarks:

8. “The interaction of the phases inside the materials was also revealed by FT-IR analysis” – I do not find any description of interaction among system components. But indeed, it would be good to show interaction among the components. Is there any bonding or interaction among them? 

9. Line 199: “Indeed, the two sharp absorption peaks (…) changed into a main broad peak (…) probably due to the increase of -OH and H-bonds in the oxidized structure.” It is not clear why more OH groups should be present in the oxidized C.

10. In the “M(OR)X” notation, X is not explained (or correctly written).

11. Full names of bacteria species should be used when firstly used in the text (Staphylococcus aureus instead of S. aureus etc.).

12. wt%, wt.% or wt. % are used in the text – please unify to the correct form.

13. Capital letters are sometimes used without justification (e.g. “Polyethylene Glycol, Caffeic Acid, Sol-Gel, and Silica).

14. “SBF (…) is a solution whose ion concentration is equal to human blood plasma” – it’s not fully correct because one may think it is equal to human blood plasma. It would be better to rephrase the sentence.

15. “Figure 5B [should probably be 4B] pointed out that at a fixed amount of silica and A (20 wt%), an increase in polyethylene glycol corresponds to an increase in the C-H twisting and C-O-C ether stretching [38,46] detected at 1240 and 1100 cm-1” – to be honest, these bands are not visible in the spectra of hybrids.

16. Figure 5 requires comparison of FTIR spectra with sample before SBF test (caption should be more precise too).

17. In Figure 6 it would be good to show sample before test (as in the case of figure 5, caption should be more precise).

Author Response

Reviewer #1:The reviewed manuscript presents results on studying properties of organic-inorganic system composed of silica, polyethylene glycol, and caffeic acid. The performed experiments are well described, most of the analyses were performed correctly and were presented in an approachable way. However, I find lack of some important results to confirm the statements and conclusions as well as lack of discussion on the investigated system in context of other research. Therefore, my suggestion to Editors would be to reject the manuscript and to the Authors to reconsider application of the material and redesign samples’ characterization to get full desired information about the material. The detailed remarks are as follow:

1. The system cannot be considered as novel material because similar materials have been already described in literature, including papers published by the Authors. Some of the presented results were also reported (for example in ref. 32)

The author thanks the reviewer for the comment, but they slightly disagree. The hybrids material presented in this paper are different from those reported in literature. Ref. 32 (now 34)  is related to our first study about the silica/caffeic acid hybrids synthesized via sol-gel route. Different from that paper, the systems are made up of 3 components: silica (as inorganic phase) and Polyethylene glycol (PEG 400) and caffeic acid (as two organic components). So, the systems here presented are new.

2. The application of the material is not clear – would it be drug delivery or regenerative medicine, or something else? In any case, the cytotoxicity tests would be obligatory. The sol-gel material is not annealed thus remaining reagents or solvents might have toxic effect on cells. 

The study presented in this paper is preliminary. This is the reason why no assays on eucaryiothic cells (fibroblasts, osteoblasts, etc.) are reported.

3. In the studies, there is missing a reference material, i.e. silica sample (S) and silica/PEG (SP). Would they have, for example, antibacterial effect?

The authors did not report about S and SP systems because they were extensively studied and already published by us in other papers [9, 10]. The antimicrobial effect of such systems are reported in [20].

4. Can this system be considered as “bioactive”? Is the material undergoing transformation in SBF or it is just the formation of HA on its surface?

The bone-bonding ability of a material is often evaluated by examining the ability of apatite to form on its surface in a simulated body fluid (SBF) with ion concentrations nearly equal to those of human blood plasma, as reported by Kokubo and Takadama in [37]. The authors stresses that the examination of apatite formation on a material in SBF is useful for predicting the in vivo bone bioactivity of a material, and the number of animals used in and the duration of animal experiments can be reduced remarkably by using this method.

5. Were the samples washed after tests in SBF? If not (as presented in the manuscript), they should contain also salts from the SBF on the surface and the EDS analysis would not show correctly the samples’ composition. What is the Ca:P ratio?

The authors thanks the comment of the review and improved the manuscript. Before sample drying, the specimen were gently washed, so EDS analysis in correctly performed avoiding the presence of some contaminants. The ratio has been reported inside the manuscript.

6. It would be needed to present tests confirming activity of oxidized caffeic acid in the investigated system (e.g. antioxidant properties).

The authors have already demonstrated in previous systems without PEG [34] that caffeic acid underwent structural modifications during the material synthesis, they did not involve the catechol moiety, still storing their scavange activity.

7. What is the meaning of “Abs(%)” shown in figure 2? Why not Absorbance? Why “zero point” starts at (0, 18.15) not (0,0)? The calibration curve together wit absorption spectra could be presented in the supplementary file.

In this graphic, the “Abs(%)” is the value of absorbance expressed in percentage. The calibration curve was then normalized. Furthermore, the use of percentages instead of concentration was intentional. The authors believe that this is more immediate to understand by the readers.

Other remarks:

8. “The interaction of the phases inside the materials was also revealed by FT-IR analysis” – I do not find any description of interaction among system components. But indeed, it would be good to show interaction among the components. Is there any bonding or interaction among them? 

We thank the Reviewer for this comment, which has helped us to ameliorate the manuscript, by adding information about the interaction between the organic and inorganic phases, as also reported in [20].

9. Line 199: “Indeed, the two sharp absorption peaks (…) changed into a main broad peak (…) probably due to the increase of -OH and H-bonds in the oxidized structure.” It is not clear why more OH groups should be present in the oxidized C.

The oxidation of caffeic acid in an acidic environment leads to the formation of species containing a large number of -OH groups [65], FT-IR is therefore able to detect the presence of hydrogen bonds. The authors, consequently, consider the sentence written in the manuscript to be justified. The authors thanks Reviewer for this suggestion, which has helped us to ameliorate result and discussion section.

10. In the “M(OR)X” notation, X is not explained (or correctly written).

The authors thanks the reviewer for the comment and the manuscript has improved as suggested.

11. Full names of bacteria species should be used when firstly used in the text (Staphylococcus aureus instead of S. aureus etc.).

The authors thanks the reviewer for the comment and the manuscript has improved as suggested.

12. wt%, wt.% or wt. % are used in the text – please unify to the correct form.

The authors thanks the reviewer for the comment and the manuscript has improved as suggested.

13. Capital letters are sometimes used without justification (e.g. “Polyethylene Glycol, Caffeic Acid, Sol-Gel, and Silica).

The authors thanks the reviewer for the comment and the manuscript has improved as suggested.

14. “SBF (…) is a solution whose ion concentration is equal to human blood plasma” – it’s not fully correct because one may think it is equal to human blood plasma. It would be better to rephrase the sentence.

The authors thanks the reviewer for the comment and the manuscript has been ameliorate as suggested.

15. “Figure 5B [should probably be 4B] pointed out that at a fixed amount of silica and A (20 wt%), an increase in polyethylene glycol corresponds to an increase in the C-H twisting and C-O-C ether stretching [38,46] detected at 1240 and 1100 cm-1” – to be honest, these bands are not visible in the spectra of hybrids.

The authors are not in agreement with the comment of the reviewer. Even if the main signal due to Si-O is very high, the shoulders present these lower peaks that affect its shape.

16. Figure 5 requires comparison of FTIR spectra with sample before SBF test (caption should be more precise too).

The authors think that is not necessary because the spectra before the analysis are reported in Figure 4.

17. In Figure 6 it would be good to show sample before test (as in the case of figure 5, caption should be more precise).

The authors thank the reviewer but they decided to not show the image before the test, since similar materials were already shown in literature [53]. The manuscript was improved with this reference as well.

Reviewer 2 Report

The manuscript of Luigi Vertuccio et al. is devoted to the preparation of the caffeic acid loaded organic-inorganic hybrid materials via sol-gel method and investigation of antimicrobial properties of these materials.

Sol-gel method is well-known procedure to obtain various materials (organic, inorganic as well as hybrids) with controllable porous structure. This method allows to functionalize the obtaining materials directly during the synthesis procedure.

The obtaining of biomaterials with enhanced antimicrobial and anti-inflammatory properties and possibility of controlled release of drugs is an important study both in terms of fundamental science and in terms of practical application.

Despite the significant advantages of the presented work, there are several comments to the authors.

1. In the introduction (lines 46 – 53) authors have mentioned only about metal alkoxides as precursors for sol-gel procedure. In fact, the hydrolysis of metal alkoxides is not the only type of sol-gel synthesis. Moreover, it also has serious disadvantages. Please provide more information.

2. Lines 112 – 114. The phrase “Finally, nitric acid (HNO3≥65%, Sigma-Aldrich, Milan, Italy) was added to the final solution to increase the polycondensation rate reactions” is not fully correct. The hydrolysis and condensation rates strongly depend on pH value. The dependency of condensation rate on pH value is monotonic. [doi:10.1016/j.jmatprotec.2007.10.060]. Moreover, it should be noted that the acidic catalysts have a greater effect on the hydrolysis reaction due to an electrophilic reaction and basic catalysts have a greater effect on the condensation reaction due to nucleophilic substitution. Please provide some additional information and discussion concerning the synthesis procedure.

Author Response

Reviewer #2: The manuscript of Luigi Vertuccio et al. is devoted to the preparation of the caffeic acid loaded organic-inorganic hybrid materials via sol-gel method and investigation of antimicrobial properties of these materials.

Sol-gel method is well-known procedure to obtain various materials (organic, inorganic as well as hybrids) with controllable porous structure. This method allows to functionalize the obtaining materials directly during the synthesis procedure.

The obtaining of biomaterials with enhanced antimicrobial and anti-inflammatory properties and possibility of controlled release of drugs is an important study both in terms of fundamental science and in terms of practical application.

Despite the significant advantages of the presented work, there are several comments to the authors.

1. In the introduction (lines 46 – 53) authors have mentioned only about metal alkoxides as precursors for sol-gel procedure. In fact, the hydrolysis of metal alkoxides is not the only type of sol-gel synthesis. Moreover, it also has serious disadvantages. Please provide more information.

The authors thanks the reviewer for the comment and improved the manuscript as suggested.

2. Lines 112 – 114. The phrase “Finally, nitric acid (HNO3≥65%, Sigma-Aldrich, Milan, Italy) was added to the final solution to increase the polycondensation rate reactions” is not fully correct. The hydrolysis and condensation rates strongly depend on pH value. The dependency of condensation rate on pH value is monotonic. [doi:10.1016/j.jmatprotec.2007.10.060]. Moreover, it should be noted that the acidic catalysts have a greater effect on the hydrolysis reaction due to an electrophilic reaction and basic catalysts have a greater effect on the condensation reaction due to nucleophilic substitution. Please provide some additional information and discussion concerning the synthesis procedure.

We thank the Reviewer for this suggestion, which has helped us to ameliorate the manuscript.

Reviewer 3 Report

The study entitled “Sol-gel synthesis of Caffeic acid entrapped in Silica/Polyethylene Glycol based organic-inorganic hybrids: Drug delivery and biological properties” by Luigi Vertuccio, Liberata Guadagno, Antonio D’Angelo, Veronica Viola, Marialuigia Raimondo, and Michelina Catauro is an interesting work related to the preparation and properties of hybrid materials with antibacterial properties. The sol-gel synthesis, bioactivity, antimicrobial response and drug delivery properties of newly SiO2-PEG matrices doped with caffeic acid are evaluated for potential biomedical applications.

The paper is well organized in different Sections (abstract, introduction, results and conclusions) and, therefore, I recommend it for publication in the applied sciences journal, but with some minor revisions. These are commented below.

1.     Although a comprehensive study of the effects that PEG and caffeic acid have on the biological and drug release responses of the hybrids is presented, it is not clear why the particular silica gel matrix composition presented was chosen. It would be interesting to analyze the influence of acid concentration and water/TEOS ratio on the structural properties of the hybrids, especially why a sub-stoichiometric hydrolysis ratio was used.

2.     To fully understand the drug-release properties it would be necessary to analyze how the textural parameters of the hybrids affect the studied properties. Therefore, adding experimental analysis using nitrogen physisorption or discussion of similar hybrid materials in the literature is convenient.

3.     It is also necessary to add a conclusion and future work to a separate section

4.  Please double check for some typos in the manuscript,(e.g. in lines 118-119 “...weight percentages of C...”, etc)

Author Response

Reviewer #3: The study entitled “Sol-gel synthesis of Caffeic acid entrapped in Silica/Polyethylene Glycol based organic-inorganic hybrids: Drug delivery and biological properties” by Luigi Vertuccio, Liberata Guadagno, Antonio D’Angelo, Veronica Viola, Marialuigia Raimondo, and Michelina Catauro is an interesting work related to the preparation and properties of hybrid materials with antibacterial properties. The sol-gel synthesis, bioactivity, antimicrobial response and drug delivery properties of newly SiO2-PEG matrices doped with caffeic acid are evaluated for potential biomedical applications.

The paper is well organized in different Sections (abstract, introduction, results and conclusions) and, therefore, I recommend it for publication in the applied sciences journal, but with some minor revisions. These are commented below.

1. Although a comprehensive study of the effects that PEG and caffeic acid have on the biological and drug release responses of the hybrids is presented, it is not clear why the particular silica gel matrix composition presented was chosen. It would be interesting to analyze the influence of acid concentration and water/TEOS ratio on the structural properties of the hybrids, especially why a sub-stoichiometric hydrolysis ratio was used.

The authors choose that ratio because this study followed a previous study in which only silica/ caffeic acid organic/inorganic hybrid materials have been characterized [34]. In this case, we added PEG at different amount and investigated the influence of it inside the new hybrids.

2. To fully understand the drug-release properties it would be necessary to analyze how the textural parameters of the hybrids affect the studied properties. Therefore, adding experimental analysis using nitrogen physisorption or discussion of similar hybrid materials in the literature is convenient.

We thank the Reviewer for this suggestion, which has helped us to ameliorate the Result and discussion sectio.

3. It is also necessary to add a conclusion and future work to a separate section

We thank the Reviewer for this suggestion, which has helped us to ameliorate Conclusion section.

4. Please double check for some typos in the manuscript,(e.g. in lines 118-119 “...weight percentages of C...”, etc)

The authors thanks the reviewer for the comment and the manuscript has improved as suggested.

Round 2

Reviewer 1 Report

The revised paper includes some improvements but it still requires corrections. The list of comments is included in the file.

Comments for author File: Comments.pdf

Author Response

Reviewer #1:

Ad. 1) “So, the systems here presented are new.” - I do not argue that the system is not new. I just want to point a small difference between “new” and “novel”.

The authors appreciate the comment of the reviewer and will take into account the differences between the words novel and new also in future papers.

Ad. 2) In the case of lack of cytotoxicity studies, please point out clearly in the discussion a possible toxic effect on cells (quite probable in the studied system as mentioned in previous review).

As reported by the authors in the previous review, the study presented in this paper is preliminary. This is the reason why no assays on eucaryiothic cells (fibroblasts, osteoblasts, etc.) are reported. To our knowledge, no literature study reports on cytotoxic effect of oxidized caffeic acid.

Ad. 3) “In the studies, there is missing a reference material, i.e. silica sample (S) and silica/PEG (SP). Would they have, for example, antibacterial effect?

The authors did not report about S and SP systems because they were extensively studied and already published by us in other papers [9, 10]. The antimicrobial effect of such systems are reported in [20].”

In such a case, please broaden the discussion in the manuscript including results published previously to describe properly and clearly the activity of hybrids.

The author agree with the comment and ameliorate the manuscript as suggested.

Ad. 4) “Can this system be considered as “bioactive”? Is the material undergoing transformation in SBF or it is just the formation of HA on its surface?

The bone-bonding ability of a material is often evaluated by examining the ability of apatite to form on its surface in a simulated body fluid (SBF) with ion concentrations nearly equal to those of human blood plasma, as reported by Kokubo and Takadama in [37]. The authors stresses that the examination of apatite formation on a material in SBF is useful for predicting the in vivo bone bioactivity of a material, and the number of animals used in and the duration of animal experiments can be reduced remarkably by using this method”

Please note that according to the current definition of “bioactivity”, formation of HA layer on the surface may not indicate glass bioactivity. Can the system actively stimulate HA formation and bone regeneration? – this question needs to be answered before calling material “bioactive”. In this regard, I would suggest to modify two sentences in the manuscript: lines 21 & 367: “the formation of a hydroxyapatite layer on hybrid surfaces, known as bioactivity,” & ” all the samples were bioactive”.

The authors thank the reviewer for the comment and have modified the manuscript according to the suggestion. The authors could not totally prove the active bones regeneration, but they proved the formation of HA granules.

Ad. 6) “It would be needed to present tests confirming activity of oxidized caffeic acid in the investigated system (e.g. antioxidant properties).

The authors have already demonstrated in previous systems without PEG [34] that caffeic acid underwent structural modifications during the material synthesis, they did not involve the catechol moiety, still storing their scavange activity.”

Is this activity mentioned in the discussion part of manuscript? How would PEG addition modify it?

The author thanks the reviewer for the comment and remember him/her that this information is already present into the manuscript in lines 86-92, so it could be redundant to report it again.

Ad. 7) “What is the meaning of “Abs(%)” shown in figure 2? Why not Absorbance? Why “zero point” starts at (0, 18.15) not (0,0)? The calibration curve together wit absorption spectra could be presented in the supplementary file.

In this graphic, the “Abs(%)” is the value of absorbance expressed in percentage. The calibration curve was then normalized. Furthermore, the use of percentages instead of concentration was intentional. The authors believe that this is more immediate to understand by the readers.”

I cannot agree that such presentation is “more immediate to understand by the readers”. On the contrary, such presentation is misleading (if not incorrect). What, for example, is the meaning of 100% Abs? Why “zero point” starts at (0, 18.15) not (0,0)? This should be explained clearly or presented in a “standard” way using absorbance values on a calibration curve.

The authors improved the manuscript as suggested.

Ad. 8) The interaction of the phases is not proved by the studies, thus, the improper information form the Abstract should be removed (lines 19-21).

The authors thanks the reviewer for the comment and improved the manuscript as suggested.

Ad. 9) “Indeed, the two sharp absorption peaks (…) changed into a main broad peak (…) probably due to the increase of -OH and H-bonds in the oxidized structure.” It is not clear why more OH groups should be present in the oxidized C.

The oxidation of caffeic acid in an acidic environment leads to the formation of species containing a large number of -OH groups [65], FT-IR is therefore able to detect the presence of hydrogen bonds.”

In reference 65, I do not find information about caffeic acid oxidation. Is oxidation leading to higher number of OH groups (I suppose, not)? Could you present the reaction? The authors are in accordance with the review but the reference regarding the OH groups in oxidized caffeic acid is [43].

Ad. 15) Figure 5B: “The authors are not in agreement with the comment of the reviewer. Even if the main signal due to Si-O is very high, the shoulders present these lower peaks that affect its shape.”

Could you please show overlapped spectra (and/or spectra in narrower range) of samples SA20 and SP12A20 for verification?

Please, find attached below as requested.

Ad. 16) “Figure 5 requires comparison of FTIR spectra with sample before SBF test (…).

The authors think that is not necessary because the spectra before the analysis are reported in Figure 4.”

Indeed, the spectrum is shown in Figure 4; however, it is difficult fir the reader to compare data in these two figures. Moreover, very low intensity of the vibrations of the PO4 groups is in contradiction to EDS and XRD results that show clearly phosphate structures – what can be the reason of low IR signal?

The IR spectra were recorder with KBr method. Once the sample is diluted in KBr, the intensity of some signals could be reduced. Moreover, the increase in sample amount result in a signal out of the range. In this case, the authors decided to  not increase the amount of sample because with 2 mg the intensity of Si-O-T was very high and there was the risk to be out of range.

Ad. 17) Figure 6 :

The authors thank the reviewer but they decided to not show the image before the test, since similar materials were already shown in literature [53]. The manuscript was improved with this reference as well.”

If the SEM image of similar system can be found in other article, please give this information clearly in the manuscript. Although, in my opinion, it would be better to present it also in figure 6 for comparison. How was the EDS analysis performed? Details on the measurement are missing in the 2.3 section.

The manuscript has been improved as suggested.

FROM REVIEW 3: “To fully understand the drug-release properties it would be necessary to analyze how the textural parameters of the hybrids affect the studied properties. Therefore, adding experimental analysis using nitrogen physisorption or discussion of similar hybrid materials in the literature is convenient.

We thank the Reviewer for this suggestion, which has helped us to ameliorate the Result and discussion section.”

In the manuscript, one may find the following information l.341-345: “According to [66], which investigated the properties of silica matrix synthesized via sol-gel route by using different TEOS:H2O ratios, a silica matrix with TEOS:H2O=1:2 ratio has a nanometric scale (12.50 nm <x< 32.01 nm), a high surface area (347.31 m2/g <x< 549.92 m2/g), a total pore volume ranging from 0.41 to 0.51 cm3/g, and isotherm hysteresis type H2. These features could explain the reason why there is a faster release of the drug in the matrices without P.”

But it is not clear how to refer theses data to the presented studies. What are the meanings of values ranges (12.50 nm <x< 32.01 nm etc.)? What are the results for the investigated system, for example before and after drug loading? Before and after addition of P?

Finally, in the whole manuscript, there is still lack of discussion of the obtained results in comparison to other systems developed for similar applications. It must be included in the text to give the reader an idea about the value and input of the research in the field of biomaterials and to distinguish the scientific article from a scientific report.

Further remarks:

Lines 46-49: “The sol-gel process is a colloidal route (…) starting from a molecular precursor (…) or colloidal particles (e.g. graphene oxide sheet, carbon nanotubes).” – can carbon nanostructures form sol-gel-derived structures? Please refer to literature data.

The ref [12] refers to the whole paragraph. The reference has been moved at the end of the paragraph.

Line 87: “made up of silica and different (weight percentages, wt%) of caffeic acid”

The authors corrected the position of the round braquets.

Line 26-28: ”The kinetic study disclosed that the hybrid materials without polyethylene glycol had faster release rates than the ones obtained without it.“ Both sentences needs correction.

The authors thank the review for the comment and ameliorate the manuscript as suggested.

1. 121: 30 °C is not called “room temperature”

The authors apologize for the incongruence. This temperature refers to the room temperature of Italian summer days. The manuscript has been improved.

l.129: “the weight percentages of C to S content” should probably use “A” not “C”

The authors thank the review for the comment and ameliorate the manuscript as suggested.

l.261: “Ca and P, with a ratio equal to 1.67” – precise which kind of ratio (molar or weight) you report

The authors thank the review for the comment and improved the manuscript as suggested.

Check lines 467-8, something is incorrect (missing reference number?)

Some numbers with decimal places have comas instead of dots.

The authors thank the review for the comment and ameliorate the manuscript as suggested.

Correct in the abstract a few notations such as “inorgan-ic”.

The authors thank the review for the comment and ameliorate the manuscript as suggested.

Reviewer 2 Report

The authors took into account all the comments

Author Response

Reviewer #2: The authors took into account all the comments.

The authors thank the reviewer for the comment.