Peptide-Based Nanogels for Pharmaceutical and Biotechnological Applications: From Fmoc-FF to Other Peptide Sequences

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review article focuses on peptide-based nanogels (NGs), with a significant portion dedicated to Fmoc-FF based nanogel formulations. The authors describe various preparation methods, characterization techniques (including DLS and CD spectroscopy), and therapeutic applications such as drug delivery is dicussed. However, the authors should improve some parts of the manuscript and correct some typos: 

Abstract

- The terminology is inconsistent. For example, “peptide-based nanogels (NGs)” suggests that “peptide” modifies “nanogels,” but the acronym “NG” is then used without the “peptide” qualifier. Clarify what NG refers to.

- In line 6, “nanogel” can be abbreviated as “NG” for brevity and consistency.

- The abstract is not well written. The authors should rewrite it, especially the final part, to improve clarity and logical flow.

Introduction

- Figure 1 and its caption need significant improvement. The authors should explain the figure’s content in more detail (e.g., the role of the aromatic ring, the reaction mechanism, etc.).

Section: 2. Fmoc-FF based Nanogel formulations

- Page 3: “Low molecular weight gelator” is defined twice in a row. Use the acronym (LMWG) at the second mention.

- Page 3: DMSO is used but never defined.

- Figure 2: Displaying the chemical structures of dox and 5-FU is not particularly relevant. It would be more informative to show experimental data, such as a synthesis scheme. Seeing only a DLS profile is a bit sad.

- Page 5: Typo: “Figure 0”. Please correct.

- Page 7: “CD spectra”, the acronym is not defined (circular dichroism). Consider including actual CD or FTIR data as figures. These would be useful for readers.

- In this section, the authors should include a table summarizing the nanomaterials described in the bibliography, covering composition, characterization methods, encapsulated drugs (loading efficiency and release profiles), and efficacy for the specific therapy.

- Regarding photothermal therapy, the authors should briefly explain the mechanism behind the ON/OFF release behavior upon irradiation.

- Figure 6A is not mentioned in the main text. Please cite it appropriately.

- Figure 6B is of very poor quality. Please replace it with a higher-resolution image.

Section: 3. Non-Fmoc-FF related nanogels

- More figures and tables are needed for many of the references provided. This would help clarify and visualize key concepts and experimental data, especially since some examples (e.g., MMPs and metal-coordinated NGs on page 11) occupy nearly an entire page. The most important examples should be included in Figure 8.

- Page 14: The authors describe a crosslinked NG prepared by UV/light radiation mediated by a Ru complex. Please provide more details on the reaction conditions, not just exposure time (6 min), but also light intensity and other relevant parameters so that readers can understand better the preparation method.

- Page 15: The zeta potential symbol is incorrectly written. Please correct it.

- Page 14: The description of His-based crosslinking activated by gamma radiation lacks detail. It is unclear how this was done (conditions, setup, etc.). Please elaborate.

- Page 14: The statement “These direct effects (ionizing radiation) are largely mitigated in aqueous solutions because water absorbs most of the radiation energy” is far from accurate. Ionizing radiation can still affect protein structure, even in the presence of water. Please revise or provide supporting evidence.

 

Author Response

This review article focuses on peptide-based nanogels (NGs), with a significant portion dedicated to Fmoc-FF based nanogel formulations. The authors describe various preparation methods, characterization techniques (including DLS and CD spectroscopy), and therapeutic applications such as drug delivery is discussed. However, the authors should improve some parts of the manuscript and correct some typos: 

Abstract

- The terminology is inconsistent. For example, “peptide-based nanogels (NGs)” suggests that “peptide” modifies “nanogels,” but the acronym “NG” is then used without the “peptide” qualifier. Clarify what NG refers to.

We thank the Reviewer for the consideration, allowing an improvement of readability and clarity. The specific point is clarified in the revised version of the manuscript both in Abstract and Introduction, explaining that the NG acronym is used for peptide-based formulations.  

- In line 6, “nanogel” can be abbreviated as “NG” for brevity and consistency.

According to the Reviewer’s suggestion, the abstract has been revised and rewritten to improve clarity, readability, and logical flow. The acronym was consequently removed.

- The abstract is not well written. The authors should rewrite it, especially the final part, to improve clarity and logical flow.

We appreciated the comment. The abstract has been revised and rewritten to improve clarity, readability, and logical flow, particularly in the final section. The revised version now presents the scope, classification, and significance of peptide-based nanogels in a more concise and coherent manner.

Introduction

- Figure 1 and its caption need significant improvement. The authors should explain the figure’s content in more detail (e.g., the role of the aromatic ring, the reaction mechanism, etc.).

We agree that the original version of Figure 1 caption was too concise, without sufficient information. We have therefore revised it. In particular, the updated version highlights the role of non-covalent forces to hydrogel matrix formation, and the consequent steps leading from peptide gelation to nanogel formation. The caption has been expanded, to guide the reader through the different stages illustrated, clarifying the relationship between the supramolecular organization of peptide gelators and the final nanogel architecture.

Section: 2. Fmoc-FF based Nanogel formulations

- Page 3: “Low molecular weight gelator” is defined twice in a row. Use the acronym (LMWG) at the second mention.

Apologizing for the repetition, it was revised and corrected.

- Page 3: DMSO is used but never defined.

The required information was added.

- Figure 2: Displaying the chemical structures of dox and 5-FU is not particularly relevant. It would be more informative to show experimental data, such as a synthesis scheme. Seeing only a DLS profile is a bit sad.

Receiving the Reviewer’s suggestion, we agree with the comment about the original Figure 2 as not sufficiently informative about the formulation process. Therefore, the figure was revised to improve its relevance and clarity by replacing the less informative panel with a schematic representation of the inverse-emulsion preparation of Fmoc-FF nanogels. In this way, the figure now better highlights the formulation strategy and provides a more useful visual support for the related discussion. We thank the Reviewer for the beneficial comment.

- Page 5: Typo: “Figure 0”. Please correct.

We apologize for the typo. It was corrected.

- Page 7: “CD spectra”, the acronym is not defined (circular dichroism). Consider including actual CD or FTIR data as figures. These would be useful for readers.

We revised the text to define ‘CD’ as circular dichroism at first mention in the text. Concerning the inclusion spectra, even if we agree that such data are informative, we chose to keep the figures focused on the general concepts and formulation strategies. The required spectra referred in the cited primary literature (open access Ref. 50 and 51).

- In this section, the authors should include a table summarizing the nanomaterials described in the bibliography, covering composition, characterization methods, encapsulated drugs (loading efficiency and release profiles), and efficacy for the specific therapy.

According to the Reviewer’s suggestion, the revised version of the manuscript contains a final summary table about Fmoc-FF related nanogels formulations (Table 1). The table includes, where available from the cited literature, information on peptide primary sequence and composition, formulation/characterization methods, encapsulated active pharmaceutical ingredient, loading or encapsulation data, release profile information, and the reported therapeutic or biotechnological efficacy. For studies in which some parameters were not explicitly reported, this has been indicated accordingly using NR (not reported).

- Regarding photothermal therapy, the authors should briefly explain the mechanism behind the ON/OFF release behaviour upon irradiation.

According to the suggestion, in the revised manuscript the mechanism of the ON/OFF release under NIR irradiation have been briefly clarified. The following sentences were included:

“Mechanistically, the ON/OFF release profile can be ascribed to the photothermal conversion of the plasmonic gold nanoparticles NIR irradiation. The 808 nm radiation can generate local heating, transiently increases the matrix permeability, thus promoting EPI diffusion. Then, once the irradiation is switched off, the thermal stimulus terminates and the release rate decreases consequently.”

- Figure 6A is not mentioned in the main text. Please cite it appropriately.

We thank the Reviewer for the comment. The inconsistency was corrected.

- Figure 6B is of very poor quality. Please replace it with a higher-resolution image.

Taking in consideration the comment, the quality of Figure 6B in the previous version was not satisfactory. We have therefore replaced it with a higher-resolution image in the revised manuscript to improve readability and figure quality.

Section: 3. Non-Fmoc-FF related nanogels

- More figures and tables are needed for many of the references provided. This would help clarify and visualize key concepts and experimental data, especially since some examples (e.g., MMPs and metal-coordinated NGs on page 11) occupy nearly an entire page. The most important examples should be included in Figure 8.

According to the comment, a Table (Table 2) concerning non-Fmoc-FF related nanogels has been added in the text as well as another figure (Figure 11), in order to have five figures for each section.

- Page 14: The authors describe a crosslinked NG prepared by UV/light radiation mediated by a Ru complex. Please provide more details on the reaction conditions, not just exposure time (6 min), but also light intensity and other relevant parameters so that readers can understand better the preparation method.

According to the comment, we have revised the text, by better describing the light-mediated crosslinking process with two sentences. To avoid introducing details not explicitly supported by the original publication, we did not add specific values for light intensity or additional setup parameters and methods.

- Page 15: The zeta potential symbol is incorrectly written. Please correct it.

Apologizing for the typo, we corrected it.

- Page 14: The description of His-based crosslinking activated by gamma radiation lacks detail. It is unclear how this was done (conditions, setup, etc.). Please elaborate.

We agree that the previous description for the His-based sequences undergoing gamma-radiation-induced crosslinking was too concise. In the revised manuscript, we have clarified that the nanogels were prepared by irradiating aqueous peptide solutions of the histidine/glycine sequences H5, H9, and H13 (at concentration of 0.1 wt.%) with γ-rays under aerated conditions at 5 kGy to obtain NGs formulation. We also added a brief crosslinking mechanistic explanation. A novel Figure (Figure 11) was included, too.

- Page 14: The statement “These direct effects (ionizing radiation) are largely mitigated in aqueous solutions because water absorbs most of the radiation energy” is far from accurate. Ionizing radiation can still affect protein structure, even in the presence of water. Please revise or provide supporting evidence.

We thank Reviewer for this important comment. Accordingly, the sentence was revised to avoid implying that direct effects are trivial and to better distinguish between direct and indirect radiation damage in aqueous media.

 

 

Reviewer 2 Report

Comments and Suggestions for Authors

Dear Authors,

The presented manuscript “Peptide-based Nanogels: Pharmaceuticals and Biotechnological Applications” is well designed and organized, easy for reading and understanding. It sheds light on the current landscape of peptide-based nanogels and their applications. The reviewed literature is relevant to the study. The article could generate ideas for future studies preparing tailored short peptides with target application.

This gives me a reason to recommend the article for publication in Pharmaceuticals in present form. I have following remarks addressed to you: You have to remove reference 84 as it is the same as ref. 45.

Author Response

Reviewer #2

Dear Authors,

The presented manuscript “Peptide-based Nanogels: Pharmaceuticals and Biotechnological Applications” is well designed and organized, easy for reading and understanding. It sheds light on the current landscape of peptide-based nanogels and their applications. The reviewed literature is relevant to the study. The article could generate ideas for future studies preparing tailored short peptides with target application.

This gives me a reason to recommend the article for publication in Pharmaceuticals in present form. I have following remarks addressed to you: You have to remove reference 84 as it is the same as ref. 45.

We sincerely appreciate the Reviewer’s positive evaluation of our manuscript and for recommending it for publication in Pharmaceuticals. We greatly value the supporting comments regarding the organization, readability, and relevance of the reviewed literature. In response to the reference remark, we apologize for the error, confirming that Reference 84 was indeed a duplicate of Reference 45. For this, Reference 84 was removed, Reference 45 recalled, and the reference list has been corrected accordingly in the revised manuscript.

 

 

Reviewer 3 Report

Comments and Suggestions for Authors

The presented article <<Peptide-based nanogels: pharmaceuticals and biotechnological applications>> by  Mariangela Rosa,  Sabrina Marino et al is the praiseworthy attempt to give the full review of nanogel applications in practice. The main attention dedicated to preparation and study of   Fmoc-FF dipeptide based nanogels. The rest peptide compositions of another different composition serve the added  knowledge regarding this special peptide. Therefore one can offer alternative title of article as <<Fmoc-FF dipeptide based nanogels:………………….>>. The evidences regarding another peptide-based gels are well fully  discussed in works cited in reference list. The manuscript needs major revision of some disadvantages.

Comment 1. The accepted classification of nanogels  dividing all nanogel compositions  on two classes is not discussed deeply. The division seems rather artificial. What  reasons to distinguish Fmoc-FF dipeptide nanogels as leading separate class.

Comment 2. Phase diagrams for gel state are not considered in manuscript.

Comment 3. Immunegenicity of injected nanogels is important issue for medical application. But this  item is not discussed.

Comment 4. Top-down methodology is not explained ( Figure 3A)

Comment 5. Figure 0. ????? (its not clear) avoiding the use of ammonium sulphate procedure…….

Comment 6. Figure 2 B :  size diamenter??? Why the measument error for two size mode fractions ( 1,3 nm; 0,8 nm)  don’t coinside in one experiment?

Comment 7. The text << Drug loading efficiency and release behaviour of NGs loaded with doxorubicin (Dox) and 5-fluorouracil (5-FU) were evaluated under physiological conditions>> don’t correspond the content of Figure 2C

Recommendation : the manuscript may be submitted after major revision and  title change.  

Comments on the Quality of English Language

no comment

Author Response

Reviewer #3

The presented article “Peptide-based nanogels: pharmaceuticals and biotechnological applications” by  Mariangela Rosa, Sabrina Marino et al is the praiseworthy attempt to give the full review of nanogel applications in practice. The main attention dedicated to preparation and study of Fmoc-FF dipeptide based nanogels. The rest peptide compositions of another different composition serve the added knowledge regarding this special peptide. Therefore, one can offer alternative title of article as “Fmoc-FF dipeptide based nanogels”. The evidence regarding another peptide-based gels are well fully discussed in works cited in reference list. The manuscript needs major revision of some disadvantages.

We appreciated the Reviewer’s suggestions. To reflect both the central role of Fmoc-FF nanogels and the inclusion in the manuscript of other relevant peptide-based nanogel systems, we have revised the title to

“Peptide-Based Nanogels for Pharmaceutical and Biotechnological Applications: From Fmoc-FF to Other Peptide Sequences.”

Comment 1. The accepted classification of nanogels dividing all nanogel compositions on two classes is not discussed deeply. The division seems rather artificial. What reasons to distinguish Fmoc-FF dipeptide nanogels as leading separate class.

We thank the Reviewer for his/her comment, allowing us deeper discussion about the systems classification. The selected categorization was not intended to imply that Fmoc-FF represents a superior or fully independent class, but rather to evidence a literature-based organizational criterion. In our review, Fmoc-FF was discussed separately due to the evidence that it is the most extensively investigated peptide building block for NGs, associated with the largest number of reported nanogel formulations, preparation strategies, and pharmaceutical applications. The remaining systems were grouped as “non-Fmoc-FF” peptide nanogels to highlight the broader structural diversity of peptide sequences explored beyond this widely studied model. We additionally clarified that the categorization is made for expository convenience, based on recurrence in the literature and not on a strict categorical separation. To avoid the impression of an artificial distinction, the text was modified in the “Introduction” section to better justify it.

Comment 2. Phase diagrams for gel state are not considered in manuscript.

As stated by the Reviewer, the phase diagrams are fundamental collection of data for defining gelation domains in relationship with formulation variables (e.g. concentration, solvent composition, pH, and temperature). However, the main aim of the present review is to focus on peptide-based nanogels, with particular attention to formulation strategies, nanoscale properties, and pharmaceutical or biotechnological applications, rather than on the fundamental phase behaviour of the parent hydrogel systems. For this reason, a detailed discussion of phase diagrams was considered outside the scope of the manuscript but recalled in a sentence, reporting the relative References (19-22 and 35)

Comment 3. Immunegenicity of injected nanogels is important issue for medical application. But this item is not discussed.

We thank the Reviewer for this important point. After carefully re-examining the cited literature, immunogenicity is generally not directly investigated in the peptide-based nanogel formulations reported in literature until now. The available data mainly concern physicochemical characterization, cytotoxicity and cellular uptake. Some experiments are related to serum stability and hemotoxicity (Reference 50 and 51), providing useful biocompatibility information. However, these data do not constitute a direct evaluation of immunogenicity. The Reviewer’s consideration was included in a sentence in “Conclusion” section to highlight specifically the evidenced point.

Comment 4. Top-down methodology is not explained (Figure 3A)

We agree that the top-down methodology should be described more clearly in the manuscript. For this, the revised text was modified to better explain this formulation strategy and improve the clarity of Figure 3A

Comment 5. Figure 0 (it’s not clear) avoiding the use of ammonium sulphate procedure

We apologize for the typo. It was corrected.

Comment 6. Figure 2 B:  size diamenter??? Why the measument error for two size mode fractions (1,3 nm; 0,8 nm) don’t coinside in one experiment?

The Figure 2B was revised according to the suggestion of Reviewer #1 by removing the DLS profile. The inconsistence was consequently eliminated.

 

Comment 7. The text << Drug loading efficiency and release behaviour of NGs loaded with doxorubicin (Dox) and 5-fluorouracil (5-FU) were evaluated under physiological conditions>> don’t correspond the content of Figure 2C

The Figure 2 was revised according to the suggestion of Reviewer #1 by removing the chemical structure of both drugs. The inconsistence was consequently eliminated.

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Thank you for the revisions. The manuscript has significantly improved and is now more appealing and engaging for readers in the field.

Reviewer 3 Report

Comments and Suggestions for Authors

I appreciate yours answers. Thank you for change of title. The title got better for essence of article. 

Comments on the Quality of English Language

no comment