Review Reports - Protein Nanocarriers: Targeted Theranostics for Cancer Treatment and Imaging

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors describe very clearly and in detail the latest improvements regarding the main proteins used as nanocarriers for targeted theranostics in cancer treatment and imaging.

Nevertheless, I have some comments / questions to address :

These protein carriers are useful primarily for active targeting, but they can also enable passive targeting ? This should be mentioned in the manuscript.
The protein nanocarriers are presented as having higher potential compared to polymer nanocarriers, among others. Does the Enhanced Permeability and Retention (EPR) effect improve significantly when using protein nanocarriers compared to polymer or metallic carriers? Some literature examples would be helpful.
How do immunogenicity and rapid clearance affect clinical translation.
Considering cost and scale-up limitations, what strategies could be used to make protein-based theranostics more commercially viable? This should be mentioned in the manuscript.

Author Response

Reviewer 1

The authors describe very clearly and in detail the latest improvements regarding the main proteins used as nanocarriers for targeted theranostics in cancer treatment and imaging.

We thank the Reviewer for positive evaluation of our work.

Nevertheless, I have some comments / questions to address:

These protein carriers are useful primarily for active targeting, but they can also enable passive targeting ? This should be mentioned in the manuscript.

The recent work focuses on active targeting mechanisms using various protein nanocarriers. However, these nanocarriers can also achieve passive targeting via the enhanced permeability and retention (EPR) effect, without the need for specific targeting ligands. We have revised the manuscript accordingly, including discussion of enhanced permeation and retention – a consequence of porous vasculature and poor lymphatic drainage in tumour microenvironments: Lines 138-141.

2.The protein nanocarriers are presented as having higher potential compared to polymer nanocarriers, among others. Does the Enhanced Permeability and Retention (EPR) effect improve significantly when using protein nanocarriers compared to polymer or metallic carriers? Some literature examples would be helpful.

In addition, we have added a comparison of protein nanocarriers with commonly used carriers, such as polymers and metallic nanoparticles, in terms of passive targeting efficiency. The related examples have been added to the revised manuscript accordingly (Lines 141-143) to clarify this point.

3. How do immunogenicity and rapid clearance affect clinical translation.

Immunogenicity and rapid clearance are critical challenges, and can lead to anti-drug antibodies, reducing therapeutic activity, altering pharmacokinetics, and causing immune reactions. In addition, rapid clearance by reticuloendothelial system, can also limit tumour accumulation, decreasing imaging sensitivity and/or therapeutic activity. Protein-based drug delivery systems can prolong drug half-life and reduce immune recognition. We have revised conclusion (Section 7, Lines 533-541).

4. Considering cost and scale-up limitations, what strategies could be used to make protein-based theranostics more commercially viable? This should be mentioned in the manuscript.

We appreciate the reviewer’s insightful comment regarding cost and scale up limitations. Several strategies may improve commercial viability including optimisation of recombinant expression systems (e.g. bacterial, yeast) to reduce production costs, use of standardised protein scaffolds to simplify manufacturing, and implementation of scalable purification technologies. We have added comments highlighted in the text (Lines 177-181) and Conclusions (Lines 544-548) in the revised manuscript to clarify this point.

Reviewer 2 Report

Comments and Suggestions for Authors

Reyhan Dilsu Colpan, Neil R. Thomas, Lyudmila Turyanska and Tracey D. Bradshaw:

Protein Nanocarriers: Targeted Theranostics for Cancer Treatment and Imaging (Review)

This manuscript gives a good summary of Protein Nanocarriers focusing to the main fields of nanocarrier-ameliorations. The manuscript is well constructed and gives valuable general overviews about the targeted receptors, nanocarriers/nanoparticles, transported dyes/drugs and applications.

The comments are as follows.

page 248. poor depth penetration, indicating the reason of limitation

page 257. I suggest changing the symbol of ICG in Fig. 2 to an abstract form of its chemical formula, to make visual difference between organic substances and compact nanoparticles)

(like this, ) //here it does not appears, see in doc., attached

line 427. 1.5- or 3 mm diameter pores of Qβ (Table 1)?

I suggest writing the whole names, instead of abbreviations, more frequently, especially when it is mentioned first time. (for example, FR, ..., in Fig.6 b the name of dyes, meaning of letters (p,q,r,s,t).

Remarks to “Conclusions and future perspectives”

I suggest involving further problems into this point. In this review the “packing protein materials” were highlighted. i) Considering biocompatibility, effect of foreign molecules (bacteriophage, horse spleen, BSA, silk spider) should be discussed. The drugs are rather widely used “model” molecules (DOX, curcumin, .) than those used in the II and III phases in clinical trials.

I suggest to give the whole reference information (titles, doi numbers)

Comments for author File: Comments.pdf

Author Response

The comments are as follows.

page 248. poor depth penetration, indicating the reason of limitation

We thank the reviewer for pointing out this important consideration, which has now been included. Lines 52-54 “Whereas fluorescence imaging agents offer high resolution, development of novel NIR imaging agents will advance depth penetration capabilities.”

page 257. I suggest changing the symbol of ICG in Fig. 2 to an abstract form of its chemical

formula, to make visual difference between organic substances and compact nanoparticles)

(like this, )

Thank you. We hope to have added clarity by (Lines 266-267) including the chemical formulae of ICG, Cy 7, Cy 5.5 and alexa fluor 680.

line 427. 1.5- or 3 mm diameter pores of Qβ (Table 1)?

Thank you for pointing out this typographical error. Please see correction and clarification of this point (Lines 444; 446-447). We qualify that this is an icosahedral capsid and that the threefold axis pores are 1.3-1.5 nm diameter.

I suggest writing the whole names, instead of abbreviations, more frequently, especially

when it is mentioned first time. (for example, FR, ..., in Fig.6 b the name of dyes, meaning of

letters (p,q,r,s,t).

We would like to clarify that the abbreviation FR was already introduced earlier in the manuscript (Line 77). We have added the whole names of dyes, and explanation of letters in Figure 6b (Lines 508-510).

Remarks to “Conclusions and future perspectives”

In the revised manuscript (Section 7, Line 533-541), we have expanded the discussion of biocompatibility concerns related to protein-based materials. We highlight the potential difference in immune responses elicited by protein nanocarriers, how they affect pharmacokinetics and systemic clearance. In addition, recombinant production strategies, protein engineering studies have been added as strategies to minimise immune responses.

The drugs are rather widely used “model” molecules (DOX, curcumin, .) than those used in the II and III phases in clinical trials.

We thank the reviewer for this observation. We respectfully clarify that doxorubicin (DOX) is not only used as a model compound in drug delivery studies, but it is also a clinically approved and widely administered chemotherapeutic agent for the treatment of many cancer types. Therefore, its inclusion in the discussed systems reflects both its established clinical relevance and its frequent use as a benchmark drug in nanomedicine research.

We agree that certain compounds, such as curcumin, are more commonly employed as model molecules in proof-of-concept studies. As this review aims to summarise the most extensively investigated cargo molecules in protein-based delivery systems, DOX appears prominently due to its dual role as both a clinically relevant drug and a widely studied reference compound in the literature.

I suggest to give the whole reference information (titles, doi numbers)

We have carefully revised the reference list and now provide complete bibliographic details, including article titles and DOI numbers, in accordance with journal guidelines.

Reviewer 3 Report

Comments and Suggestions for Authors

This review article, titled "Protein Nanocarriers: Targeted Theranostics for Cancer Treatment and Imaging," provides a comprehensive overview of the current state of protein-based nanoplatforms for combined cancer diagnosis and therapy. It details various naturally derived and engineered protein assemblies—including albumin, lipoproteins, ferritin, viral protein capsids, and silk proteins—and discusses how they can be functionalized to target overexpressed receptors on cancer cells. The article also explores the integration of diverse imaging modalities (MRI, PET, NIR) and the importance of using advanced 3D preclinical models to bridge the translational gap in oncology.

The article is exceptionally well-structured and timely, addressing a critical need for safer and more effective "theranostic" devices in personalized oncology. The authors have Seamlessly integrated complex topics with material science and clinical imaging technologies. Additionally, the authors also provide a high-quality, informative figures that effectively summarize complex interactions between nanocarriers, ligands, and imaging probes. The article covers a wide range of protein sources, from endogenous ferritin to plant-derived viral capsids and silk proteins, highlighting both their unique advantages and current limitations.

Minor Comments for the Authors

While the manuscript is of high quality, the following minor points should be addressed to further enhance its impact:

Safety and Regulatory Context: In the section discussing Gd-based contrast agents for MRI, the authors mention they have been withdrawn due to safety concerns like nephrogenic systemic fibrosis.This needs more explanations and some examples.

Clinical Translation Challenges: While silk proteins and lipoproteins are presented as promising, you correctly note limitations like batch-to-batch inconsistency. Adding a brief sentence on how recent advances in recombinant protein production might mitigate these issues would provide a more forward-looking perspective.

Refining Nomenclature: Ensure consistent use of abbreviations. For instance, you define "H-Ft" and "AFt" but sometimes use "Ft" more generally. Standardizing these throughout the specific case studies (Section 6) would improve readability.

Author Response

Minor Comments for the Authors

While the manuscript is of high quality, the following minor points should be addressed to further enhance its impact:

We have expanded this section, supporting the statement that Gd-based contrast agents have been withdrawn from clinical use (Line 248-253, Reference 100) with the specific example of brain accumulation of Gd-based agent (Line 250-253, Reference 101).

We thank the Reviewer for this constructive suggestion. In the revised manuscript (Line 177-181 for lipoproteins, Line 230-232 for silk proteins), we have added a brief statement highlighting how recent advances in recombinant protein production and controlled expression systems may help mitigate batch-to-batch variability and improve scalability. This addition provides a more forward-looking and translational perspective.

We thank the reviewer for the suggestion regarding nomenclature consistency. We intentionally maintained distinctions among “H-Ft” (Heavy-chain of ferritin),” L-Ft” (Light-chain of ferritin), “Ft” (Ferritin, H+ L mix with iron) and “AFt”(Apoferritin, H+L mix without iron) in the studies (Section 6), as each abbreviation refers to slightly different protein constructs or contexts. We believe that preserving these distinctions improves accuracy and helps readers clearly differentiate between the various forms discussed.

Reviewer 4 Report

Comments and Suggestions for Authors

I appreciate this review; it was very well performed, and the references are very current.

Author Response

I appreciate this review; it was very well performed, and the references are very current.

We sincerely thank the reviewer for this positive and encouraging comment. We are pleased that the review was found to be thorough and that the references are considered current and relevant.