Development of Chitosan-Carbon Dot Hybrid Nanoemulsomes for MEIS2 Inhibitor Delivery and Bioimaging in Colorectal Cancer

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors This report relates to improving chemotherapy for colorectal cancer. Abstract: will readers know what MEIS is? How is ‘drug encapsulation efficiency known to three significant figures? Line 85: change ‘following’ to ‘follows’. Why are names of some agents capitalized? Line 96 needs a space between ‘10' and ‘min’.  Line 99: what and elsewhere: the % sign comes after the number, not before. Line 103: leave a space after ‘30'. Line 105: what is ‘DI’? Line 103" change ‘added’ to ‘in’. The ‘%’ sign shoiuld come after the number, not before. Line 129: change ‘at’ to ‘in’. Before resubmission, the entire manuscript needs to be inspected by someone more familiar with English usage. An example of what needs to be dealt with occurs in line 136 ‘and waited 12 hours’. An editor will know what to do. How does the MTS assay assess viability (line 141)? Only clonogenic studies reliably test for viability. What does the MTS actually measure? This should be indicated. What assay was used to assess ‘cell cycle’ (section 2.9)? Colonony formation assay (section 2.10) is a reliable approach. In Fig. 3: only the clonogenic assays reliably indicate survival of tumor cells.  In the colonly formation procedure (section 2.10), cells were treated with assorted reagents for 24 hours. Such an exposure time can readily be carried out in vitro, but how could a 5 μM level of an inhibitor be maintained for 24 or 48 hours in vivo?  The discussion points out that MEISi is unstable in aqueous media. What is the ‘non-soluble salt’ (lines 269-270)?   The major issues with this report are [1] only clonogenic studies reliably report on tumor death; [2] it is unlikely that a significant drug level can be maintained for 24 hours in vivo.   Comments on the Quality of English Language

Generally adequate but there are regions (pointed out in the review) that need editing. 

Author Response

Comment 1: 

This report relates to improving chemotherapy for colorectal cancer. Abstract: will readers know what MEIS is? How is ‘drug encapsulation efficiency known to three significant figures? Line 85: change ‘following’ to ‘follows’. Why are names of some agents capitalized? Line 96 needs a space between ‘10' and ‘min’.  Line 99: what and elsewhere: the % sign comes after the number, not before. Line 103: leave a space after ‘30'. Line 105: what is ‘DI’? Line 103" change ‘added’ to ‘in’. The ‘%’ sign shoiuld come after the number, not before. Line 129: change ‘at’ to ‘in’. Before resubmission, the entire manuscript needs to be inspected by someone more familiar with English usage. An example of what needs to be dealt with occurs in line 136 ‘and waited 12 hours’. An editor will know what to do. How does the MTS assay assess viability (line 141)? Only clonogenic studies reliably test for viability. What does the MTS actually measure? This should be indicated. What assay was used to assess ‘cell cycle’ (section 2.9)? Colonony formation assay (section 2.10) is a reliable approach. In Fig. 3: only the clonogenic assays reliably indicate survival of tumor cells.  In the colonly formation procedure (section 2.10), cells were treated with assorted reagents for 24 hours. Such an exposure time can readily be carried out in vitro, but how could a 5 μM level of an inhibitor be maintained for 24 or 48 hours in vivo?  The discussion points out that MEISi is unstable in aqueous media. What is the ‘non-soluble salt’ (lines 269-270)?   The major issues with this report are [1] only clonogenic studies reliably report on tumor death; [2] it is unlikely that a significant drug level can be maintained for 24 hours in vivo.  

Response 1: 

The MTS assay was employed to evaluate changes in metabolic activity as an indicator of proliferative capacity rather than direct cell death. The primary objective of this assay was to compare the metabolic impact of CDChitosan and CDChitosanMEISi and to determine an appropriate concentration range for subsequent mechanistic analyses. As reported, concentrations above 7 μM were not well tolerated, guiding the selection of 5 μM for further experiments.

We did not conclude tumor cell death solely based on MTS data. To assess long-term survival more reliably, clonogenic assays were performed (Section 2.10, Figure 3). These experiments demonstrated substantial suppression of colony formation following MEISi-2 treatment, supporting a sustained anti-proliferative effect beyond short-term metabolic inhibition. Importantly, the empty nanocarrier produced colony numbers comparable to the untreated control, indicating that the observed long-term suppression is primarily attributable to MEIS inhibition rather than carrier-associated effects.

Regarding cell cycle analysis (Section 2.9), flow cytometry was conducted using propidium iodide (PI) staining. The methodological description has been clarified and typographical errors have been corrected in the revised manuscript (Section 2.9, line 154).

As correctly noted by the reviewer, maintaining a stable molar concentration for 24–48 hours in vivo cannot be directly extrapolated from in vitro exposure conditions. However, drug release studies demonstrated sustained release (~30% within 24 hours), supporting controlled availability of MEISi-2 from the nanoemulsion system under physiological conditions. The present study is designed as an in vitro proof-of-concept investigation to demonstrate controlled release behavior and biological activity. Comprehensive in vivo pharmacokinetic and biodistribution analyses will be addressed in future studies.

Reviewer 2 Report

Comments and Suggestions for Authors

The present paper, entitled "Development of chitosan-carbon dot hybrid nanoemulsomes for MEIS2 inhibitor delivery and bioimaging in colorectal cancer" explored the development of a multifunctional hybrid nanoemulsome carrier by integrating boron-silane-doped Carbon dots with chitosan via glutaraldehyde crosslinking, encapsulating a MEIS inhibitor for colorectal cancer therapy. The research design was systematic and the data was comprehensive. This work is highly innovative, particularly in its dual approach of drug delivery and bioimaging, and is suitable for the journal. The manuscript should be revised by authors according to the following comments before publication.

Specific remarks:

1. In Introduction, the authors mentioned that an "advanced carrier system is necessary" due to the physicochemical limitations of MEIS inhibitors. However, the text only briefly mentions chitosan and carbon dots without explicitly stating the unique advantages of this specific hybrid nanoemulsome design compared to other common nanocarriers such as liposomes or PLGA nanoparticles for this specific drug. It is suggested to supplement relevant literature for comparative analysis, and clarify the superiority of this hybrid system to highlight the rationality of the material selection.
2. In 2.2 and 2.3, the synthesis protocols describe using a "0.45µm pore-sized PES filter" to remove aggregates. Given that the final particle size of the drug-loaded system is reported as 322.8 nm, which is relatively close to the pore size, the authors need to clarify if there was any significant yield loss during this filtration step or if the polydispersity index suggests a population of particles larger than the pore size was lost.
3. In Figure 3 (A and B), the bar charts displaying cell proliferation results lack statistical significance indicators such as asterisks. Although error bars are present, the graphs do not explicitly show whether the differences between the Control, CDChitosan, and CDChitosanMEISi groups are statistically significant. Please add these markers to the charts for clarity.
4. In the Materials and Methods section, the manuscript describes various assays but omits a dedicated subsection on statistical analysis. The authors should add a paragraph specifying the statistical tests employed, and the software used to process the data.
5. In Figure 2D, the AFM images are presented without clearly visible scale bars. Please update the figure to include clear scale indicators.
6. In 3.2, the results indicate that MEISi-2 loading increased the particle size significantly from 150.9 nm to 322.8 nm. The authors should expand the discussion to address how this doubling in size might influence the passive targeting capability via the EPR effect or cellular uptake efficiency compared to the smaller empty carriers.
7. In 2.3, the phrase "Larger and big aggregates" is redundant and should be corrected. Furthermore, the manuscript requires careful proofreading to correct typos like "Gluteraldehyde" and inconsistent formatting in unit notations, such as the difference between "10.000 xg" and "14,000xg".
8. It is recommended to cite the following references, European Cells and Materials,2024,47, 152-169. https://doi.org/10.22203/eCM.v047a11. European Cells and Materials,2025,50,20-32. https://doi.org/10.22203/eCM.v050a02. European Cells and Materials, 50, 87-108 , https://doi.org/10.22203/eCM.v050a06.

 

From the above, I think this manuscript can be accepted after revision since the research topic is clinically relevant and the experimental results regarding the hybrid carrier are encouraging.

Author Response

Comments and Suggestions for Authors

The present paper, entitled "Development of chitosan-carbon dot hybrid nanoemulsomes for MEIS2 inhibitor delivery and bioimaging in colorectal cancer" explored the development of a multifunctional hybrid nanoemulsome carrier by integrating boron-silane-doped Carbon dots with chitosan via glutaraldehyde crosslinking, encapsulating a MEIS inhibitor for colorectal cancer therapy. The research design was systematic and the data was comprehensive. This work is highly innovative, particularly in its dual approach of drug delivery and bioimaging, and is suitable for the journal. The manuscript should be revised by authors according to the following comments before publication.

Response: We sincerely thank the reviewer for the positive and encouraging evaluation of our work. We appreciate the recognition of the novelty and systematic design of our study, particularly the dual functionality of the hybrid nanoemulsome platform for MEIS inhibitor delivery and bioimaging. We have carefully revised the manuscript in line with all comments and believe that these revisions have further strengthened the clarity and scientific rigor of the work.

 

Specific comments:

1. In Introduction, the authors mentioned that an "advanced carrier system is necessary" due to the physicochemical limitations of MEIS inhibitors. However, the text only briefly mentions chitosan and carbon dots without explicitly stating the unique advantages of this specific hybrid nanoemulsome design compared to other common nanocarriers such as liposomes or PLGA nanoparticles for this specific drug. It is suggested to supplement relevant literature for comparative analysis, and clarify the superiority of this hybrid system to highlight the rationality of the material selection.

Response 1: We thank the reviewer for this insightful and constructive comment. An explanatory paragraph has been added to the Introduction to clarify the rationale for selecting the chitosan–carbon dot hybrid nanoemulsion system, explicitly discussing its unique advantages over conventional nanocarriers such as liposomes and PLGA nanoparticles and its particular suitability for MEISi-2 delivery, supported by relevant literature.

1. In 2.2 and 2.3, the synthesis protocols describe using a "0.45µm pore-sized PES filter" to remove aggregates. Given that the final particle size of the drug-loaded system is reported as 322.8 nm, which is relatively close to the pore size, the authors need to clarify if there was any significant yield loss during this filtration step or if the polydispersity index suggests a population of particles larger than the pore size was lost.

Response 2: We thank the reviewer for this thoughtful and important comment. In response to Comment 2, the potential impact of the 0.45 µm PES filtration step on yield has been clarified by additional analysis: UV–Vis measurements showed only a minor decrease (~4–6%) in absorbance intensity after filtration (Figure S5), indicating minimal material loss. This slight reduction is attributed to the removal of occasional larger aggregates rather than the loss of the main nanoparticle population. The more noticeable decrease in absorbance beyond 400 nm further supports the elimination of aggregated fractions contributing to light scattering. Importantly, the characteristic absorption peaks associated with MEISi-2 remained unchanged, with no detectable wavelength shift, indicating that filtration did not significantly affect the drug-associated spectral profile and that the structural and functional integrity of the nanoemulsion system was preserved.

1. In Figure 3 (A and B), the bar charts displaying cell proliferation results lack statistical significance indicators such as asterisks. Although error bars are present, the graphs do not explicitly show whether the differences between the Control, CDChitosan, and CDChitosanMEISi groups are statistically significant. Please add these markers to the charts for clarity.

Response 3: We thank the reviewer for this helpful and constructive comment. Statistical analyses of the cell proliferation data have been added using one-way ANOVA followed by Dunnett’s multiple comparisons test. The Statistical Analysis subsection has also been updated to clearly specify the software and methods used.

 

1. In the Materials and Methods section, the manuscript describes various assays but omits a dedicated subsection on statistical analysis. The authors should add a paragraph specifying the statistical tests employed, and the software used to process the data.

Response 4: We thank the reviewer for this helpful and constructive comment. Statistical analyses of the cell proliferation data have been added using one-way ANOVA followed by Dunnett’s multiple comparisons test. The Statistical Analysis subsection has also been updated to clearly specify the software and methods used.

1. In Figure 2D, the AFM images are presented without clearly visible scale bars. Please update the figure to include clear scale indicators.

Response 5: We thank the reviewer for this valuable observation. The AFM images in Figure 2D have been revised to include enlarged and clearly visible scale bars, improving both clarity and visual accuracy.

 

1. In 3.2, the results indicate that MEISi-2 loading increased the particle size significantly from 150.9 nm to 322.8 nm. The authors should expand the discussion to address how this doubling in size might influence the passive targeting capability via the EPR effect or cellular uptake efficiency compared to the smaller empty carriers.

Response 6: We thank the reviewer for this insightful comment. In response to Comment 6, the Discussion section has been expanded with an additional paragraph addressing how the increase in particle size following MEISi-2 loading may influence passive tumor targeting via the enhanced permeability and retention (EPR) effect, as well as its potential implications for cellular uptake efficiency compared with the smaller empty carriers.

 

1. In 2.3, the phrase "Larger and big aggregates" is redundant and should be corrected. Furthermore, the manuscript requires careful proofreading to correct typos like "Gluteraldehyde" and inconsistent formatting in unit notations, such as the difference between "10.000 xg" and "14,000xg".

Response 7: We thank the reviewer for the careful reading and constructive suggestions. In response to Comment 7, the redundant phrase “larger and big aggregates” has been corrected, typographical errors such as “Gluteraldehyde” have been revised, and unit notations have been standardized throughout the manuscript to ensure consistency and clarity.

1. It is recommended to cite the following references, European Cells and Materials,2024,47, 152-169. https://doi.org/10.22203/eCM.v047a11. European Cells and Materials,2025,50,20-32. https://doi.org/10.22203/eCM.v050a02. European Cells and Materials, 50, 87-108 , https://doi.org/10.22203/eCM.v050a06.

Response 8: We thank the reviewer for this helpful recommendation. The suggested references from European Cells and Materials have been carefully reviewed and cited in the manuscript where they were relevant and appropriate, thereby strengthening the contextual background and supporting the discussion of our findings; references not directly pertinent were not included to avoid unnecessary citation.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors MDPI Life 399 V2 Feb 27 Abstract line 14 should read: ‘. . inhibitors have a restricted application in anti-cancer . . ‘ Will readers know what FTIR and DLS (line 20) mean? Line 32 should read ‘Colorectal cancer (CRC). . . ‘. Line 35 should begin with ‘proteins have shown . . ‘. What does ‘in a pro-metastatic factor’ mean? What is ‘stemness’ (line 40)? Line 44: the word ‘its’ should be replaced with ‘their’, since the word ‘inhibitors’ follows in line 45.  What does ‘can be interacted’ mean (line 51)? This likely means ‘can interact’. Line 53 should read ‘construction of hybrid . . ‘ Where is the ‘tradition’ (line 55)? What is ‘passivating’ (line 61)? Line 64 should read binding *of* chitosan. What is ‘salting out tendency’ (line 74)? In lineS 76 and 79, we see ‘Span80', but in the next line, this becomes ‘span 80'.  Line 105: what was ‘then filtered’? The word ‘with’ should be replaced with ‘through’.  Capitalization is often random. For example, in line 108, ‘chitosan’ is not capitalized, but EDTA and Acetic Acid are. In line 114, the first usage of ‘milliQ’ is not capitalized, but in the second appearance, this becomes ‘Milli-Q’. What does ‘stirring under vacuum presure (line 119) mean? Line 121 reads ‘For control system also contains . . ‘ Does this mean that ‘Controls used the same detergent system . . . ‘? Line 125, it appears that ‘use’ should be replaced with ‘used’. Line 126 should indicate ‘filtered through a 0.45 . . ‘. Lines 133-136 need to be rewritten. In their present form, details are difficult to deduce. What does ‘with using Varioscan LUX using kinetic mode’ mean? What does ‘at medium force were dissolving’ mean” Why is ‘Complete’ capitalized in line 135? The word ‘at’ in line 138 should be replaced with something more appropriate, e.g., in a solution of DMEM . . . ‘. Why does ‘%’ come before ‘0.25' in line 140? What does ‘were measured’ mean (line 142)? In line 145, the correct format is ‘and incubated overnight’.  How does the MTS assay assess ‘viability”? Viability can only be unambiguously determined by clonogenic assays. Line 159 should read ‘70%’. What is ‘the PE channel (line 162-3'? The phrase ‘and waited 24 hours’ (line 165) should be changed. It appears that the DMEM medium was replaced after treatment. This section, along with the rest of the report, needs to be inspected and edited by someome more familiar with English usage. Line 183: what is ‘fluorescence capacity’? This appears to be a fluorescence emission spectrum. Line 186 likely should read ‘to produce a strong blue luminescence signal . . ‘. Will readers remember what APTES and BA mean (line 190)? Line 200: if the word ‘differ’ is to be used, it should be preceded by ‘structures’. The inner filter effect (lines 219-220) only becomes pertinent if the concentration of the fluorophore is so high that photons are absorbed before they provide a signal. Line 224 reads ‘cellular internalization . . in the cells’. Where else could this occur? In line 231, we see ‘150.9', but in line 232: 322,8. Replace the comma with a period. Line 244: ‘complete DMEM with serum’ can be replaced by ‘the presence of serum’. How was ‘proliferation’ detected in Fig. 3? Panel E shows poor disbursal of cells. In such assays there should be a uniform pattern of colonies. When there are regions where cells are not uniformly disbursed, an accurate colony count is not possible. In the Discussion section, ‘oncogenic’ (line 279) means generating tumors. How can a protein both generate tumors and be ‘regulatory’?     Comments on the Quality of English Language

Generally adequate but there are regions (pointed out in the review) that need editing. 

Author Response

Comments 1: MDPI Life 399 V2 Feb 27 Abstract line 14 should read: ‘. . inhibitors have a restricted application in anti-cancer . . ‘ Will readers know what FTIR and DLS (line 20) mean? Line 32 should read ‘Colorectal cancer (CRC). . . ‘. Line 35 should begin with ‘proteins have shown . . ‘. What does ‘in a pro-metastatic factor’ mean? What is ‘stemness’ (line 40)? Line 44: the word ‘its’ should be replaced with ‘their’, since the word ‘inhibitors’ follows in line 45.  What does ‘can be interacted’ mean (line 51)? This likely means ‘can interact’. Line 53 should read ‘construction of hybrid . . ‘ Where is the ‘tradition’ (line 55)? What is ‘passivating’ (line 61)? Line 64 should read binding *of* chitosan. What is ‘salting out tendency’ (line 74)? In lineS 76 and 79, we see ‘Span80', but in the next line, this becomes ‘span 80'.  Line 105: what was ‘then filtered’? The word ‘with’ should be replaced with ‘through’.  Capitalization is often random. For example, in line 108, ‘chitosan’ is not capitalized, but EDTA and Acetic Acid are. In line 114, the first usage of ‘milliQ’ is not capitalized, but in the second appearance, this becomes ‘Milli-Q’. What does ‘stirring under vacuum presure (line 119) mean? Line 121 reads ‘For control system also contains . . ‘ Does this mean that ‘Controls used the same detergent system . . . ‘? Line 125, it appears that ‘use’ should be replaced with ‘used’. Line 126 should indicate ‘filtered through a 0.45 . . ‘. Lines 133-136 need to be rewritten. In their present form, details are difficult to deduce. What does ‘with using Varioscan LUX using kinetic mode’ mean? What does ‘at medium force were dissolving’ mean” Why is ‘Complete’ capitalized in line 135? The word ‘at’ in line 138 should be replaced with something more appropriate, e.g., in a solution of DMEM . . . ‘. Why does ‘%’ come before ‘0.25' in line 140? What does ‘were measured’ mean (line 142)? In line 145, the correct format is ‘and incubated overnight’.  How does the MTS assay assess ‘viability”? Viability can only be unambiguously determined by clonogenic assays. Line 159 should read ‘70%’. What is ‘the PE channel (line 162-3'? The phrase ‘and waited 24 hours’ (line 165) should be changed. It appears that the DMEM medium was replaced after treatment. This section, along with the rest of the report, needs to be inspected and edited by someome more familiar with English usage. Line 183: what is ‘fluorescence capacity’? This appears to be a fluorescence emission spectrum. Line 186 likely should read ‘to produce a strong blue luminescence signal . . ‘. Will readers remember what APTES and BA mean (line 190)? Line 200: if the word ‘differ’ is to be used, it should be preceded by ‘structures’. The inner filter effect (lines 219-220) only becomes pertinent if the concentration of the fluorophore is so high that photons are absorbed before they provide a signal. Line 224 reads ‘cellular internalization . . in the cells’. Where else could this occur? In line 231, we see ‘150.9', but in line 232: 322,8. Replace the comma with a period. Line 244: ‘complete DMEM with serum’ can be replaced by ‘the presence of serum’. How was ‘proliferation’ detected in Fig. 3? Panel E shows poor disbursal of cells. In such assays there should be a uniform pattern of colonies. When there are regions where cells are not uniformly disbursed, an accurate colony count is not possible. In the Discussion section, ‘oncogenic’ (line 279) means generating tumors. How can a protein both generate tumors and be ‘regulatory’?   

Response 1: We sincerely thank the reviewer for the careful reading of our manuscript and for the detailed comments regarding language, clarity, and scientific rigor. The suggested revisions significantly improved the overall quality, precision, and readability of the manuscript. All language-related issues have been carefully addressed throughout the revised version.

We thank the reviewer for important observation about Inner Filter Effect.  We agree that classical inner filter effects typically occur at high fluorophore concentrations. In our system, however, the absorbance remained between 3–4 AU even after dilution and filtration, as shown in Figure 2A and Supplementary Figure S5, indicating high optical density. Such optical density is sufficient to attenuate excitation and emission photons. Therefore, we have revised the manuscript to refer to “inner filter–like effects associated with high optical density and scattering” for clarity.

We thank the reviewer for this important clarification about MTS. We agree that clonogenic assays represent the gold standard for assessing long-term survival and proliferative capacity. The MTS assay measures cellular metabolic activity via reduction of tetrazolium salt by metabolically active cells and therefore reflects relative metabolic activity rather than direct clonogenic survival. To avoid ambiguity, the terminology in the manuscript has been revised accordingly (e.g., “proliferation” replaced with “metabolic activity (MTS assay)” where appropriate). In addition, colony formation analysis was performed to evaluate long-term proliferative capacity.

We appreciate the reviewer’s observation on the Colony Formation . Cells were seeded at a defined density (5000 cells/well) and carefully distributed to allow uniform attachment. Minor regional density variations may occur during long-term incubation due to biological growth dynamics and medium exchange. Colony quantification was performed across the entire well area under identical imaging conditions. The marked reduction in colony density and overall colony coverage in MEISi-2 and CDChitosanMEISi groups was clearly observable and, supporting the reliability of the assay.

We agree that the wording required clarification. MEIS proteins are transcriptional regulators that modulate gene expression programs involved in cell cycle progression, epithelial–mesenchymal transition (EMT), and s cancer stem cell–like. However, when dysregulated or overexpressed, MEIS proteins may exert oncogenic effects in colorectal cancer. The sentence has been revised accordingly to avoid ambiguity.