This manuscript by Shoeb Ahmed and his co-workers presents a study on the effect of biomixing temperature on chitooligosaccharide (COS)-based hydrogels for controlled drug release. The study is well-structured, incorporating comprehensive characterization methods such as FTIR, SEM, XRD, DSC-TGA, and swelling kinetics. The results provide valuable insights into temperature-dependent drug delivery applications. However, several areas need refinement to improve clarity, scientific rigor, and presentation.
Major Strengths
Relevance and Novelty
• The study addresses an important area in drug delivery, specifically optimizing hydrogel formulations for targeted and controlled drug release.
• The impact of biomixing temperature on hydrogel properties and drug release has been explored systematically.
Comprehensive Characterization
• The manuscript employs a range of analytical techniques (FTIR, SEM, XRD, DSC-TGA) to validate findings.
• The swelling behavior and drug release kinetics models provide meaningful interpretations.
Scientific Impact and Application
• The work has potential applications in developing temperature-responsive drug delivery systems, particularly for colonic drug delivery.
• The study effectively demonstrates the role of polymer interactions in controlling drug release.
Major Issues
Clarity and Organization Issues
Abstract: The abstract is concise but could use a clearer structure. While it summarizes the findings, it does not explicitly state the significance or broader applications of the research.
Introduction: The introduction is informative but could be more focused. It presents background information, but it is somewhat broad, especially in the early paragraphs. While the introduction covers hydrogel-based drug delivery, the transition to the study's specific objectives is abrupt. Clearly state the research gap and the hypothesis tested.
Scientific Rigor and Data Interpretation Issues
XRD Analysis (Figure 6)
• The claim that 4°(D) hydrogel has higher amorphous content than N(D) needs quantification.
DSC-TGA Analysis (Figure 7)
• The thermogram interpretation could be improved.For instance: "The shift in exothermic peaks upon drug incorporation indicates potential hydrogen bonding between vancomycin and the hydrogel matrix, modifying its thermal stability."
Swelling Behavior and LCST (Figure 8)
• The claim that PEGDA reduces LCST should be substantiated with prior literature or additional data.
Drug Release Kinetics (Figure 9)
• While the Korsmeyer-Peppas model is used, there is no discussion on the mechanism of diffusion. Please explain what the “n” values indicate in terms of drug transport behavior (Fickian vs. non-Fickian diffusion).
Minor Issues
Use of acronyms should be standardized throughout the text. Also, figures need better annotation. For example, Figure 3 should have arrows marking key morphological differences in SEM images.Also try to make uniform boldness of scalebar within the SEM imgaes. The font size in figure legends is too small — consider improving readability.

The authors have submitted a revised version of the manuscript titled "Effect of Biomixing Temperature on Chitooligosaccharide-Based Hydrogels for Controlled Drug Release", addressing several of the concerns raised in the initial review. The revised manuscript reflects a sincere effort to improve both the scientific clarity and overall presentation. The topic remains relevant to the field of drug delivery, and the findings provide insightful contributions to the development of temperature-responsive hydrogel systems.
While the majority of the previous comments have been adequately addressed, a few critical points still require further attention and clarification.

1. Relevance and Novelty
   • The study continues to address a timely topic in drug delivery, specifically evaluating the impact of biomixing temperature on chitooligosaccharide (COS)-based hydrogel formulations for controlled release.
   • The revised manuscript maintains a systematic approach to examining temperature influence and presents potentially valuable insights into formulation optimization.
2. Comprehensive Characterization
   • The authors have retained a diverse set of analytical methods (FTIR, SEM, XRD, DSC-TGA, swelling kinetics) to support their findings.
   • Drug release kinetics models remain relevant and are supported by experimental data.
3. Scientific Impact and Application
   • The research holds continued promise for developing thermoresponsive hydrogels applicable to colonic or targeted drug delivery systems.
   • The revision has improved overall coherence in the interpretation of drug-polymer interactions.

Remaining Concerns
Although most of the previous comments have been taken care of, the following areas still require consideration:
1. XRD Analysis (Figure 6)
• Issue: The claim regarding the amorphous nature of the 4°(D) hydrogel remains qualitative.
• Suggestion: Quantify the crystalline/amorphous ratio using crystallinity index analysis or peak integration to validate the claim and strengthen the argument.
2. Drug Release Kinetics (Figure 9)
• Issue: Although the Korsmeyer-Peppas model is applied, interpretation of the “n” value remains insufficient.
• Suggestion: Include a brief discussion on what the “n” value indicates in terms of release mechanism (e.g., Fickian, anomalous, or case-II transport) and relate it to the structural properties of the hydrogel matrix.

Minor Issues
• Acronyms: Acronyms like COS, PEGDA, and LCST are inconsistently defined or reintroduced without clarification. Please standardize their usage and define them upon first appearance in each major section.
• Figure Annotations: Figures such as Figure 3 (SEM) should include annotations (arrows or circles) to highlight differences in surface morphology. This will aid interpretation.
• Figure Legends: Font size in figure legends has improved slightly but remains difficult to read in print. Please increase font size for better readability.

Additional Recommendations
• Language and Grammar: Minor grammatical and typographical errors persist. A professional English proofreading service is recommended to enhance clarity.
• Conclusion: The conclusion section could be strengthened by clearly stating the contribution of this work to the field and suggesting potential follow-up studies such as in vivo evaluation or formulation scale-up.

Overall Recommendation: The manuscript is scientifically sound and presents valuable data; however, a few scientific and editorial revisions are still necessary before final acceptance.

The authors have submitted a revised manuscript titled "Effect of the Temperature of Biomixing on the pH/Temperature Sensitive Controlled Drug Release of a Chitooligosaccharide-Based Hydrogel". I have carefully evaluated the resubmitted version in light of the comments provided during the previous review cycle.

Major Revisions Addressed:

• XRD Analysis: The authors have included quantitative crystallinity index data and crystalline/amorphous ratios, thereby strengthening their claim regarding the amorphous nature of the 4°(D) hydrogel.
• Drug Release Kinetics: The interpretation of the Korsmeyer–Peppas model has been clarified, with an adequate discussion of the “n” value and its relevance to the drug release mechanism.
• Figure Annotations: SEM images have been improved with appropriate annotations (arrows and circles) to highlight morphological features and drug loading differences.
• Conclusion: The conclusion has been revised to better articulate the study’s significance and suggests directions for future research, including in vivo studies and broader exploration of biomixing temperatures.

Minor Issues (Partially Addressed):
• Acronym Consistency: While most acronyms are now defined upon first use, a few still lack consistent introduction in later sections.
• Figure Legend Readability: Font size improvements are not explicitly verifiable from the document provided.
• Language and Grammar: Minor grammatical errors persist; the manuscript would benefit from final proofreading by a native English speaker or professional editing service.

Recommendation:
The authors have addressed all major scientific concerns raised during the initial review, and the manuscript now presents a coherent and well-supported study with enhanced clarity and scientific rigor. I recommend acceptance pending minor editorial revisions, particularly language polishing and consistency in terminology.