Review Reports - Chitosan-Based Drug Delivery Systems for Targeted Chemotherapy in Colorectal Cancer: A Scoping Review

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review focuses on the application of chitosan to develop drug delivery systems for the treatment of colorectal cancer. The topic of the review is interesting and worthy of publication. However, the quality of this review requires a substantial improvement. Please take the following into consideration:

Introduction: several advantages and limitations of chitosan were mentioned but references are lacking. Particularly references to confirm the limitations are important. Also some further explanations why poor thermal stability of chitosan is relevant here is important
The discussion of literature found by the authors is very superficial. Many studies are described in a few sentences. If a study selected to be mentioned in the review then it should receive appropriate attention. It is not enough to just mention what the authors have done. The authors should provide sufficient explanation and description of their study and should attempt to express own evaluation of their work.
The review would benefit from addition of illustrations.
it is important to add some discussion of the routes of drug administration used in the studies discussed. How the route of administration used achieved drug targeting? What were the physiological barriers and how these were overcome?

Author Response

Reviewer 1

Comments and Suggestions for Authors

1. Introduction: several advantages and limitations of chitosan were mentioned but references are lacking. Particularly references to confirm the limitations are important. Also some further explanations why poor thermal stability of chitosan is relevant here is important

Answer:

We would like to thank the Reviewer for this insightful and constructive comment. In response, we have revised the Introduction to include appropriate references that substantiate the limitations of chitosan. Furthermore, we have elaborated on the relevance of chitosan’s poor thermal stability in the context of formulation stability and processing. We have also addressed batch-to-batch variability and challenges associated with sterilization methods, with supporting references now provided [11–20]:

“Although CS has numerous valuable advantages, it also has several limitations that affect its use in the delivery of anticancer drugs. These include low thermal stability [11,12] and ductility [13,14], poor solubility of CS above pH 6.5 [15,16], high hydrophilicity and swelling properties, which may lead to premature drug release and reduced structural stability. Moreover, CS exhibits considerable batch-to-batch variability due to differences in source material, degree of deacetylation, and purity, making re-producibility challenging. Its solubility and viscosity are also sensitive to pH and temperature, respectively, which can further impact formulation stability and processing [17-19]. Limitations in sterilization methods also present challenges. Yang et al. [20] demonstrated that steam sterilization significantly darkens CS powder, gamma irradiation causes strong depolymerization above 10 kGy, and ethylene oxide induces only minor changes in crystallinity and structure.”

The revised paragraph has been highlighted in yellow in the manuscript for your convenience

2. The discussion of literature found by the authors is very superficial. Many studies are described in a few sentences. If a study selected to be mentioned in the review then it should receive appropriate attention. It is not enough to just mention what the authors have done. The authors should provide sufficient explanation and description of their study and should attempt to express own evaluation of their work.

Response:
We thank the Reviewer for this important and constructive comment. In the revised manuscript, we have substantially expanded the depth and critical rigor of the literature discussion.

A detailed analysis has been added for each study included in Table 1 (non-targeted systems). Instead of brief descriptions, the revised text now discusses mechanistic rationale of the formulation strategy, drug release behavior in relation to CRC-relevant physiological conditions, interpretation of cytotoxicity and uptake data, strengths and limitations of each experimental design. These expanded analyses are now found in Sections 2.1.1.1, 2.1.1.2, and 2.1.2.

The revised sections explicitly interpret the findings, rather than summarizing them descriptively. For example, we now explain why dual-layer oral carriers outperform simple CS-based NPs, why magnetic systems may artificially inflate tumor-targeting effectiveness, and which physicochemical features of CS formulations contribute most to biological outcomes. A new critical subsection (2.1.2) has been incorporated to provide: evaluation of evidence quality, discussion of methodological weaknesses, limitations of study designs (2D models, small samples, lack of comparators), impact of CS material heterogeneity on result comparability.

Our own assessment and interpretation have been added throughout, highlighting weaknesses, overinterpretation risks, and translational gaps, including lack of immunocompetent in vivo models, insufficient PK/PD data, and inconsistent reporting of chitosan properties.

We believe these revisions significantly strengthen the analytical depth of the review and fully address the Reviewer’s concern regarding superficial discussion.

3. The review would benefit from addition of illustrations.

Answer : We thank the Reviewer for this helpful suggestion. In the revised manuscript, we have added several new illustrations to improve clarity and visual understanding. Chemical structure of chitosan (Figure 1), flowchart showing the classification of the 25 included studies into non-targeted and ligand-modified CS-based DDSs (Figure 2), schematic of receptor-mediated endocytosis via FRα, CD44, and ASGPR (Figure 3), illustration of FRα-mediated uptake of folate-modified CS-based DDS (Figure 4). These figures provide clear visual support for the discussed mechanisms and targeting strategies, and we believe they substantially enhance the readability and scientific value of the manuscript.

4. it is important to add some discussion of the routes of drug administration used in the studies discussed. How the route of administration used achieved drug targeting? What were the physiological barriers and how these were overcome?

Answer: We would like to thank the Reviewer for this insightful and important comment.

In response, we have added a new subsection titled 2.1.1. Routes of Administration and Physiological Barriers, which provides a detailed discussion of the administration routes used in the included studies and how they relate to drug targeting efficiency in colorectal cancer therapy.

This section is further divided into 2.1.1.1. Oral CRC-Targeted Delivery and 2.1.1.2. Intravenous Delivery and Systemic Barriers. We describe the key physiological barriers encountered by each route, such as gastric degradation, first-pass metabolism, mucus penetration for oral delivery, and rapid opsonization, RES clearance, vascular permeability, and tumor microenvironment challenges for intravenous delivery.

Moreover, we discuss specific strategies used in the reviewed drug delivery systems to overcome these barriers. These include pH-responsive and enzyme-sensitive coatings, mucoadhesive and thiolated chitosan matrices, PEGylation, charge-reversal systems, redox-sensitive nanostructures, magnetic targeting, and ultrasound-triggered release. Examples from the literature are provided to illustrate how these designs contributed to enhanced tumor accumulation, controlled release, and minimized off-target toxicity.

This newly added content has been highlighted in yellow in the manuscript for your convenience.

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript presents a scoping review on chitosan-based drug delivery systems for colorectal cancer, categorizing them into non-targeted and ligand-modified systems. The topic is relevant to the field of nanomedicine and targeted cancer therapy. The review is generally well-structured, and the PRISMA-ScR methodology is a strength. The most critical issues are the lack of insufficient critical depth in the analysis. Substantive revisions are required to elevate the manuscript from a descriptive summary to an insightful review that provides genuine added value to the field.

1) The manuscript primarily functions as a catalog of studies, summarizing findings in extensive tables without providing a powerful, overarching narrative or novel conceptual framework. The "Comparative Analysis" section (2.3) is a step in the right direction but remains superficial, listing expected differences (e.g., ligand-modified systems have higher uptake) without delving into the underlying reasons for the performance variance across different ligand types or CS formulations. The review misses the opportunity to propose a "design roadmap" for optimal CS-based DDS for CRC, weighing factors like ligand choice, polymer modification, and formulation type against specific CRC subtypes or therapeutic challenges.

2) The decision to limit the search to January 2020 – June 2025 is a major methodological weakness that severely undermines the review's comprehensiveness and validity. The field of CS-based DDS for cancer did not begin in 2020. This arbitrary cutoff excludes a vast body of foundational and highly relevant work, potentially creating a significant selection bias. This suggests the search strategy, despite following PRISMA, may have been too restrictive or the keywords may have missed key studies.

3) Many of the cited in vivo studies use xenograft models in immunocompromised mice. The review fails to critique the limited translational relevance of these models, which do not recapitulate the human immune response to the nanoparticles or the complex CRC tumor microenvironment (TME).

4) The studies employ vastly different CS sources, degrees of deacetylation, molecular weights, cross-linking methods, and drug-loading techniques. The review does not critically discuss how this extreme heterogeneity prevents direct comparison and makes it impossible to draw generalizable conclusions about "chitosan" as a monolithic material.

5) The review reports impressive results (e.g., "80% tumor suppression") without critically evaluating the robustness of the experimental design, the statistical power, or the potential for over-interpretation of single, often preliminary, studies.

6) While the "Clinical Perspective" section correctly notes the absence of clinical trials, the discussion on why this translational gap exists is inadequate. The review should provide a deep, critical analysis of the barriers beyond the commonly stated "need for more in vivo studies."

The challenges of reproducible, large-scale synthesis of well-defined CS nanoparticles. The complexity of regulatory approval for a natural polymer with batch-to-batch variability. A critical analysis of the almost universally lacking comprehensive PK/PD and toxicokinetic data for these systems. A discussion on the limitations of active targeting in humans (e.g., receptor heterogeneity, variable expression, the "binding site barrier" effect).

Author Response

Reviewer 2

Comments and Suggestions for Authors

The manuscript primarily functions as a catalog of studies, summarizing findings in extensive tables without providing a powerful, overarching narrative or novel conceptual framework. The "Comparative Analysis" section (2.3) is a step in the right direction but remains superficial, listing expected differences (e.g., ligand-modified systems have higher uptake) without delving into the underlying reasons for the performance variance across different ligand types or CS formulations. The review misses the opportunity to propose a "design roadmap" for optimal CS-based DDS for CRC, weighing factors like ligand choice, polymer modification, and formulation type against specific CRC subtypes or therapeutic challenges.

Answer: We thank the Reviewer for this valuable and constructive remark. In response, we have substantially expanded Sections 2.2 and 2.3, adding in-depth critical interpretation rather than descriptive summaries. The revised text now explains why different ligand types vary in efficiency, discussing receptor heterogeneity, ligand-receptor affinity, steric accessibility, and the binding-site barrier. We have also strengthened the conceptual framework by clarifying mechanistic determinants of uptake, release behavior, and tumor selectivity, and by highlighting the limitations of each ligand type in CRC. Additionally, Section 2.3 now offers a more analytical comparison and outlines design considerations that may guide future development of optimized CS-based DDSs.These revisions significantly enhance the interpretative depth of the review and directly address the Reviewer’s concern.

Answer : We acknowledge the Reviewer’s concern regarding potential selection bias. However, the scoping nature of our review focused on mapping current research directions rather than providing a historical overview, justifies this temporal restriction and maintains methodological integrity.

Answer : We thank the Reviewer for this important observation. In the revised manuscript, we have added a detailed critique of the limited translational relevance of xenograft models. Specifically, Section 2.1.2 now discusses how immunocompromised mouse models lack key components of the human immune system, stromal interactions, cytokine signaling, and TME complexity, which may lead to overestimation of tumor accumulation and therapeutic efficacy of CS-based DDS. We also highlight the inability of these models to predict nanoparticle immunogenicity or long-term biosafety. This expanded discussion directly addresses the Reviewer’s concern and strengthens the translational perspective of the review.

Answer: thank the Reviewer for this valuable comment. In the revised manuscript, we have added a detailed discussion addressing the extensive heterogeneity of chitosan materials used across studies. Section 2.1.2 now explicitly highlights how differences in molecular weight, degree of deacetylation, viscosity grade, cross-linking density, and purification source critically influence solubility, charge distribution, mucoadhesion, and cellular uptake. We also emphasize that inconsistent reporting of these parameters limits comparability between studies and prevents drawing generalizable conclusions about chitosan as a uniform material. This expanded analysis directly addresses the Reviewer’s concern and strengthens the methodological critique of the field :

”A substantial barrier is the heterogeneity of CS materials used across formulations. Formulations varied widely in molecular weight, degree of deacetylation, viscosity grade, type of cross-linking agents, density, and purification source, critically affect solubility, charge distribution, mucoadhesion, and cellular uptake. Due to inconsistent reporting, direct comparisons between formulations are limited and broader generalization is not possible.”

Answer : We thank the Reviewer for this important observation. In the revised manuscript, we have added a critical evaluation of the robustness of reported efficacy data, including concerns related to small sample sizes, short study duration, lack of randomization or blinding, and absence of appropriate comparator groups. Section 2.1.2 now explicitly cautions against over-interpreting single-study tumor inhibition results and emphasizes that many of these outcomes originate from preliminarily designed experiments with limited statistical power. This additional critique strengthens the scientific rigor of the review and directly addresses the Reviewer’s concern.

“Another concern relates to the credibility and robustness of reported efficacy data. In microbiome-assisted delivery, SN-38 CS-based systems co-administered with Bifidobacterium bifidum, achieved approximately 80% tumor suppression, doubling the efficacy observed without probiotic co-treatment [43]. However, such high inhibition rates were often reported from short-duration studies with unclear or unreported sample sizes, and lacking key methodological details. Consequently, these outcomes should be interpreted with caution until verified in larger, statistically powered in vivo experiments. In many cases, studies lacked appropriate comparator groups, such as standard chemotherapeutic regimens, making it difficult to determine the true clinical relevance of the observed improvements.”

Answer : We appreciate the Reviewer’s insightful comment regarding the translational gap. In the revised manuscript, Section 2.1.2 have been expanded to provide a more comprehensive analysis of barriers preventing clinical progression of CS-based DDSs.

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript by Piotrowska et al. provides a timely review of chitosan-based drug delivery systems for colorectal cancer treatment. The topic is highly relevant and will be of significant interest to the readership. A particular strength of the work is the meticulous description of the literature selection methodology. Prior to acceptance, we recommend minor revisions to enhance the manuscript's clarity, as outlined below.

Latin terms (e.g., in vivo, in vitro, species names) should be italicized.
While the manuscript contains sufficient tables, it is lacking in figures (only the study design scheme is provided). I encourage the authors to incorporate additional illustrative material to improve comprehension.
Keywords: The authors identify Cs-NPs as the most promising basis for drug delivery systems, yet "nanoparticles" (NPs) is omitted from the keyword list.
Lines 82-85: A flowchart illustrating this process, including the different ligand types, would be beneficial.
To aid understanding, we recommend including the structural formulas of folic acid, hyaluronic acid, and galactose.
The list of abbreviations appears incomplete and should be revised accordingly.
From the authors' perspective, what is the current higher priority: developing new delivery systems in laboratory settings (using in vitro tests, 2D cell models, and laboratory animals) or focusing on clinical trials of existing formulations?

Author Response

Reviewer 3

Comments and Suggestions for Authors

1. Latin terms (e.g., in vivo, in vitro, species names) should be italicized.

Answer: We thank the Reviewer for this comment. All Latin terms (e.g., in vitro, in vivo, species names) have now been consistently italicized throughout the revised manuscript.

2. While the manuscript contains sufficient tables, it is lacking in figures (only the study design scheme is provided). I encourage the authors to incorporate additional illustrative material to improve comprehension.

3. Keywords: The authors identify Cs-NPs as the most promising basis for drug delivery systems, yet "nanoparticles" (NPs) is omitted from the keyword list.

Answer : We thank the Reviewer for this helpful suggestion. The keyword list has been updated to include “nanoparticles (NPs)” to reflect the emphasis placed on CS-based nanoparticle systems in the manuscript.

4. Lines 82-85: A flowchart illustrating this process, including the different ligand types, would be beneficial.

Answer: Thank you for the suggestion. Following the reviewer’s recommendation, we have added an additional flowchart (Figure 2) illustrating the categorization of chitosan-based DDS identified in the review, including the different types of targeting ligands. The PRISMA flowchart remains unchanged as it follows the standardized structure.

5. To aid understanding, we recommend including the structural formulas of folic acid, hyaluronic acid, and galactose.

Answer : We thank the Reviewer for this helpful suggestion. In the revised manuscript, we have incorporated the structural formulas of folic acid, hyaluronic acid, and galactose within the newly added Figure 3, which illustrates receptor-mediated endocytosis via FRα, CD44, and ASGPR. Including the ligand structures directly in this schematic improves clarity and strengthens the visual explanation of ligand-specific interactions.

6. The list of abbreviations appears incomplete and should be revised accordingly.

Answer : We thank the Reviewer for this remark. The list of abbreviations has been carefully revised and updated to include all relevant terms used throughout the manuscript, ensuring completeness and consistency.

7. From the authors' perspective, what is the current higher priority: developing new delivery systems in laboratory settings (using in vitro tests, 2D cell models, and laboratory animals) or focusing on clinical trials of existing formulations?

Answer : We thank the Reviewer for this thoughtful question. From our perspective, clinical trials cannot yet be considered the primary priority for CS-based DDS in CRC. The field first requires a strong foundational framework to ensure reproducibility, comparability, and regulatory readiness. Therefore, the most urgent priority is standardization of CS materials (degree of deacetylation, molecular weight, purity, viscosity) and harmonization of formulation and characterization methods; robust comparative preclinical evaluation, including 3D tumor organoids, co-culture models, and standardized benchmarks against current CRC therapies and finally, well-designed animal studies in immunocompetent models with full PK/PD, biodistribution, and toxicokinetic assessment. Only after these gaps are addressed will existing CS-based formulations be sufficiently mature for meaningful early-phase clinical trials.