Possible Mechanisms of mRNA-LNP Degradation: A Comprehensive Review

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This review introduced degradation mechanisms and key factors of mRNA-LNP. However, the paper is not well organized.

1. Figure 1 in Introduction shows the instability factors across the mRNA-LNP workflow. But the text is just focused on the mRNA-LNP formulation. Therefore, the Introduction needs to be modified.
2. This paper just analyzed the degradation. It is not enough for a Review. Some strategies to overcome these degradation problems should be provided, which have been developed a lot and attracted more attentions. For example, as Table 2 shows, the storage temperature of Comirnaty is -70°C while that of mRNA-1273 is -20°C. How is this storage technique developed?
3. It is improper to use “physical degradation” to describe the behaviors such as aggregation, mRNA leakage. Therefore, it is suggested to use the word “instability” to replace “degradation”.

Author Response

We would like to thank the reviewer for his detailed and constructive comments. Please see below a point-by-point responses to the comments of reviewer-1 along modification underlined in the revised version of the manuscript.

This review introduced degradation mechanisms and key factors of mRNA-LNP. However, the paper is not well organized.

Comment 1: Figure 1 in Introduction shows the instability factors across the mRNA-LNP workflow. But the text is just focused on the mRNA-LNP formulation. Therefore, the Introduction needs to be modified.

Response: We thank the reviewer for this observation. A new paragraph has been added at the end of the Introduction that explicitly walks through every instability factor showed in Figure 1, including the upstream factors (enzymatic degradation, thermal and pH stress, incomplete 5′ capping, poly(A)-tail shortening, and process-related impurities) that were previously absent from the text. Each factor is now cross-referenced to the section where it is treated mechanistically: capping and poly(A) integrity in Section 4.1.1, and process impurities in Section 4.4.4. The Introduction therefore now covers the full scope of the figure - upstream, downstream, formulation, and storage/transport. Figure 1 has also been modified to include the missing points.

 

Comment 2: This paper just analyzed the degradation. It is not enough for a Review. Some strategies to overcome these degradation problems should be provided, which have been developed a lot and attracted more attentions. For example, as Table 2 shows, the storage temperature of Comirnaty is -70°C while that of mRNA-1273 is -20°C. How is this storage technique developed?

 

Response: We fully agree. A dedicated new Section 5, “Stabilization Strategies to Overcome Instability,” has been added. Rather than listing generic strategies, this section anchors each mitigation in a named product, regulatory filing, or primary study. The revised Table 2 now shows how those constraints have since been relaxed - refrigerated shelf life is now up to 10 weeks for Comirnaty and up to 60 days for Spikevax in the updated 2025–2026 product labels [References 4,7 in the manuscript]. A new summary Table 4 maps each degradation pathway from Section 4 to a mitigation strategy and a real-world example.

 

Comment 3: It is improper to use “physical degradation” to describe the behaviors such as aggregation, mRNA leakage. Therefore, it is suggested to use the word “instability” to replace “degradation”.

 

Response: We agree with this distinction. Throughout the revised manuscript, “physical degradation” has been replaced with “physical instability” from Line 359-362 when describing aggregation, fusion, leakage, and structural rearrangement.

 

Reviewer 2 Report

Comments and Suggestions for Authors

Major Comments

1. Figure 2 provides an overview of mRNA vaccine design; however, the schematic oversimplifies the immune response by depicting only antibody production as the final outcome. Since mRNA vaccines induce both humoral and cellular immunity, the figure and legend should be revised to provide complete information. In addition, the phrase "Antibodies, to target a specific pathogen" is grammatically incorrect and should be reworded for clarity.

2. In section 3, the stated molar ratios of LNP components appear overly generalized. Since lipid compositions vary among different mRNA-LNP formulations, the authors should clarify that these values are representative examples rather than universal ratios, or provide additional references supporting the stated ranges.

3. Figure 3 provides a useful overview of mRNA degradation pathways; however, it may oversimplify the mechanisms involved. The authors should clarify whether the schematic is intended to depict only the major degradation pathways or consider discussing additional degradation processes reported in the literature. The figure should be expanded into a more mechanistic schematic.

4. In section 4.2.1, the example describing reduced mRNA half-life in yeast under heat stress reflects intracellular mRNA turnover rather than degradation of formulated mRNA-LNP vaccines during storage. The relevance of this example to vaccine stability is unclear and should be better justified or replaced with data directly related to mRNA-LNP formulations.

5. The review does not adequately discuss the role of ionizable lipid pKa on formulation stability. The apparent pKa of the ionizable lipid is a critical parameter governing both endosomal escape efficiency and colloidal stability, and its alteration upon degradation is a key instability mechanism that is absent from this review.

6. The review focuses heavily on degradation mechanisms but provides limited discussion of solutions. A dedicated section discussing potential stabilization strategies would significantly improve the manuscript.

7. A major limitation of the review is the lack of comprehensive discussion regarding the analytical methods used to characterize mRNA-LNP integrity, degradation, and product quality. To enhance the practical value of the review, the authors should include a dedicated section summarizing commonly used analytical techniques, their applications, advantages, and limitations. Such a section would provide readers with a more comprehensive understanding of how mRNA-LNP degradation, structural integrity, particle characteristics, and biological activity are evaluated, thereby substantially improving the translational and practical relevance of the review.

Minor Comments

1. Language & Grammar

The abstract states that ‘the drawback of the instability of mRNA-LNP make their usage dependent on ultra-cold chain systems’ – this sentence is grammatically awkward and contains a subject-verb agreement error.

The sentence “Degradation of mRNA-LNP involves two separate keyways, including degradation of the mRNA itself and degradation of the lipid nanoparticles (LNPs) delivery system” should be revised for clarity and grammar. In particular, “keyways” appears to be incorrect terminology and should be replaced with “pathways” or “mechanisms.”

2. Table 2 contains outdated information

The storage data for Pfizer-BioNTech and Moderna have been updated multiple times since the initial approvals. The authors should update storage conditions using the most current product labels and regulatory guidance.

This review article addresses an important and clinically relevant topic in the mRNA vaccine field. The breadth of coverage and the organizational structure are commendable. However, the manuscript currently reads more like an introductory summary than a comprehensive critical review. Greater emphasis on mechanistic insight, analytical characterization methods, stabilization strategies, and recent advances in mRNA-LNP formulation science is needed. Upon successful revision addressing all major and minor comments, the manuscript has the potential to be a valuable contribution to the field.

 

Author Response

We would like to thank the reviewer for his detailed and constructive comments. Please see below a point-by-point responses to the comments of reviewer-1 along modification underlined in the revised version of the manuscript.

Major Comments

1. Figure 2 provides an overview of mRNA vaccine design; however, the schematic oversimplifies the immune response by depicting only antibody production as the final outcome. Since mRNA vaccines induce both humoral and cellular immunity, the figure and legend should be revised to provide complete information. In addition, the phrase "Antibodies, to target a specific pathogen" is grammatically incorrect and should be reworded for clarity.

Response: We have corrected this. Revised Figure 2 retains the original left-to-right flow (virus → mRNA → LNP/syringe → cell → immune output) but now mentions humoral and cellular immunity. The figure legend has been rewritten accordingly: “…host cells translate the mRNA into the target antigen, which is presented via MHC-I and MHC-II pathways to prime both humoral (antibody) and cellular (CD8⁺ and CD4⁺ T-cell) immunity against the pathogen.” The grammatical fragment “Antibodies, to target a specific pathogen” has been removed.

 

2. In section 3, the stated molar ratios of LNP components appear overly generalized. Since lipid compositions vary among different mRNA-LNP formulations, the authors should clarify that these values are representative examples rather than universal ratios, or provide additional references supporting the stated ranges.

 

Response: A clarifying sentence has been added immediately from Line 138-142 after the stated ratios in Section 3: “These molar ratios (ionizable lipid 40–50%, phospholipid 10–15%, cholesterol 35–45%, PEG-lipid 1–3%) are representative of the licensed COVID-19 products and related four-component systems rather than universal values; the optimal ratio is formulation- and lipid-dependent and is determined empirically for each construct [25,26].”

 

3. Figure 3 provides a useful overview of mRNA degradation pathways; however, it may oversimplify the mechanisms involved. The authors should clarify whether the schematic is intended to depict only the major degradation pathways or consider discussing additional degradation processes reported in the literature. The figure should be expanded into a more mechanistic schematic.

 

Response: Revised Figure 3 retains the central LNP-with-four-radiating-arrows layout of the original but now makes each quadrant mechanistic. Specifically: (i) mRNA chemical degradation now shows the 2′-OH in-line transesterification through a 2′,3′-cyclic-phosphate intermediate leading to chain scission, oxidation via ROS/aldehyde adducts, exo and endonuclease routes, and decapping/poly(A)-tail loss; (ii) LNP chemical degradation shows ester hydrolysis of ionizable/helper lipids, lipid peroxidation generating reactive aldehydes that form mRNA adducts, and N-oxide/impurity formation with consequent pKa shift; (iii) physical instability shows freeze–thaw → ice stress → fusion, aggregation, mRNA leakage, and PEG-lipid desorption; (iv) environmental stressors show temperature (Arrhenius acceleration), pH shifts, light/photo-oxidation, and moisture as a hydrolysis driver. The legend now states explicitly that the schematic depicts the major, experimentally supported pathways and that these routes are interdependent.

4. In section 4.2.1, the example describing reduced mRNA half-life in yeast under heat stress reflects intracellular mRNA turnover rather than degradation of formulated mRNA-LNP vaccines during storage. The relevance of this example to vaccine stability is unclear and should be better justified or replaced with data directly related to mRNA-LNP formulations.

 

Response: Thank you for pointing this issue. In the revised manuscript we removed  mRNA half-life in yeast under heat stress., We have added a directly relevant formulated-product example from Line 260-269. Mantri et al. showed that LNP encapsulation slows mRNA chemical degradation by up to approximately 9-fold compared with naked mRNA under matched thermal stress.

 

5. The review does not adequately discuss the role of ionizable lipid pKa on formulation stability. The apparent pKa of the ionizable lipid is a critical parameter governing both endosomal escape efficiency and colloidal stability, and its alteration upon degradation is a key instability mechanism that is absent from this review.

 

Response: A new sub-section 4.4.2(a), “Ionizable-lipid apparent pKa and instability,” has been added to the LNP key-factors discussion. This sub-section covers: (i) the TNS fluorescence assay as the standard measurement method [82]; (ii) the empirically optimal pKa ranges (~6.2–6.5 for hepatic delivery, ~6.6–6.9 for intramuscular immunogenicity), with the specific values for ALC-0315 (~6.09) and SM-102; and (iii) the critical point for this review that apparent pKa is not fixed but shifts upon hydrolysis or oxidation of the ionizable-lipid headgroup (e.g., N-oxide formation, ester cleavage), simultaneously degrading colloidal stability and endosomal-escape efficiency even when particle size appears unchanged. This provides a direct mechanistic link between the chemical degradation discussed in Section 4.3 and functional potency loss, and it is revisited in Section 6 (analytical methods) as a quality attribute requiring dedicated monitoring.

 

6. The review focuses heavily on degradation mechanisms but provides limited discussion of solutions. A dedicated section discussing potential stabilization strategies would significantly improve the manuscript.

 

Response: We fully agree. A dedicated new Section 5, “Stabilization Strategies to Overcome Instability,” has been added. The section is organized by target pathway (buffer/excipient optimization, lipid and RNA engineering, solid-state formulation, process controls), grounded in named products and primary studies, and concludes with a summary Table (table 4) mapping each degradation pathway to its strategy and evidence.

 

7. A major limitation of the review is the lack of comprehensive discussion regarding the analytical methods used to characterize mRNA-LNP integrity, degradation, and product quality. To enhance the practical value of the review, the authors should include a dedicated section summarizing commonly used analytical techniques, their applications, advantages, and limitations. Such a section would provide readers with a more comprehensive understanding of how mRNA-LNP degradation, structural integrity, particle characteristics, and biological activity are evaluated, thereby substantially improving the translational and practical relevance of the review.

 

Response: . A new Section 6, “Analytical Methods for Assessing mRNA–LNP Integrity and Quality,” has been added. It is organized by quality attribute (6.1, mRNA integrity; 6.2, encapsulation efficiency; 6.3, particle size and morphology; 6.4, lipid impurities, adducts, and pKa; 6.5, biological potency; 6.6, the need for orthogonal characterization) and also documents who has actually used each method: Mantri et al.’s CE-vs-HPLC comparison [77], Packer et al.’s IP-RP-HPLC/LC-MS identification of mRNA–lipid adducts [55,78], Birdsall et al.’s DNPH-LC-MS quantification of ALC-0315 aldehydes [57], Hermosilla et al.’s particulate analysis of in-use Comirnaty/Spikevax [16], and SCIEX BioPhase 8800 multi-capillary CGE for manufacturer release testing [79]. A new summary Table 5 groups methods by attribute, documented user, and key limitation. The section closes with the key caution that physical integrity and biological potency can decouple, so orthogonal characterization is mandatory.

 

Minor Comments

 

1. Language & Grammar

 

The abstract states that ‘the drawback of the instability of mRNA-LNP make their usage dependent on ultra-cold chain systems’ – this sentence is grammatically awkward and contains a subject-verb agreement error.

The sentence “Degradation of mRNA-LNP involves two separate keyways, including degradation of the mRNA itself and degradation of the lipid nanoparticles (LNPs) delivery system” should be revised for clarity and grammar. In particular, “keyways” appears to be incorrect terminology and should be replaced with “pathways” or “mechanisms.”

Response: Both corrected. The abstract sentence now reads: “The instability of mRNA–LNP products make their use dependent on ultra-cold-chain systems.” The word “keyways” has been replaced with “pathways”: “Degradation of mRNA–LNP proceeds through two distinct pathways: degradation of the mRNA itself and degradation of the LNP delivery system.” A full language and grammar pass has been performed on the revised manuscript.

 

2. Table 2 contains outdated informationThe storage data for Pfizer-BioNTech and Moderna have been updated multiple times since the initial approvals. The authors should update storage conditions using the most current product labels and regulatory guidance.

This review article addresses an important and clinically relevant topic in the mRNA vaccine field. The breadth of coverage and the organizational structure are commendable. However, the manuscript currently reads more like an introductory summary than a comprehensive critical review. Greater emphasis on mechanistic insight, analytical characterization methods, stabilization strategies, and recent advances in mRNA-LNP formulation science is needed. Upon successful revision addressing all major and minor comments, the manuscript has the potential to be a valuable contribution to the field.

 Response: Table 2 has been replaced with current label data reflecting the updated 2025–2026 formulations: Comirnaty −90 to −60 °C frozen, up to 10 weeks at 2–8 °C; Spikevax −50 to −15 °C frozen, up to 60 days at 2–8 °C. The original entries (CureVac CVnCoV development-stage data, BNT162b2 duplicating Comirnaty, and the now-superseded 6-month durations at −70/−20 °C) have been removed.

Reviewer 3 Report

Comments and Suggestions for Authors

The present review addresses an important topic, since mRNA-LNP stability remains one of the key challenges for the development and global deployment of mRNA-based therapeutics and vaccines. The manuscript is in general well organized and covers many of the principal degradation pathways affecting both mRNA and lipid nanoparticle components. However, the following issues need to be addressed before it can be considered for publication.

1. Figure 1 shows several instability factors (e.g., incomplete capping, poly(A) tail shortening, process-related impurities) which should be discussed in the text in the manuscript.
2. Figure 2 appears only indirectly related to degradation mechanisms. It would be good to include a diagram summarizing analytical methods used to evaluate mRNA-LNP stability.
3. The section 2 on mRNA structure is largely descriptive and contains well-known My suggestion is to focus more on structural features that directly influence degradation and stability rather than general mRNA biology.
4. Discussion of oxidative degradation in the section 4.1.3 (page 5): please distinguish between experimentally demonstrated degradation mechanisms and those that are currently hypothetical or inferred from broader RNA chemistry studies.
5. Table 1 is descriptive. I recommend to include the underlying degradation mechanism, impact on product quality, and potential mitigation strategies.
6. Discussion of temperature effects (page 6): The discussion relies mainly on storage recommendations for commercial vaccines. Please incorporate a mechanistic discussion of how temperature affects mRNA integrity, lipid degradation, and biological potency.
7. In page 7 please provide additional evidence for the statement that UV light penetrates LNPs and directly damages encapsulated mRNA.
8. pH and LNP stability (pH 7-9): The effects of pH on mRNA degradation are discussed, but the impact of pH on ionizable lipid behavior and nanoparticle structure should be also addressed.
9. In page 10 please expand the discussion of reactive lipid impurities and their role in mRNA modification and potency loss, since it is a very important point.
10. There is substantial overlap between Sections 4.2 and 4.4 regarding temperature, pH, moisture, and freeze-thaw effects. Please reduce repetitions.
11. The conclusion (page 10- 11) summarizes the discussion but remains mainly descriptive. My suggestion is to provide a more critical perspective on the unresolved challenges and future directions for improving mRNA-LNP stability.

Author Response

We would like to thank the reviewer for his detailed and constructive comments. Please see below a point-by-point responses to the comments of reviewer-3 along modification underlined in the revised version of the manuscript.

Comments and Suggestions for Authors

The present review addresses an important topic, since mRNA-LNP stability remains one of the key challenges for the development and global deployment of mRNA-based therapeutics and vaccines. The manuscript is in general well organized and covers many of the principal degradation pathways affecting both mRNA and lipid nanoparticle components. However, the following issues need to be addressed before it can be considered for publication.

Comment 1: Figure 1 shows several instability factors (e.g., incomplete capping, poly(A) tail shortening, process-related impurities) which should be discussed in the text in the manuscript.

Response: Addressed. The new Introduction paragraph now explicitly discusses each of these factors. Incomplete 5′ capping, poly(A)-tail shortening, and process-related impurities (residual enzymes, template DNA, dsRNA, trace metals) and cross-references where each is treated later in the manuscript. Capping and poly(A) integrity in Section 4.1.1, and process impurities in Section 4.4.4.

 

Comment 2: Figure 2 appears only indirectly related to degradation mechanisms. It would be good to include a diagram summarizing analytical methods used to evaluate mRNA-LNP stability.

Response: We agree that a methods-focused figure adds more value than the general vaccine-design schematic. While we have retained and corrected Figure 2, we have added a new section “Analytical Methods for Assessing mRNA–LNP Integrity and Quality” and a table summarizing the analytical methods for mRNA–LNP characterization, grouping techniques by the quality attribute they assess (integrity, encapsulation, size/morphology, lipid degradants/pKa, potency).

Comment 3: The section 2 on mRNA structure is largely descriptive and contains well-known My suggestion is to focus more on structural features that directly influence degradation and stability rather than general mRNA biology.

Response: Section 2 has been condensed and refocused. The revised opening eliminates textbook mRNA biology and instead centers on the structural features that directly govern stability: (i) the 5′ cap as the single most important determinant of 5′-end stability; (ii) poly(A)-tail length as a tunable stability parameter (~50 nt minimum, ~150 nt diminishing returns); (iii) UTR secondary structure and GC content as modulators of in-line hydrolysis susceptibility; and (iv) modified nucleosides (m1ψ) improving translation but not conferring chemical stability against hydrolysis. General statements about ribosome recruitment and codon optimization have been trimmed to focus on the stability implications.

 

Comment 4: Discussion of oxidative degradation in the section 4.1.3 (page 5): please distinguish between experimentally demonstrated degradation mechanisms and those that are currently hypothetical or inferred from broader RNA chemistry studies.

Response: A clarifying paragraph has been added at the end of Section 4.1.3 from Line 220-225 that explicitly separates the two categories. The experimentally demonstrated pathway is the indirect route: lipid peroxidation and N-oxide breakdown generate reactive aldehydes that form covalent adducts on mRNA cytosine residues, as demonstrated by Packer et al. using LC-MS with structural confirmation [55]. The inferred pathway is the direct oxidation of nucleobases by dissolved ROS or trace transition metals, which is chemically plausible and supported by broader RNA/DNA radiation- and oxidation-chemistry studies [49] but has not been directly demonstrated for formulated mRNA–LNP products. The text now explicitly flags the latter as “inferred from broader RNA chemistry” rather than presenting it as established for mRNA vaccines.

Comment 5: Table 1 is descriptive. I recommend to include the underlying degradation mechanism, impact on product quality, and potential mitigation strategies.

Response: Table 1 has been expanded from a two-column descriptive format to a four-column table with the columns: Factor, Underlying Mechanism, Impact on Product Quality, and Mitigation. All ten factors are now accompanied by their mechanistic basis (e.g., “Arrhenius-accelerated backbone hydrolysis; lipid oxidation; phase transitions” for temperature), their impact (e.g., “loss of full-length mRNA; aggregation; potency loss”), and specific mitigation strategies (e.g., “frozen/cold chain; lyophilization; thermostable lipids”). The mitigation column cross-references the new Section 5 for detailed discussion.

Comment 6: Discussion of temperature effects (page 6): The discussion relies mainly on storage recommendations for commercial vaccines. Please incorporate a mechanistic discussion of how temperature affects mRNA integrity, lipid degradation, and biological potency.

Response: The temperature discussion in Section 4.2.1 now leads with mechanism rather than storage labels from Line 259-268. The revised text explains that temperature accelerates mRNA degradation primarily through Arrhenius-governed phosphodiester backbone hydrolysis and through destabilization of secondary structure (less negative ΔG of folding), exposing single-stranded regions to nucleases. For the LNP, elevated temperature accelerates ester bond cleavage in ionizable and helper lipids and drives peroxidation of unsaturated tails, causing potency decline. The storage recommendations are now presented as the practical consequence of these mechanisms, not as the explanation itself. A formulated-product benchmark from Mantri et al. has been added to replace the over-reliance on the yeast intracellular-decay example.

 

Comment 7: In page 7 please provide additional evidence for the statement that UV light penetrates LNPs and directly damages encapsulated mRNA.

Response: We have revised the paragraph. The revised text in Section 4.2.2 from Line 273-290 now states that the better-supported mechanism is ROS-mediated photo-oxidation. UV and high-energy visible wavelengths generate reactive oxygen species that oxidize nucleobases and lipids, causing strand breaks and adducts. The statement that UV “penetrates LNPs and directly damages encapsulated mRNA” has been replaced with: “Whether UV penetrates intact LNPs to damage encapsulated mRNA directly is less firmly established than this ROS-mediated route. The practical recommendation is amber or opaque vials and minimized light exposure.

Comment 8: pH and LNP stability (pH 7-9): The effects of pH on mRNA degradation are discussed, but the impact of pH on ionizable lipid behavior and nanoparticle structure should be also addressed.

Response: The effects of pH on mRNA and LNP is discussed now. This has been addressed through two additions. First, the existing Section 4.4.2 on pH and buffer composition already discusses pH-induced phase transitions (SAXS, DLS evidence) and ionization-dependent aggregation propensity; we have strengthened this by connecting it explicitly to the lamellar–inverse-hexagonal phase behaviour that underlies endosomal escape. Second, the new sub-section 4.4.2 on apparent pKa directly explains how pH governs the ionizable lipid’s protonation state, surface charge, phase behaviour, and therefore both colloidal stability and delivery function. The two sub-sections together now provide a comprehensive picture of pH effects on both mRNA and LNP components.

 

Comment 9: In page 10 please expand the discussion of reactive lipid impurities and their role in mRNA modification and potency loss, since it is a very important point.

Response: This is now elaborately explained in Section 4.4.4 has been expanded with the mechanistic discussion. The expanded text explains that aldehydes and N-oxides from ionizable-lipid synthesis or oxidative aging form covalent adducts on mRNA nucleobases (notably cytosine) and phosphate groups. Packer et al. demonstrated these adducts block translation using LC-MS with structural confirmation [55]. Birdsall et al. quantified aldehyde levels above 0.05% in approximately 30% of ALC-0315 batches by DNPH derivatization–LC-MS, correlating impurity load with mRNA degradation [57]. Even sub-percent impurity levels cause disproportionate potency loss because a single adduct on a long transcript can abolish full-length translation. We concluded the paragraph by motivating >99% lipid purity specifications, antioxidant/chelator inclusion (cross-referencing Section 5.2), and routine LC-MS monitoring (cross-referencing Section 6.4).

 

Comment 10: There is substantial overlap between Sections 4.2 and 4.4 regarding temperature, pH, moisture, and freeze-thaw effects. Please reduce repetitions.

Response: Thank you for pointing this out. We worked on these sections to remove all the duplications between these two sections.

 

Comment 11: The conclusion (page 10-11) summarizes the discussion but remains mainly descriptive. My suggestion is to provide a more critical perspective on the unresolved challenges and future directions for improving mRNA-LNP stability.

Response: We found this comment so helpful to make a meaningful conclusion. The conclusion is now fully rewritten covering unresolved challenges and future directions for improving mRNA-LNP stability.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have adequately addressed my comments, and the manuscript has been significantly improved. I have no further comments and believe the manuscript is suitable for publication.

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for addressing all comments.