Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript describes the development of a novel library of thioester-containing ionizable lipids (TAILs) designed to improve the delivery and safety profile of RNA therapeutics. The study is exceptionally thorough, moving from rational molecular design to sophisticated in vivo models.

A particular strength of this work is the identification of the lead candidate CP-LC-1272, which demonstrates superior endosomal escape and biodegradability compared to the gold-standard SM-102. Furthermore, the successful application in a tRNA delivery model for protein restoration highlights the broad potential of this platform. The data regarding long-term storage stability after lyophilization is also a highly valuable addition for the pharmaceutical science community. This is a high-quality contribution to the field of non-viral delivery systems.

Author Response

We would like to thank the reviewers for their careful reading of our manuscript and for their constructive and thoughtful comments. We appreciate the opportunity to revise and improve the manuscript in response to their feedback. Please find our point-by-point responses in the attached Word document.

Author Response File: Author Response.docx

Reviewer 2 Report

Comments and Suggestions for Authors

The authors synthesized a library of ionizable lipids for use as RNA carriers within lipid nanoparticles. Among the synthesized compounds, the most active substances were selected, whose properties as RNA carriers were thoroughly studied by the authors. Not only the transfection efficiency but also potential toxicity limitations of the new nanoparticles were determined. It should be noted that in addition to testing transfection using mRNA, the authors demonstrated the effectiveness of the identified candidate ionizable lipid for tRNA delivery. A distinctive feature of the ionizable lipids synthesized by the authors is the introduction of a thioether bond linking two fragments of the structure into the whole lipid molecule. However, the authors did not explain why it was necessary to introduce a more hydrolytically unstable bond between the components they selected. Hydrolysis of this bond also results in the formation of unnatural lipid-like structures (metabolite A and the second component), which may be toxic if repeated administration is required. This aspect of the study requires further clarification.

Minor remarks

In the Materials and Methods of section 2.3, it is necessary to indicate the main points of the synthetic schemes, provide points to the schemes, and provide a list of the synthesized compounds with purity characteristics.

#151 “After adjusting to a concentration of 100 ug/mL RNA, LNPs were lyophilized…”  Describe the procedure (dilute, concentrate, solvent?) Where there any crioprotectors?

#167-168

mRNA encapsulation efficiency was determined via Quant-IT® Ri-167 bogreen assay. [Reference!]  To measure apparent pKa of LNPs, 6-(p-toluidinyl)naphthalene-2-sulfonic 168 acid (TNS) assay [Reference!] was performed

#198 “The appearance of metabolite A and B was monitored using HPLC-MS”
 Please, specify the structure of each metabolite.

#371-374

“All LNP formulations exhibited favorable physicochemical characteristics, including  high encapsulation efficiencies, appropriate particle sizes with low polydispersity, and  suitable surface charges and apparent pKa values consistent with effective delivery performance. Detailed measurements are provided in Supplementary Table S1.”

Please. Provide these data for the most active compounds ( e.g. CP-LC-1272, CP-LC-1539, and CP-LC-1545 ) as a table within the main manuscript text.

Figure 2 E. The statistical data (mean ± standard deviation ) for  all three datasets are absent. Is the difference between values statistically significant?

Figure 4 D. The same remark. In the capture authors pointed out “Data represent mean ± SD (n = 3 biologically independent 435 samples).#375” but at the picture theses data are absent.

# 596 “Notably, our optimized freeze-drying process enabled the 596 production of lyophilized LNPs that retained full biological activity for at least six months when stored at 4 °C “ [ Please, specify characteristics of “optimized freeze-drying process”.

A significant number of sources in the list of references lack DOI values  (e.g. 1-9, 11-13).

In the Supplementary materials, it is necessary to provide the characteristics of all reagents used (purity, manufacturer, country).

For synthesized compounds, purity must be indicated.

Author Response

We would like to thank the reviewers for their careful reading of our manuscript and for their constructive and thoughtful comments. We appreciate the opportunity to revise and improve the manuscript in response to their feedback. Please find our point-by-point responses in the attached Word document.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The manuscript presents the design and evaluation of a library of thioester-containing ionizable lipids intended to improve endosomal escape and biodegradability of lipid nanoparticles for mRNA and tRNA delivery. The study combines synthetic chemistry, structure–activity relationship (SAR) screening, and in vivo evaluation to identify CP-LC-1272 as a promising lead candidate. The topic is timely and relevant given the rapid development of RNA therapeutics and the need for safer, more biodegradable lipid nanoparticle systems. However, the main weakness of the work is the limited depth of mechanistic validation supporting the central claim that thioester incorporation improves endosomal escape and biodegradability. While the authors present performance comparisons and biodegradation observations, the mechanistic link between the thioester chemistry and the observed biological outcomes is largely inferred rather than directly demonstrated. My specific comments are as follows;

1. The central claim of the manuscript is that thioester incorporation into ionizable lipids enhances endosomal escape and biodegradability. However, the evidence presented is largely indirect. The manuscript attributes improved intracellular delivery to the acid-lability of the thioester bond, but no direct mechanistic experiments demonstrating enhanced endosomal membrane destabilization or escape are shown. Measurements such as endosomal escape assays, pH-dependent degradation kinetics within cellular compartments, or imaging-based tracking of endosomal release would significantly strengthen the argument. Without such mechanistic validation, it is difficult to determine whether the improved delivery is truly caused by the thioester linker or by other structural changes.
2. Another concern relates to the scope and rigor of the structure–activity relationship analysis. Although a lipid library was synthesized and screened, the manuscript does not sufficiently justify how many variants were evaluated or whether the chemical diversity of the library was broad enough to draw strong conclusions about the role of thioester linkers. The SAR discussion appears somewhat descriptive rather than quantitatively analytical. A more systematic comparison of headgroups, linker chemistry, and hydrophobic tail composition would help clarify whether the observed improvements are specifically attributable to the thioester motif.
3. The in vivo data supporting the superiority of CP-LC-1272 also require deeper analysis. The manuscript compares the lead candidate with existing lipid nanoparticle systems such as commonly used ionizable lipids. Critical parameters such as dosing equivalence, particle size distribution, encapsulation efficiency, and pharmacokinetics are not discussed in sufficient detail. Without a comprehensive comparison under identical experimental conditions, it is difficult to determine whether the reported improvements are statistically meaningful.
4. Another important limitation concerns biodegradability claims. While the manuscript suggests that thioester bonds confer rapid degradation and reduce lipid accumulation, the presented data do not fully characterize degradation pathways. Demonstrating biodegradation in vivo typically requires detailed pharmacokinetic studies, metabolite identification, or long-term biodistribution analysis. The current results provide suggestive evidence but do not fully establish improved safety or reduced accumulation.
5. The restoration of protein expression in the premature stop-codon model is promising, but the manuscript would benefit from additional controls, replication across different mutation contexts, or evaluation in more physiologically relevant models.
6. Potential stability issues of thioester bonds during formulation, storage, or systemic circulation are not addressed. Since thioesters can be chemically labile, it is important to evaluate whether premature hydrolysis could affect nanoparticle stability or delivery efficiency.
7. Some methodological details are insufficiently described. For example, the preparation conditions for lipid nanoparticles, including mixing ratios, buffer systems, and purification methods are not mentioned clearly. Additionally, information regarding the number of biological replicates and statistical tests used should be included in all the results.
8. The discussion section also tends to describe results instead of interpreting them in the context of broader lipid nanoparticle design. A closer comparison to previously reported biodegradable ionizable lipids and alternative linker chemistry would help to contextualize this work in the literature.
9. The figures showing lipid structures and degradation pathways may also be improved for better clarity. For instance, schematic representations showing how thioester cleavage facilitates biodegradable fragments would help readers understand better.
10. The writing is generally good, but there are some grammatical inconsistencies and typing errors throughout the document that need to be corrected during revision. The sentences in the introduction and discussion are too long and have multiple clauses, which is not easy to read. The sentences should be shorter and simpler.

Overall, the manuscript addresses an important problem in RNA delivery and introduces an interesting lipid design strategy. However, stronger mechanistic evidence, more rigorous comparative analysis, and improved clarity in the presentation of experimental details are necessary to support the conclusions.

 

Author Response

We would like to thank the reviewers for their careful reading of our manuscript and for their constructive and thoughtful comments. We appreciate the opportunity to revise and improve the manuscript in response to their feedback. Please find our point-by-point responses in the attached Word document.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

The revised version is satisfactory.

Author Response

Thank you