Kim and co-workers described a new piperazine-derived ionizable lipid nanoparticles for mRNA delivery to health lung and fibrotic lungs through systemic injection. They proposed that the degradable 244cis lipid nanoparticles has lower immunogenicity and enhaced mRNA delivery than SM-102, another benchmark for vaccine studies. The results for transfected cell types in the lung is interesting, however, the novelty is still a problem as DOTAP has been widely used on LNP formulations for mRNA delivery to the lung. Several issues should also be addressed before publications;
1. The authors need to prove that 244cis can improve membrane fluidity, rather than just reporting that unsaturated lipid can improve endosomal escape, as amine head can also influence this property.
2. SM-102 LNP system is used for vaccine application, which needs a potential immunogenicity for vaccine, so the MCP-1 level for SM-102 is reasonable. But 244cis has higher MCP-1 level than MC-3, so it is overclaim to say that 244cis has lower immunogenicity for mRNA delivery as different LNP formulations have different application directions. SM-102 may not be a good benchmark, C12-200 LNP with DOTAP may be another choice.
3. Line 131-138 showed 244cis induce MCP-1 secretion at 320 pg/mL, while SM-102 for 950 pg/mL for 0.1 mpk hEPO mRNA injection. Bur in line 221-225, the author demonstrated 220 pg/mL with DOTAP and 170 pg/mL with DOPE for 0.5 mpk fluc mRNA injection. I am so confused by these results, and suspect the authenticity of the data.
4. Piperazine-drived lipid has been published by Dahalman lab in 2022 nature comme, so this finding is not so exciting. In their previous studies, Adv. Funct. Mater.2023, 33, 2209432, they report another lipid 246C10 for lung delivery, this lipid is also based on piperazine, therefore highlight piperazine lipids is not novel enough.
5. In Line 165, why are you using the ICR mice here?
6. What is the transfection of cells when there is only one injection of 0.3 mpk Cre mRNA? does it totally different from repeated dosage?
7. It is not possible to know the structure of 244cis and (Z)-non-2-en-1-yl 9-bromononanoat are correct if there is no NMR spectrum for them.
8. More safety data of lung should be added if the author want to demonstrate the safety of their LNP system, some important index like immune cell population in the health lung (as positive charged lipids sometimes induce local inflammtory), and blood oxygen level for demonstrating if the lung has been afftected after delivery.

The authors addressed all of my concerns, I recommend its publications as this version.