LNPs are usually composed of phospholipids/cationic lipids/cholesterol/pegylated lipids. Almost all previous studies have focused on optimizing the structure of cationic lipids, but the types and structures of commercial phospholipids currently used are single. However, phospholipids have their unique advantages. Phospholipids have the same structure as biological membranes and have strong endosomal membrane fusion and endosomal escape potential. Therefore, new phospholipids with controllable structures can be flexibly designed by chemical means to obtain a more efficient LNPs system. Provides a new idea. The author speculates that a zwitterionic head composed of a tertiary amine and a phosphate group, combined with a tail with three hydrophobic alkyl chains, will facilitate the escape of phospholipid endosomes. In the acidic endosomal environment, the tertiary amine is protonated, and the phospholipid forms a smaller zwitterionic head and a larger multi-hydrophobic chain tail, which is inserted into the phospholipid membrane to form a cone structure, which promotes the membrane to transform into a hexagonal crystal phase. Achieve endosomal escape. At a neutral physiological pH, the negatively charged iPhos is difficult to insert into the endosomal membrane, which ensures the low toxicity of the iPhos material. In order to verify this theory, the author designed and synthesized 572 iPhos phospholipids, which contain various amphiphilic heads and different numbers of hydrophobic chain tails. The in vitro structure-activity relationship results and membrane phase transition studies have effectively supported the above theory.
Next, the authors further screened for mRNA delivery in vivo. The structure-activity relationship in vivo reveals very interesting conclusions. The structure of iPhos not only affects transfection efficiency in vivo, but also controls organ selectivity. The results prove that: 1) iPhos containing one ionizable tertiary amine, one negatively charged phosphate group and three hydrophobic alkyl chains has the highest efficiency; 2) the length of the alkyl chain on one side of the tertiary amine controls the efficiency of mRNA delivery in vivo, 8 ~ 10 The carbon chain length iPhos has the highest efficiency in vivo; 3) The length of the alkyl chain on one side of the phosphate group determines the organ selectivity. iPhos with a length of 9 to 12 carbon chain delivers mRNA to the liver for expression, and iPhos with a length of 13 to 16 carbon chain delivers mRNA to the spleen for expression . This interesting phenomenon indicates that regulating the chemical structure of iPhos can achieve organ selectivity, which is of reference significance for the design of other delivery systems.
Using SORT technology for reference, the author focused on the optimized iPhos 9A1P9, assisted with various lipids, and realized organ-specific mRNA delivery on the premise of ensuring high mRNA delivery efficiency: 9A1P9 bound zwitterionic lipids (DOPE) ), which mediates the expression of mRNA in the spleen; 9A1P9 binds to ionizable cationic lipids (MDOA, DODAP or 5A2-SC8) so that mRNA is expressed in the liver; 9A1P9 binds to non-ionizable cationic lipids (DOTAP or DDAB) to achieve mRNA is expressed in the lungs. It is worth mentioning that 9A1P9-5A2-SC8 iPLNPs have reached the upper limit of chemiluminescence detection by small animal in vivo imaging systems at a dose as low as 0.05 mg/kg Fluc mRNA. The mRNA delivery efficiency of 9A1P9 is 40 to 965 times that of commercial phospholipids DOPE and DSPC. Then, the author used FDA-approved DLin-MC3-DMA LNPs (siRNA drug Onpattro) as a positive control, and found that 9A1P9-5A2-SC8 iPLNPs delivered mRNA in vivo with 13 times the efficiency of DLin-MC3-DMA LNPs. These research results fully prove the excellent performance of iPhos in mRNA delivery.
Subsequently, the authors used liver-targeted 9A1P9-5A2-SC8 iPLNPs and lung-targeted 9A1P9-DDAB iPLNPs to conduct applied research on mRNA delivery and gene editing. The author chose tdTomato (tdTom) transgenic mice. When the termination signal before the tdTom gene is cleaved by Cre enzyme or CRISPR/Cas9, the expression of red fluorescent protein will be activated for easy detection. The author injected a single intravenous injection of 9A1P9-5A2-SC8 iPLNPs and 9A1P9-DDAB iPLNPs with Cre mRNA to tdTom mice, and measured the editing efficiency of different cell types by flow cytometry. The editing efficiency of liver parenchymal cells was as high as 91%, and the editing efficiency of lung endothelial and epithelial cells reached 34% and 20%, respectively. These results provide a basis for mRNA-dependent organ-specific treatment.
The author further applied organ-targeted iPLNPs to CRISPR/Cas gene editing to co-deliver Cas9 mRNA and sgRNA. After an intravenous administration, liver-targeted 5A2-SC8 iPLNPs and lung-targeted 9A1P9-DDAB iPLNPs successfully edited the liver and lungs of tdTom mice, maintaining organ specificity. Next, the authors tested the ability of iPLNPs to target-edit endogenous genes (PTEN). In C57BL/6 mice, after a single intravenous injection of iPLNPs loaded with Cas9 mRNA and sgPTEN, not only was successfully observed organ-selective gene editing, but the lung-targeted 9A1P9-DDAB iPLNPs had a high gene editing efficiency of 28.3 in the lungs. %. Finally, the authors verified the clinical transformation potential of the iPLNPs system: 1) Mass production of iPLNPs using microfluidic technology, and maintaining high mRNA delivery efficiency and organ specificity; 2) iPLNPs can be administered repeatedly and maintaining high transfection efficiency ; 3) iPLNPs have very low toxicity in vivo.
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