Liu Shuai's research group at Zhejiang University has developed a single component cationic lipid mRNA delivery carrier that targets splenic T cells after intravenous administration

Four component lipid nanoparticles（LNPs）It has been widely used in clinical trials of the marketed COVID-19 9mRNA vaccine and a large number of mRNA therapies, demonstrating their effectiveness in delivering mRNA to the liver and muscles. The extrahepatic targeting distribution of mRNA drugs is of great significance for the treatment of various diseases affecting non liver organs, especially the specific mRNA delivery targeting the spleen and T cells, which provides a remarkable option for cancer immunotherapy and induction of antiviral immune responses.

However, targeting mRNA delivery to extrahepatic tissues remains a significant challenge that limits its application development to a large extent. The current spleen targeting technology often comes at the cost of reduced transfection efficiency or therapeutic efficacy, resulting in lower overall mRNA expression levels in the spleen compared to the liver when using novel delivery vectors.

On April 18, 2024, fromLiu Shuai's research group from the School of Pharmacy, Zhejiang UniversityInApplied Chemistry International EditionPublished an article titled“One-Component Cationic Lipids for Systemic mRNA Delivery to Splenic T Cells”The research paper. This study proposes that,Cationic lipids rich in secondary aminesCan be used as an independentSingle component carrierEffectively deliver mRNA in vitro and in vivo.In addition, these carriers help to effectively deliver mRNA to splenic T cell subsets after intravenous administration, demonstrating great potential for immunotherapy applications. This simple strategy extends the utility of liposomes in extrahepatic mRNA delivery, providing new insights for the development of carriers beyond LNP to further advance the field of precision mRNA therapy.

As an important component of most four component LNP carriers, numerous studies have designed cationic lipids containing tertiary amines（An-R-T）It is usually synthesized through amino and epoxy analogues or acrylic esters. By carefully controlling the reaction temperature and molar ratio of epoxy ring opening and Michael addition starting materials, cationic lipids rich in secondary amine groups can be obtained（An-R-S）（图1B、1C）And using this designed ionizable cationic lipid as a single component carrier to prepare cationic lipid/mRNA nanoparticles（CLNs）.

Figure 1. Design and synthesis of single component cationic lipid carriers

In vitro screening experiments showed that,Rich amine cationic lipid(AnEP-S and An Ac-S)Mediated strong mRNA expression and minimal cytotoxicity, the best candidate drug exhibited better performance than tertiary amine lipids(An EP-T and An Ac-T)4 orders of magnitude higher delivery efficiency.Compared with ALC-0315 LNPs, TE-EP10-S CLNs with better efficiency showed a 43 fold enhancement.

On the binding/encapsulation of mRNA, tertiary amine liposomes only showed a low mRNA binding rate of less than 60%, while the mRNA binding rate of a single secondary amine lipid reached 95%(Figure 2D)The size of CLN prepared by pipettes is relatively large, and relying on microfluidic devices can effectively reduce particle size,The optimal size is maintained at100 nm左右Due to the high content of secondary amine groups and hydrophobic tails in An-R-S lipids, their electrostatic and hydrophobic interactions with mRNA ensure the stability of CLNs, even at 4 ℃ in 10% fetal bovine serum（FBS）After incubation in serum for 7 days, CLNs under this design remained stable(Figure 2E).

Hemolysis determined by red blood cell rupture and fluorescence resonance energy transfer（FRET）Analysis of lipid fusion showsThere is no significant difference in endosome escape between two types of rich amine lipids(Figure 2G)Confocal fluorescence imaging confirmed the effective escape of optimized secondary amine CLNs from the endosome(Figure 2H)On the contrary, the carrier corresponding to tertiary amines mediates limited endosome escape due to lower cellular uptake. These results highlight the breakthroughs made by single component ionizable cationic lipids in overcoming various delivery barriers. In addition, CLN based on secondary amines is superior to tertiary amines due to enhanced mRNA binding and cellular uptake.

Figure 2. Structure Activity and Physicochemical Properties Analysis of Single Component Cationic Lipid CLN

The study further evaluated the organ targeting of CLNs in C57BL/6 mice by intravenous injection at a dose of 0.25 mg/kg(Figure 3A)Unlike LNPs that primarily target the liver, single component ionizable cationic lipids mainly mediate mRNA expression targeting the spleen.

TE-EP8-S hasTwo hydrophobic tailsandFive intermediate amino groupsThe obtained CLNs showed the highest mRNA expression in the spleen(Figure 3B)The expression of luciferase mediated by it is several orders of magnitude higher in the spleen and increases in a dose-dependent manner.

Figure 3. In vivo mRNA delivery of single component CLNs

The Ai9 mouse model introduced with loxp Cre recombinant system can induce the expression of tdTomato in the presence of Cre recombinant protein to delete the transcription termination signal box（stop cassettes）Experiments have shown that TE-EP8-S CLNs loaded with Cre mRNA effectively promote protein expression in the spleen, with nearly 6% of T cells transfected after intravenous administration(Figure 4B, C).

Figure 4. TE-EP8 Scln mediated translation of splenic T cell mRNA

The single component cationic lipid proposed in this study is directly used for carrier constructionMRNA drugsProvides another delivery option and has the potential to deliver mRNA to extrahepatic tissues. The single component carrier method based on secondary amines proposed in this article can be extended to a wide range of cationic lipids and polymer carriers, opening up new research directions for further development of mRNA delivery technology and applications.