Journal: Nature Communications

Article Title: Structure-guided design of endosomolytic chloroquine-like lipid nanoparticles for mRNA delivery and genome editing

doi: 10.1038/s41467-025-59501-y

Figure Lengend Snippet: A library of Clls was constructed by combining three distinctive modules (the scaffold, linker, and tail) extracted from chloroquine and ionizable lipids. The lead Cll (CF3-2N6-UC18) identified from the library, together with auxiliary lipids (DOPE, cholesterol, and PEGylated lipids) spontaneously self-assembles into ecoLNPs in the presence of mRNA molecules after formulation optimization using CCD. ecoLNPs administered through multiple administration routes show potent mRNA delivery in vivo. The proposed mechanism of action of ecoLNPs as an endosomolytic mRNA delivery vehicle involves (i) the proton sponge effect and (ii) saposin B (sapB)-promoted membrane disruption. DOPE 1,2-dioleoyl- sn -glycero-3-phosphoethanolamine, CCD Central Composite Design, IV intravenous injection, SC subcutaneous injection, IM intramuscular injection, SCJ subconjunctival injection, SR subretinal injection, IVS intravesical instillation.

Article Snippet: At 24 h after plating, cells were treated with ecoLNPs containing 200 ng of β-gal mRNA (TriLink).

Techniques: Construct, Formulation, In Vivo, Membrane, Disruption, Injection

Journal: Nature Communications

Article Title: Structure-guided design of endosomolytic chloroquine-like lipid nanoparticles for mRNA delivery and genome editing

doi: 10.1038/s41467-025-59501-y

Figure Lengend Snippet: a The z-average hydrodynamic size distribution (left panel) and representative transmission electron microscopy images (right panel) of ecoLNPs. b In vitro delivery performance of ecoLNPs during storage at 4 °C. ecoLNPs containing 200 ng of eGFP mRNA were stored at 4 °C for different storage times and their in vitro delivery performance was estimated by the intensity of green fluorescence originating from ecoLNPs-treated 293T cells. Scale bar, 50 µm. c Susceptibility of ecoLNPs-encapsulated eGFP mRNA to serum or RNase A. Free eGFP mRNA was used as a control group. The green and red triangles indicate the location of bands of ecoLNPs-encapsulated and free mRNA, respectively. d Hemolytic activity induced by different concentrations of ecoLNPs at pH of 5.4 and 7.4 ( n = 3 biological replicates, two-way ANOVA with Tukey’s multiple comparison test). The concentration of ecoLNPs used for assessment of in vitro delivery activity was defined as 1×. e Dose-response and time-course analyses of 293T cells exposure to eGFP mRNA-loaded ecoLNPs. MFI, mean fluorescence intensity. f A comparison of eGFP mRNA delivery efficiency of ecoLNPs with Lipofectamine 2000 (Lipo2k) and Lipofectamine MessengerMAX (LipoMMAX) in 293T (left panel) and THP-1 cells (right panel) by flow cytometry. PBS-treated cells were used to differentiate positive and negative events. g A comparison of β-gal mRNA delivery efficiency of ecoLNPs with Lipo2k and LipoMMAX in 293T cells by in situ β-galactosidase staining (top panel; scale bar, 100 µm) and a comparison of mCherry mRNA delivery efficiency by fluorescence imaging (bottom panel; scale bar, 50 µm). h Quantitative analysis of both β-gal mRNA and mCherry mRNA delivery efficiency using a colorimetric method (left panel) and flow cytometry (right panel), respectively ( n = 3 biological replicates, one-way ANOVA with Tukey’s multiple comparison test). MFI, mean fluorescence intensity. Fluorescent images of 3D cells treated with eGFP mRNA- ( i , top panel), mCherry mRNA- ( i , bottom panel), or dual mRNA- ( j ) loaded ecoLNPs. Scale bar, 50 μm. For all relevant panels, data are presented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: At 24 h after plating, cells were treated with ecoLNPs containing 200 ng of β-gal mRNA (TriLink).

Techniques: Transmission Assay, Electron Microscopy, In Vitro, Fluorescence, Control, Activity Assay, Comparison, Concentration Assay, Flow Cytometry, In Situ, Staining, Imaging

Journal: Nature Communications

Article Title: Structure-guided design of endosomolytic chloroquine-like lipid nanoparticles for mRNA delivery and genome editing

doi: 10.1038/s41467-025-59501-y

Figure Lengend Snippet: a Effects of inhibitors and temperature on the cellular uptake of ecoLNPs. MFI of each group was determined by flow cytometry and normalized to that of the “no inhibitor” group. Data are presented as mean ± SEM ( n = 2 biological replicates for 4 °C, n = 3 biological replicates for the remaining groups, one-way ANOVA with Tukey’s multiple comparison test). b A comparison of chemical structures between CF3-2N6-UC18 and PNT-2N6-UC18. Representative confocal microscopy images of HeLa cells treated with Cy5 mRNA (red)-loaded ecoLNPs for tracking ecoLNP-mediated endosomal escape via co-localization analysis ( c ) and the calcein leakage assay ( e ). Over a dozen of images were taken for each group and the most representative image was presented. Endo-lysosomal tracker (green) and Hoechst (blue) were used to stain endo-lysosomes and nucleus ( c ). Calcein (green) leakage was used to evaluate the endosomal escape capacity of co-incubated molecules ( e ). Free Cy5 mRNA and Cy5 mRNA-loaded PNT-2N6-UC18 LNPs served as control groups. The red fluorescence signals in the PNT-2N6-UC18 LNPs-treated group appeared weak at high magnification (63× oil immersion objective lens), largely owing to their punctate distribution. Scale bar, 10 µm. Quantitative analysis of fluorescent colocalization of Cy5 and endo-lysosomal tracker fluorescence ( d ) in ( c ), or calcein fluorescence ( f ) in ( e ) across white arrows (30 µm). The maximum fluorescence intensity was defined as 100% in each panel. g Molecular docking of CF3-2N6-UC18 (sticks in cyan) or PNT-2N6-UC18 (sticks in gray) to the sapB dimer binding pocket (surface representation in green). Chloroquine (sticks in oranges) in complex with sapB was used as a reference. The hydrophobic residues (M61, M65, and L73 from one monomer; E35 and R38 from the second monomer) were labeled in yellow and blue, respectively. The residue E69 involved in the potential hydrogen-bonding interaction with chloroquine was labeled in magenta. Inset, the interaction details of chloroquine or lipids with sapB. Single-letter abbreviations for amino acid residues are as follows: E, Glu; L, Leu; M, Met; and R, Arg. Source data are provided as a Source Data file.

Article Snippet: At 24 h after plating, cells were treated with ecoLNPs containing 200 ng of β-gal mRNA (TriLink).

Techniques: Flow Cytometry, Comparison, Confocal Microscopy, Staining, Incubation, Control, Fluorescence, Binding Assay, Labeling, Residue

Journal: Nature Communications

Article Title: Structure-guided design of endosomolytic chloroquine-like lipid nanoparticles for mRNA delivery and genome editing

doi: 10.1038/s41467-025-59501-y

Figure Lengend Snippet: a Representative in vivo and ex vivo bioluminescent images of C57BL/6 mice 4 h post-injection of ecoLNPs at a FLuc mRNA dose of 0.5 mg kg −1 for IV ( n = 2), SC ( n = 2), and IM injection ( n = 4), 200 ng for SCJ ( n = 3) and SR ( n = 3) injection, and 0.25 mg kg -1 for IVS ( n = 3) instillation. 1, heart; 2, liver; 3, spleen; 4, lung; 5, kidney; 6, lymph node; 7, eye. Quantification of luminescence at the IM injection sites of living mice ( b ) and in ex vivo tissues ( c ) 4 h post-IM injection of FLuc mRNA-loaded ecoLNPs or SM-102 LNPs. Data are presented as mean ± SEM ( n = 4, unpaired, two-tailed Student’s t test). d The percentage of luminescence for each ex vivo tissue from ( c ). e , f Schematic showing the activation of tdTomato expression in Ai9 transgenic mice by delivering Cre mRNA or co-delivering Cas9 mRNA and gRNA simultaneously targeting the three repeats of the loxP-flanked stop cassette locus. RBE recombinase binding element, PAM protospacer adjacent motif, TGS third-generation sequencing. g Ex vivo fluorescent images of Ai9 mice 7d post-injection of ecoLNPs at a Cre mRNA dose of 0.5 mg kg −1 via intramuscular injection ( n = 3 legs). Legs injected with PBS were used for comparison. 1, heart; 2, liver; 3, spleen; 4, lung; 5, kidney; 6, lymph node; 7, hind leg. h Representative ex vivo fluorescent images of Ai9 mice 14d post-injection of two doses of ecoLNPs at a total RNA (Cas9 mRNA:gRNA = 3:1, wt:wt) dose of 1 mg kg −1 via intramuscular injection ( n = 3 legs). 1, heart; 2, liver; 3, spleen; 4, lung; 5, kidney; 6, lymph node; 7, hind leg. i tdTomato expression (red) in frozen sections of injected muscle and lymph nodes from ( g ) and ( h ). Nuclei were stained by DAPI (blue). Scale bar, 20 μm for lymph node, 50 μm for injected muscle. j Verification of Cre mRNA-mediated genome editing by gel electrophoresis. Source data are provided as a Source Data file.

Article Snippet: At 24 h after plating, cells were treated with ecoLNPs containing 200 ng of β-gal mRNA (TriLink).

Techniques: In Vivo, Ex Vivo, Injection, Two Tailed Test, Activation Assay, Expressing, Transgenic Assay, Binding Assay, Sequencing, Comparison, Staining, Nucleic Acid Electrophoresis