A naturally derived lipopeptide lipid nanoparticle platform enabling multiple nucleic acids delivery

Figures

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  Fig. 1. Design and formulation of RmHnC-DOPE lipopeptides and four-component LNPs. a) Synthesis of lipopeptides through the thiol-ene reaction between DOPE-Mal and cysteine residue at the C-terminal of RmHnC peptides (RmHnC, where m represents the number of the arginine residue, n indicates the number of the histidine residue, with m = 1–5 and n = 1–9). b) Representative chemical structures of helper lipids used in LNP formulation: β-sitosterol, DOPE, and DMPE-PEG2000. c) Modular formulation strategy combining RmHnC-DOPE lipopeptides with helper lipids and nucleic acids to generate a library of RmHnC-DOPE LNPs.

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  Fig. 2. Physicochemical characterization of RmHnC-DOPE LNPs. a-c) Comparison of particle zeta potential, size and stability for 13 RmHnC-DOPE and SM102 LNPs, each encapsulating PCSK9 siRNA. Stability was evaluated during long-term storage at 4 °C in DI water. d) Particle size and PDI of R3H7C-DOPE, R4H6C-DOPE, R5H5C-DOPE, and SM102 LNPs encapsulating eGFP pDNA, measured using DLS. e) Titration curves of R3H7C-DOPE, R4H6C-DOPE, and R5H5C-DOPE. f) Cryo-TEM images of R3H7C-DOPE, R4H6C-DOPE, R5H5C-DOPE, and SM102 LNPs, scale bar = 50 nm.

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  Fig. 3. Functional performance of RmHnC-DOPE LNPs. a) Relative PCSK9 mRNA expression in HEPA1-6 cells treated with LNPs encapsulating PCSK9 siRNA or naked siRNA controls (24-well plate, 50 pmol per well, 24-h incubation; n ≥ 3 biological replicates). Gene expression was normalized to GAPDH (housekeeping gene) and data are presented as mean ± standard deviation (SD). b) Fluorescence microscopy images and flow cytometry analysis of eGFP expression in HepG2 cells treated with eGFP pDNA-loaded R3H7C-DOPE, R4H6C-DOPE, R5H5C-DOPE, SM102, Lipofectamine 2000, scale bar = 1 mm. c) Quantitative flow cytometry analysis showing the percentage of eGFP pDNA-expressing HepG2 cells and mean fluorescence intensity (MFI). d) Firefly luciferase (fluc) activity in HepG2 cells transfected with fluc pDNA-loaded LNPs, Lipofectamine 2000, or naked pDNA controls (mean ± SD). e) Quantitative analysis of eGFP mRNA-expressing cells from flow cytometry. f) Fluorescence microscopy images of HepG2 cells expressing eGFP after treatment with eGFP mRNA-loaded LNPs, Lipofectamine 2000, scale bar = 200 μm. g) Fluorescence microscopy images and flow cytometry analysis of eGFP expression in A549 cells treated with eGFP…

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  Fig. 4. Transfection mechanism of siRNA RmHnC-DOPE LNPs. a) Cellular uptake of Cy5-labeled siRNA (20 pmol/well, 24-well plate) in HepG2 cells after the 4 h treatment with R3H7C-DOPE, R4H6C-DOPE, R5H5C-DOPE, or SM-102 LNPs. Red fluorescence indicates internalized siRNA. Scale bar: 10 μm; b) Inhibitor pretreatment (4 °C, CPZ, MβCD, or EIPA) in HepG2 cells transfected with Cy5-labeled siRNA-loaded LNPs. Uptake quantified by flow cytometry (4 h post-transfection); c) PCSK9 mRNA suppression in Bafilomycin A1-treated (1h) HEPA1-6 cells after delivery of PCSK9 siRNA via LNPs. Data normalized to GAPDH (mean ± SD, n ≥ 3).

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  Fig. 5. In vivo biodistribution and transgene expression efficiency of siRNA RmHnC-DOPE LNPs in C57BL/6 mice. a) Organ accumulation of Cy5-labeled siRNA 2 h post-IV injection of R3H7C-DOPE, R4H6C-DOPE, R5H5C-DOPE, or SM102 LNPs, imaged via a PE Spectrum small animal in vivo imaging system. b) Quantitative analysis of tissue distribution.

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  Fig. 6. Hepatic PCSK9 silencing, LDL receptor upregulation, and tissue-specific siRNA knockdown efficacy following RmHnC-DOPE LNP-mediated PCSK9 siRNA delivery in C57BL/6 mice C57BL/6 mice were administered with siRNA-loaded lipid nanoparticles via intravenous injection to evaluate gene silencing efficacy and tissue targeting profiles. a) Comparative analysis of relative PCSK9 mRNA levels in mouse livers 7 days following IV administration of PCSK9 siRNA formulated with R3H7C-DOPE, R4H6C-DOPE, R5H5C-DOPE, or SM102 LNPs. Data normalized to GAPDH expression (mean ± SD, n ≥ 4). b) Long-term PCSK9 mRNA suppression kinetics over 28 days post-IV injection, comparing PCSK9 siRNA R3H7C-DOPE LNPs (selected based on their superior 7-day efficacy demonstrated in panel a) with SM102 LNPs. Data normalized to GAPDH expression (mean ± SD, n ≥ 4). c) Representative immunofluorescence microscopy images showing LDL receptor expression (green fluorescence) in liver tissue sections from PCSK9 siRNA R3H7C-DOPE-treated mice (28 days post-treatment, left panel) and untreated control mice (right panel), demonstrating sustained LDL receptor upregulation resulting from PCSK9 silencing. Cell nuclei were…

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  Fig. 7. Expression efficacy of firefly luciferase plasmid DNA and mRNA LNPs in BALB/c mice. a) pFluc expression 24 h post-IV injection. Group 1 represents naked pDNA control, Group 2 represents pDNA-SM102, and Group 3 represents pDNA-R5H5C-DOPE. b) firefly luciferase mRNA expression 6 h post-IV injection. Group 1 represents naked mRNA control, Group 2 represents mRNA-SM102, and Group 3 represents mRNA-R5H5C-DOPE. The first column in each group serves as the blank control, followed by firefly luciferase expression levels in heart, liver, spleen, lung, and kidney, demonstrating the extrahepatic targeting potential of different carriers. Quantitative assessment of pulmonary bioluminescence intensity (normalized to blank controls) following firefly luciferase pDNA (c) and mRNA (d) administration. Data represent mean ± SD, n = 4.

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  Fig. 8. Toxicity assessment of siRNA LNPs in mice 28 days post-IV injection. a-h) Blood biochemical analysis, including a) Total bilirubin (TBL); b) Albumin (ALB); c) Total protein (TP); d) Blood urea nitrogen (BUN); e) Uric acid (UA); f) Serum creatinine (CR); g) Aspartate aminotransferase (AST); h) Alanine aminotransferase (ALT). i) Body weight changes in this batch of mice over 28 days post-IV injection. j) H&E staining of major organs from mice 28 days post-IV injection, Scale bar: 50 μm. k) Quantification of inflammatory cytokines IL-1β, IL-6 in serum via ELISA.

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  Fig. 9. Toxicity assessment of pDNA LNPs in C57BL/6 mice 24 h post-IV injection. a-h) Blood biochemical analysis, including a) Total bilirubin (TBL); b) Albumin (ALB); c) Total protein (TP); d) Blood urea nitrogen (BUN); e) Uric acid (UA); f) Serum creatinine (CR); g) Aspartate aminotransferase (AST); h) Alanine aminotransferase (ALT). i) Body weight changes in this batch of mice over 24 h. j) H&E staining of major organs from mice 24 h post-IV injection of LNPs, Scale bar: 50 μm. k) Magnified image of the spleen, Scale bar: 312 μm