Comprehensive Evaluation of Lipid Nanoparticles and Polyplex Nanomicelles for Muscle-Targeted mRNA Delivery

Figures

• 

  Figure 1. Schematic illustration of FLuc mRNA-loaded polyplex nanomicelle (A) and LNP (B) when administered by HLV or IM injection (C).

• 

  Figure 2. Luciferase expression profile after IM or HLV administration of mRNA delivered by LNP or nanomicelle. (A) Representative IVIS images of luciferase expression. Color spectrum represents the intensity of the bioluminescence. (B) Time course of luciferase expression observed at 8 h, 24 h, 48 h, one week, and two weeks after administration. (C) Luciferase assay performed on muscles at the end of the IVIS experiments. Data are presented as mean ± SEM, n = 3. *** p < 0.001, ** p < 0.01, * p < 0.05. One-way ANOVA followed by Tukey’s honest significance test.

• 

  Figure 3. Evaluation of organ toxicity by luciferase assay and blood biochemistry. Luciferase assay was performed on the livers (A) and kidneys (B) collected 24 h after administration (n = 3). (C) Blood biochemistry tests on blood samples collected 1 and 5 days after administration (n = 3 and n = 6 for the control group). Grey bands represent the referential normal range indicated by the animal supplier (Japan SLC Inc.) and previous reports. Data are presented as mean ± SEM. *** p < 0.001, ** p < 0.01, * p < 0.05. One-way ANOVA followed by Tukey’s honest significance test.

• 

  Figure 4. Histological changes in the muscle 2 and 5 days after administration of LNP or nanomicelle at 4× (A) and 20× (B) magnifications. Arrowheads indicate centrally nucleated fibers. (C) Levels of CK and LDH in the blood samples collected 1 and 5 days after administration (n = 3 and n = 6 for the control group). Data are presented as mean ± SEM. ** p < 0.01, one-way ANOVA followed by Tukey’s honest significance test.

• 

  Figure 5. Average fiber area (A), total fiber area (B), and muscle mass of the muscles collected at 2 and 5 days after administration (n = 3 and n = 6 for the control group). For (A), 200 to 250 fibers from each mouse were analyzed. Data are presented as mean ± SEM. **** p < 0.0001, ** p < 0.01. Student’s t-test was performed for (A), and one-way ANOVA followed by Tukey’s honest significance test was performed for (B,C).

• 

  Figure 6. Representative images of EBD staining in the muscle tissue ((A), upper panel), muscle transverse section ((A), lower panel), and muscle cross-section under a fluorescence microscope (B). Samples were collected 2 days after HLV or IM administration of Fluc mRNA-loaded LNP or nanomicelle.

• 

  Figure 7. Relative mRNA expression of inflammatory cytokines IL-6 (A), IL-1β (B), and TNF-α (C) 24 h post administration. Results were normalized to the expression levels of β-actin. Data are presented as mean ± SEM, n = 3. ** p < 0.01, * p < 0.05. One-way ANOVA followed by Tukey’s honest significance test.

• 

  Figure 8. Pain behavior evaluated by the incapacitance test. The distinct difference in weight distribution between the injected and noninjected hindlimb can be interpreted as the animal’s adaptation to pain. Data are presented as mean ± SEM, n = 3. * p < 0.05, one-way ANOVA followed by Tukey’s honest significance test.