Table 1.
LNP Components and characteristics.
Fig 1.
(A) Schematic of an LNP listing all major components. (B) Bar graph showing the size and PDI of one set of particles when percentage of PEG is increased and modulated against cholesterol. (C) Bar graph showing size and PDI of one set of particles when percentage of PEG is increased and modulated against DSPC. DSPC-1,2-distearoyl-sn-glycero-3-phosphocholine, DMG-PEG2k-1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000, MC3-Dlin-MC3-DMA, PDI-polydispersity index.
Fig 2.
The effects of LNP size and cholesterol modifications post-subretinal injection.
(A) Representative images demonstrating luciferase activity in the eye 24 hours post-injection. (B) Luciferase activity plotted for each group over time. Data are presented as mean ± SD. A two-way ANOVA, Tukey’s multiple comparisons test was used for analysis. *p<0.05. n = 3–6. (C) Representative bright field (top) and tdTomato (bottom) fundus images for each group taken 7 days post-injection. (D) Quantification of tdTomato intensity, represented as a fold change compared to PBS. Data are presented as mean ± SD. An ordinary one-way ANOVA, with Tukey’s correction for multiple comparisons test was used for comparisons. *p<0.05. n = 3–6. (E) Representative confocal images of immunohistochemistry showing RFP expression in the RPE for all groups. n.s.—not significant, LNP- lipid nanoparticle, RFP-red fluorescent protein, RPE-retinal pigment epithelium, ONL- outer nuclear layer, INL-inner nuclear layer, GCL- ganglion cell layer.
Fig 3.
The effects of LNP size and DSPC modifications post-subretinal injection.
(A) Representative images demonstrating luciferase activity in the eye 24 hours post-injection. (B) Luciferase activity plotted for each group over time. Data are presented as mean ± SD. A two-way ANOVA, Tukey’s multiple comparisons test was used for analysis. *p<0.05. n = 3–6. (C) Representative bright field (top) and tdTomato (bottom) fundus images for each group taken 7 days post-injection. (D) Quantification of tdTomato intensity, represented as a fold change compared to PBS. Data are presented as mean ± SD. An ordinary one-way ANOVA, with Tukey’s correction for multiple comparisons test was used for comparisons. n = 3–6. (E) Representative confocal images of immunohistochemistry showing RFP expression in the RPE for all groups. n.s.—not significant, LNP- lipid nanoparticle, RFP-red fluorescent protein, RPE-retinal pigment epithelium, ONL- outer nuclear layer, INL-inner nuclear layer, GCL- ganglion cell layer.
Fig 4.
LNP-mediated RPE expression is not dependent on apoE or the MERTK receptor.
(A) Representative images demonstrating luciferase activity in C57BL6 and apoE-/- eyes 24 hours post-injection. (B) Luciferase activity plotted for both groups at 24 hours post-injection. Data are presented as mean ± SD. An unpaired t-test was used for analysis. n = 4. (C) Representative confocal images of immunohistochemistry showing mCherry expression in the RPE of C57BL6, apoE-/- and Mertk-/- mice. n.s.—not significant, PBS-phosphate buffered saline, LNP- lipid nanoparticle, RPE-retinal pigment epithelium, ONL- outer nuclear layer, INL-inner nuclear layer, GCL- ganglion cell layer.
Fig 5.
The effects of LNP size and cholesterol modifications post-intravitreal injection.
(A) Representative images demonstrating luciferase activity in the eye 24 hours post-injection. (B) Luciferase activity plotted for each group over time. Data are presented as mean ± SD. A two-way ANOVA, Tukey’s multiple comparisons test was used for analysis. *p<0.05. n = 3–6. (C) Representative bright field (top) and tdTomato (bottom) fundus images for each group taken 7 days post-injection. (D) Quantification of tdTomato intensity, represented as a fold change compared to PBS. Data are presented as mean ± SD. An ordinary one-way ANOVA, with Tukey’s correction for multiple comparisons test was used for comparisons. n = 3–6. (E) Representative confocal images of immunohistochemistry showing RFP expression in the Müller glia, the ONH and the TM for all groups. n.s.—not significant, LNP- lipid nanoparticle, RFP-red fluorescent protein, ONL- outer nuclear layer, INL-inner nuclear layer, GCL- ganglion cell layer, ONH-optic nerve head, TM-trabecular meshwork, CB-ciliary body.
Fig 6.
The effects of LNP size and DSPC modifications post-intravitreal injection.
(A) Representative images demonstrating luciferase activity in the eye 24 hours post-injection. (B) Luciferase activity plotted for each group over time. Data are presented as mean ± SD. A two-way ANOVA, Tukey’s multiple comparisons test was used for analysis. *p<0.05. n = 3–6. (C) Representative bright field (top) and tdTomato (bottom) fundus images for each group taken 7 days post-injection. (D) Quantification of tdTomato intensity, represented as a fold change compared to PBS. Data are presented as mean ± SD. An ordinary one-way ANOVA, with Tukey’s correction for multiple comparisons test was used for comparisons. *p<0.05. n = 3–6. (E) Representative confocal images of immunohistochemistry showing RFP expression in the Müller glia, the ONH and the TM. n.s.—not significant, LNP- lipid nanoparticle, RFP-red fluorescent protein, ONL- outer nuclear layer, INL-inner nuclear layer, GCL- ganglion cell layer, ONH-optic nerve head, TM-trabecular meshwork, CB-ciliary body.