Targeted Delivery of Cell Softening Micelles to Schlemm's Canal Endothelial Cells for Treatment of Glaucoma

Abstract

Increased stiffness of the Schlemm's canal (SC) endothelium in the aqueous humor outflow pathways has been associated with elevated intraocular pressure (IOP) in glaucoma. Novel treatments that relax this endothelium, such as actin depolymerizers and rho kinase inhibitors, are in development. Unfortunately, these treatments have undesirable off-target effects and a lower than desired potency. To address these issues, a targeted PEG-b-PPS micelle loaded with actin depolymerizer latrunculin A (tLatA-MC) is developed. Targeting of SC cells is achieved by modifying the micelle surface with a high affinity peptide that binds the VEGFR3/FLT4 receptor, a lymphatic lineage marker found to be highly expressed by SC cells relative to other ocular cells. During in vitro optimization, increasing the peptide surface density increased micellar uptake in SC cells while unexpectedly decreasing uptake by human umbilical vein endothelial cells (HUVEC). The functional efficacy of tLatA-MC, as measured by decreased SC cell stiffness compared to non-targeted micelles (ntLatA-MC) or targeted blank micelles (tBL-MC), is verified using atomic force microscopy. tLatA-MC reduced IOP in an in vivo mouse model by 30-50%. The results validate the use of a cell-softening nanotherapy to selectively modulate stiffness of SC cells for therapeutic reduction of IOP and treatment of glaucoma.

Keywords: controlled delivery; endothelium; glaucoma; latrunculin; nanoparticle.

Figure 1.

The design of PEG-b-PPS micelles that target SC cells in the anterior segment of the eye. A, B) Human SC cells express Flt4/VEGFR3. A) Representative confocal microscopy images of SC endothelial cells that were stained with anti-FLT4 antibody to confirm presence of FLT4 on the cell surface. Cells were stained with Hoescht 33342 (blue) and FLT4 (red) B) Flow cytometry data comparing median fluorescence intensity (MFI) of SC cells and HUVECs stained with anti-FLT4 antibodies. Data shown as mean±SEM. Significance determined by unpaired t-test (****p<0.0001). C) Schematic representation of peptide-displaying micelles. D) The peptide targeting construct consisting of targeting peptide, PEG spacer and palmitoleic acid tail. E) LCMS spectra of the purified Flt4-targeting peptide construct. F) Peptide loading efficiency of PEG 6 peptide construct into PEG-b-PPS micelles at various molar ratios as determined by tryptophan fluorescence measurements. Data shown as mean ±SD, N=3 technical replicates. G) STEM micrograph of negatively stained PEG-b-PPS micelles (MC) displaying the peptide targeting construct (5%) (200,000X magnification). H) Synchrotron small angle x-ray scattering (SAXS) plots for Blank micelles, and micelles displaying the targeting peptide at 1% and 5% molar ratios. A core shell model (solid line) was fit to the data (gray points). The core radius (rc), shell thickness (rt), and total MC radius (r) is displayed together with the chi square (X²) value for the final model fit.

Figure 2.

The micellar display of a FLT4-binding peptide targets the delivery of Lat A to SC endothelial cells in vitro. A-C) The FLT4-targeting peptide enhances micelle uptake by SC endothelial cells and decreases uptake by HUVEC. A) Nanoparticle formulation abbreviations. B) unloaded micelles, C) micelles loaded with latrunculin (LatA). Cells were incubated for 1 h with Alexa 555 labeled micelles incorporated with various molar ratios (1%, 3%, 5%) of peptide to micelle. After 1 h incubation, cells were washed, harvested and fixed for analysis via flow cytometry. Median fluorescence intensity (MFI) was measured to quantify uptake of the various formulations by both cell types. Data shown as mean ± SEM (n=3 biological replicates). In B, significance determined by one way ANOVA for each of the two cell types with post hoc Tukey’s multiple comparisons test (‡‡‡‡ p<0.0001, ‡p<0.05). In B, C)Two way ANOVA was used to evaluate differences between all groups with post hoc Sidak’s multiple comparisons test (****p<0.0001, *p<0.05). D-E) Targeted delivery of Lat A decreases the stiffness of human SC cells. D) Representative images taken during AFM measurements of cells treated for 2 hours with BL-MC, tBL-MC (0.97 mg/mL), ntLatA-MC, or tLatA-MC (0.05 μM Lat A, 0.97 mg/mL); AFM tip is at top of each panel. E) Stiffness of SC cells after 2 h was determined by AFM; data shown is geometric mean ± SD ($\ge$5 measurements/condition). In E), significance determined by ANOVA with post hoc Tukey’s multiple comparisons test (*p<0.05).

Figure 3.

Targeted delivery of latrunculin reduces IOP in mouse eyes. A) Illustrative overview of experiments which consisted of 2 different IOP measurement schedules. Trial #1 and Trial #2 IOP timepoints shown with black and grey arrows respectively. B) Trial 1 results. The first trial consisted of 2 μL intracameral injection of BL-MC or tLatA MC (40 mg/mL, 5% Peptide, 17 μM Lat A) in 5 mice. IOP was measured prior to injection, and after 24 h and 48 h. C) Trial 2 results. 2 μL of BL-MC, ntLatA-MC, or tLatA-MC (15.5 μM Lat A, 40 mg/mL 5% peptide) micelles were injected into 1 eye of 5 mice each. IOP was measured prior to injection and at three timepoints during a 48 h time course. Intraocular pressure (IOP) is measured by rebound tonometry (TonoLab; Icare). Data shown are mean ± SEM (n=5). Trial #1 significance determined by unpaired t-test (*p<0.003). Trial #2 significance determined by ANOVA with post hoc Tukey’s multiple comparisons test (*p<0.03).