Fig 1.
Preparation of PLGA microspheres.
(A) Schematic for preparation of TPCA-1/PLGA microspheres. (B) Microscopic images of microparticles loaded with TPCA-1. Scale bar, 10 μm. (C) Size distribution of TPCA-1/PLGA microparticles. The mean diameter is 2.4 μm.
Fig 2.
Encapsulation efficiency and in vivo drug release profiles.
(A) Encapsulation efficiency of TPCA-1 by a solvent-evaporation process is around 80%. (B) Choroid/RPE/retina TPCA-1 release profile at 1, 7, 14, and 21 days post retrobulbar administration of TPCA-1/PLGA (0.8 mg TPCA-1/10 mg PLGA in 100 μL PBS) microparticles. Data expressed as mean±SD, n = 3 (individual experimental mice).
Fig 3.
Assessment of retinal histology and visual function.
(A-C) H&E-stained images of the retinas from untreated mice (A), mice injected with sham-loaded PLGA microparticles (10 mg PLGA in 100 μL PBS) (B), and mice injected with TPCA-1-loaded PLGA (PLGA/TPCA-1) microparticles (0.8 mg TPCA-1/10 mg PLGA in 100 μL PBS) (C). The tissues were collected at 7 days after treatment. (D) The quantitative comparison of the cell layers in the outer nuclear layer (ONL) among untreated mice, mice treated with PLGA or PLGA/TPCA-1 at 7 days post treatment. Data expressed as mean±SD, n = 3 (individual experimental mice). (E) Visual acuity measured by OKR from eyes treated with PLGA or PLGA/TPCA-1 in comparison with untreated control eyes. Data expressed as mean±SD. N = the number of eyes tested.
Fig 4.
Histological and flow cytometric analyses of liver, spleen, and T cell populations.
(A) H&E-stained sections of liver (A, C, and E) and spleen (B, D, and F) tissues on day 7 after PLGA (10 mg PLGA in 100 μL PBS) (A and B) or PLGA/TPCA-1 (0.8 mg TPCA-1/10 mg PLGA in 100 μL PBS) (C and D) retrobulbar injection. The eyes without any injection were used for control (E and F). (G-I) Flow cytometry analysis of CD4+ and CD8+ single-positive and CD4+/CD8+ double-positive T cell populations in thymus (G) and spleen (H). Apoptotic assessment of the TCR+ lymphocytes was performed on splenocytes using flow cytometry for annexin V exposure on the cell surface (I). Data expressed as mean±SD, n = 3 for each group.
Fig 5.
Effects of retrobulbar-injected TPCA-1-loaded PLGA microparticles on laser-induced CNV formation.
(A-C) Representative images of fluorescein angiogram were taken from the eyes receiving no injection (A), PLGA (10 mg PLGA in 100 μL PBS) injection (B), and PLGA/TPCA-1 (0.8 mg TPCA-1/10 mg PLGA in 100 μL PBS) injection (C) at 7 days post drug treatment and laser injury. Laser burn was performed in the eyes immediately after retrobulbar injection. (D) Quantification of the fluorescein-leakage score was measured as described in Materials and Methods. (E-G) Representative images of laser-induced CNV stained by isolectin B4. The eyes were injected with PLGA (F) or PLGA/TPCA-1 (G) in comparison with eyes receiving no injection (E). The images were taken and calculated at 7 days after laser injury. (H) Area of CNV in each eye under the indicated conditions, measured from the isolectin B4 staining. The images in E-G were taken and calculated at 7 days after laser injury. “Untreated” represents the results from the eyes without any injection. Data is expressed as mean±SEM.; n = the number of mice used for each condition. The statistical analysis performed for all comparisons was one-way anova followed by post hoc Bonferroni t-tests using ProStat Ver5.5. **p < 0.01, ***p < 0.005.
Fig 6.
Effects of TPCA-1-loaded PLGA microparticles on macrophage recruitment to the laser lesions.
Retrobulbar injection of 100-μL microparticle suspensions containing either PLGA (10 mg) alone or PLGA loaded with TPCA-1 (10 mg/0.8 mg) was performed on the mice at age of 8-week-old, and followed immediately by laser burn of Bruch’s membrane. After 24, 48, or 72 hours, mice were euthanized, and the eyes were dissected and fixed in 4% PFA. The posterior eye cups with attached RPE layer were washed with 1xPBS, stained with F4/80 antibody, and flat-mounted for fluorescent imaging. (A) The representative flat-mount images show that F4/80-stained macrophages appear to have migrated to CNV lesions at 24, 48, or 72 hrs post-laser. Images 1–8 of the laser lesion area on the whole-mounted eye cups were taken at low magnification, and the images 9–14 were taken at higher-magnification. The white arrows indicate optic disks. The scale bar in image A1 represents the magnification of images A1–8, and the scale bar in A9 represents the magnification of A9–14. Scale bar, 250 μm. (B) The number of anti-F4/80-stained macrophages in the choroid/RPE surface were counted and expressed as mean±SD, n = 4 eyes for each condition. Statistics were performed using a two-tailed Student’s t-test to compare the values between the PLGA and PLGA/TPCA-1-treated eyes. ***p < 0.005. (C) mRNA expression of Vegfa and Ccl2 determined via qPCR. RNA samples were collected from the choroid/RPE tissues of control and treated mice. The CT method normalized to Gapdh was used to analyze relative changes in gene expression. The relative expression (arbitrary units) is expressed as a ratio. Data are expressed as mean±SD, n = 3 experiments. Statistics were performed using a two-tailed Student’s t-test to compare the values between the PLGA- and PLGA/TPCA-1- treated eyes. *p < 0.05, ***p < 0.005.