Diffusion of macromolecules through sclera.

PURPOSE: To quantify the in vitro permeability coefficient over different topographical locations of porcine sclera to macromolecules with different molecular weight. METHODS: Fresh equatorial and posterior superotemporal porcine sclera was mounted in a two-chamber diffusion apparatus, and its permeability to fluorescein isothiocyanate (FITC)-conjugated dextrans ranging in molecular weight from 40 kDa to 150 kDa was determined by fluorescence spectrophotometry. The sclera was processed as frozen sections and viewed with a fluorescence microscope. The thickness of the area and the thickness that macromolecules enriched in the surface of sclera were measured. RESULTS: The permeability coefficient (Pc) of porcine sclera to macromolecules was significantly higher (40 kDa, p = 0.028; 70 kDa, p = 0.033; 150 kDa, p = 0.007) in equatorial region than posterior, which could be attributed to the significant difference of thickness (p < 0.001, Kruskal-Wallis) between them. Moreover, linear regression indicated a significant negative relationship (40 kDa, p < 0.001; 70 kDa, p = 0.015; 150 kDa, p < 0.001) between scleral permeability coefficient and thickness. Also, Pc declined significantly with increasing molecular weight (MW, p < 0.001, Kruskal-Wallis). The area that the macromolecules enriched in the scleral surface was thicker for those with larger MW (p < 0.001, Kruskal-Wallis). The maximum MW and size for equatorial and posterior superotemporal scleral tissue were 185.01 KDa and 180.42 KDa, 9.92 nm and 9.67 nm, respectively. CONCLUSIONS: The permeability coefficient of porcine sclera has a significant negative relationship with scleral thickness and MW of macromolecules. Larger macromolecules are more likely to accumulate in scleral surface. The difference between topographical locations may have pharmacokinetic implications when considering transscleral diffusion of macromolecules.

Diffusion of macromolecule through retina after experimental branch retinal vein occlusion and estimate of intraretinal barrier.

The disposition and diffusion knowledge of intravitreally injected macromolecule drugs through retina in pathological condition is crucial but the related studies are absent. Retinal edema is a common pathological change of fundus diseases and retinal vein occlusion (RVO) pig model were established to emulate it. FITC-dextrans of various molecular weights were dissolved in RPMI-1640 solutions and the rate of transretinal diffusion was determined with a spectrophotometer. Theoretical maximum size of molecule (MSM) was calculated by extrapolating the trend-linear relationship with the diffusion rate. In separate experiments to determine the sites of barrier to diffusion, FITC-dextrans were applied to either the inner or outer retinal surface, processed as frozen sections, and viewed with a fluorescence microscope. Paired-Samples T test was used to compared the diffusion rate of dextrans of the both eyes of one pig. The MSM in RVO tissues and normal tissue was 6.5+/-0.39 nm and 6.18+/-0.54 nm respectively (t=4.143, P=0.0001). FITC-dextrans applying to inner retinal surface, 4.4 kDa dextran were largely arrested at inner nuclear layer (INL). The INL of the 19.6-71.2 kDa dextran diffusion retina section became dark and the nerve fiber layer (NFL) and inner plexiform layer got brighter. As for 150 kDa dextran, the NFL was bright and the other layers were dark. FITC-dextrans applying to outer retinal surface, most dextrans were blocked before outer nuclear layer (ONL). In summary, ONL and INL may act as bottle-neck barriers to diffusion of macromolecules. Compared with normal neuroretina, the MSM of fresh edema retina after RVO increased limitedly.