Relationship of Stokes Radius to the Rate of Diffusion across Bruch's Membrane

Department of Ophthalmology and Vision Science, University of Arizona, Tucson, Arizona 85724, USA.
Investigative ophthalmology & visual science (Impact Factor: 3.4). 06/2011; 52(7):4907-13. DOI: 10.1167/iovs.10-6595
Source: PubMed


To determine the effect of Stokes radius (R(S)) on the diffusion of molecules through Bruch's membrane (BM), and to establish a system suitable for the analysis of diffusion through small (<2 mm(2)) samples of BM.
Porcine BM/choroid (BM/Ch) was mounted in a modified Ussing chamber. A concentration gradient was simultaneously established for four tracers with R(S) values ranging from <1.0 to 6.15 nm. Samples were collected from both chambers at various time points up to 36 hours and the amount of each tracer was determined using quantitative gel exclusion chromatography. The integrity of samples was determined using scanning electron microscopy.
BM/Ch mounted in the chamber exhibited no obvious damage even after 36 hours in the chamber. Flux was significantly (P < 0.05) greater in the BM to Ch direction than that in the Ch to BM direction for only two of the tracers: cytosine and RNase A. Flux also was dependent on R(S); cytosine, the smallest tracer (R(S) < 1 nm), exhibited the greatest flux and ferritin (R(S) = 6.15 nm) the least. Permeability coefficients for each tracer were determined and exhibited a power relationship with R(S).
Flux was dependent on the direction of the concentration gradient and the R(S) of the individual tracers. We have successfully demonstrated that quantitative gel exclusion chromatography can be used to follow diffusion of a mixture of tracers across BM/Ch, and that we can measure flux across BM/Ch preparations with an exposed surface area as small as 1.8 mm(2).

Download full-text


Available from: Astrid Zayas-Santiago, Aug 20, 2014
    • "Additionally, a number of reports describing in-vivo and ex-vivo transport experiments on the diffusion behavior of small and large molecules in the direction of VH to choroid [19] [20] [21] [22] as well as choroid to VH [23] are available. However, ex-vivo transport experiments are limited in the duration of the experiment to a few hours only, in order to maintain tissue integrity (retina/choroid). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The stability of protein therapeutics during the residence time in the vitreous humor (VH) is an important consideration for intra ocular treatment and can possibly impact therapeutic efficacy and/or treatment intervals. Unavailability of the reliable Ex-vivo intravitreal (ExVit) model to estimate protein stability following IVT has driven the research focus to develop such model which can facilitate protein stability estimation before in-vivo experiments. In this manuscript, we have developed and evaluated three ExVit models, namely, ExVit static, semi-dynamic and dynamic. These models were utilized and compared when studying the in-vitro stability of model protein formulations under simulated intraocular conditions using porcine vitreous humor (VH). The ExVit static model exhibited significant precipitation and aggregation of proteins, most likely due to pH change occurred in the VH after isolation. The semi-dynamic model assessed was composed of two compartments i.e., VH- and buffer-compartment which has effectively stabilized the pH of the VH and facilitated the migration of VH degradation products. However, some limitations related to investigation of long-term protein stability were also observed with semi-dynamic model. The dynamic model developed, was comprised of three diffusion controlling barriers (two diffusion controlling membranes and a gel-matrix), which allowed modulation of the diffusion rate of macromolecules. The ability of dynamic model to modulate protein retention time in the VH will overcome the challenges faced by the semi-dynamic model such as long-term stability evaluation. Copyright © 2015 Elsevier B.V. All rights reserved.
    No preview · Article · May 2015 · European journal of pharmaceutics and biopharmaceutics: official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik e.V
  • [Show abstract] [Hide abstract]
    ABSTRACT: Purpose: Retinal pigment epithelium (RPE) transplantation is a promising strategy for the treatment of dry age-related macular degeneration (AMD). However, previous attempts at subretinal RPE cell transplantation have experienced limited success due to poor adhesion, organization, and function on aged or diseased Bruch's membrane. Instead, cell-based strategies may benefit from a synthetic scaffold that mimics the functions of healthy Bruch's membrane to promote the formation of a functional RPE monolayer while maintaining metabolite exchange between the vasculature and outer retina. Methods: This study evaluated the behavior of human RPE on nanopatterned porous poly(ε-caprolactone) (PCL) film as a potential scaffold for therapeutic transplantation. Fetal human RPE (fhRPE) was cultured on porous PCL, nonporous PCL, or Costar porous polyester transwells for up to 8 weeks and assessed using light microscopy, fluorescent microscopy, transepithelial resistance, quantitative PCR, ELISAs, and phagocytosis assays. Results: fhRPE on porous PCL displayed improved markers of maturity and function compared with both porous polyester transwells and nonporous PCL, including pigmentation, increased cell density, superior barrier function, up-regulation of RPE-specific genes, and polarized growth factor secretion. Conclusions: This study indicates that porous PCL is an attractive scaffold for RPE transplantation. In addition to being biocompatible with the subretinal space, porous PCL also allows for trans-scaffold metabolite transport and significantly improves RPE cell behavior compared to nonporous PCL or porous polyester transwells.
    No preview · Article · Feb 2014 · Investigative ophthalmology & visual science