Transscleral-RPE permeability of PEDF and ovalbumin proteins: implications for subconjunctival protein delivery.
OBJECTIVE
To investigate the in vitro, ex vivo, and in vivo transscleral-retinal pigment epithelium (RPE) permeability of PEDF and structurally related proteins for the exploration of novel routes of protein delivery to the retina.


METHODS
Monkey RPE cells were cultured on permeable supports to separate apical and basal compartments. Porcine scleral tissue was placed in Ussing chambers to separate uveal and orbital compartments. Transepithelial resistance and voltage were measured by an electrical resistance system, and paracellular tracer flux was evaluated with trypan blue. Subconjunctival administration in rat eyes was by injections of soluble protein or by implantation of polyvinyl alcohol devices containing protein. Fluorescein-conjugated (Fl-) PEDF and ovalbumin were determined by spectrofluorometry, laser scanning, immunoblotting, epifluorescence, and confocal microscopy. Permeability was assessed by fluoresceinated-protein flux.


RESULTS
Transepithelial resistance, impermeability to trypan blue, and confocal microscopy confirmed functional and structural tight junction formation of RPE cells cultured on permeable supports. Full-length Fl-PEDF and Fl-ovalbumin proteins diffused through RPE cell monolayers from either the apical or basal side. Fl-ovalbumin diffused through scleral tissues at constant rates. Subconjunctival Fl-PEDF or Fl-ovalbumin administration in vivo revealed movement of full-length protein into the choroid and retina as early as 1 hour.


CONCLUSIONS
The sclera and RPE were permeable in vitro, ex vivo, and in vivo to PEDF and ovalbumin proteins. These large proteins can traverse through the sclera-RPE to reach the retina. In addition, these data prompt the proposal that subconjunctival protein delivery may represent a feasible and minimally invasive route for PEDF administration in the clinic.
Amaral
Juan
J
Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-0706, USA.
Fariss
Robert N
RN
Campos
Maria M
MM
Robison
W Gerald
WG
Kim
Hyuncheol
H
Lutz
Robert
R
Becerra
S Patricia
SP
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D003228:Conjunctiva / Q000378:metabolism; D015194:Diffusion Chambers, Culture; D016503:Drug Delivery Systems; D004553:Electric Conductivity; D017097:Electric Impedance; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000378:metabolism*; D019793:Fluorescein / Q000378:metabolism; D015590:Fluorophotometry; D008253:Macaca mulatta; D018613:Microscopy, Confocal; D009414:Nerve Growth Factors / Q000378:metabolism*; D010047:Ovalbumin / Q000378:metabolism*; D010539:Permeability; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D051381:Rats; D011914:Rats, Inbred BN; D012590:Sclera / Q000378:metabolism*; D015843:Serpins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013552:Swine; D000099174:Pigment Epithelium-Derived Factor
D005136:Eye Proteins; D019793:Fluorescein; D009414:Nerve Growth Factors; D010047:Ovalbumin; D015843:Serpins; D000099174:Pigment Epithelium-Derived Factor