Effect of Membrane Porosity on CNTF Delivery Using the NT-501 Micronized Device

Abstract

Purpose:: To investigate an increase in membrane porosity of the micro NT-501 devices on the rate of CNTF delivery to the eye.

Methods:: The molecular weight cut off for PES 14 (clinical membranes) and PES 1000 was determined using fluorescently tagged dextrans. Devices were manufactured using standard protocols and held at 37°C in closed packages containing 37 mls Endo-SFM. Devices were pulsed for CNTF release in 1 ml of Endo-SFM for 24 hours during the testing period. CNTF release from devices was quantified using a commercial ELISA kit (R&D Systems). Cell metabolism was determined using the CCK-8 assay (Dojindo). Histological examination of the devices was performed using standard hematoxylin and eosin staining techniques. Apoptosis was monitored by the incorporation of fluorescein 12-dUTP using the Dead End Flurometric TUNEL Assay (Promega). Electron microscopy of membranes was performed using standard SEM techniques. Rabbits were implanted with 4 week old devices for 4 and 12 weeks. Antibodies to RPE cells were quantified by Elisa.

Results:: The molecular weight cutoff for the PES 14 and PES 1000 are 45,000 and >200000 Da, respectively. In vitro analysis of devices manufactured using PES 1000 membranes showed an increased delivery of CNTF over the testing period compared to the clinical devices. Histological examination of the devices made from either PES 1000 or clinical membrane showed that the majority of the cells from each test device were healthy. Very few cells from devices manufactured with either membrane appeared to be picnotic or unhealthy during the testing period. Examination of cells from devices treated with mitomycin C appeared to be less healthy (as indicated by darkly staining, condensed nuclei). TUNEL analysis showed that a small percentage of cells (<5%) in each test device analyzed were apoptotic as compared to devices that had been treated with mitomycin C. Rabbits tolerated the implanted device well during the implantation period and examination of the host sensitization did not show a presence of antibodies against RPE cells. In vivo device performance is currently being evaluated.

Conclusions:: The PES 1000 membrane can be employed to deliver larger doses of therapeutic molecules or molecules of larger size without observing adverse effects.

Keywords: protective mechanisms • growth factors/growth factor receptors • cell survival