Terminable cell-encapsulating drug delivery device for sustained intraocular glial cell-derived neurotrophic factor (GDNF) delivery

Abstract

Encapsulated-cell therapy (ECT) is an attractive approach to treat sight-threatening diseases through sustained delivery of therapeutics secreted from immunoisolated cells. Although collagen-alginate (CAC) scaffold integrates the merits of collagen and alginate, and alginate-poly-l-lysine (AP) polyelectrolytic coating improves the properties of alginate systems, the properties and performance of CAC and AP-CAC ECT systems are not well-understood. Also, sustained delivery of GDNF, a potent neurotrophic factor with a short half-life, via ocular ECT has not been investigated. Moreover, most reported ECT systems do not have externally controllable functions.

Aiming to develop externally controllable ECT devices for prolonged ocular applications, this study investigated the relationships between: (1) the factors controlling CAC-based matrix properties, (2) the interactions between cell, collagen, alginate and AP-coating, and (3) the long-term stability and functionality of these systems. The effectiveness of Tet-on inducible Caspase 8 (Casp8) termination control for safe in vivo applications was also examined.

Our findings indicated that cell growth kinetics and microstructural organization of CAC-based ECT systems were tunable by factors controlling collagen and alginate gelation, including alginate concentration, molecular weight and viscosity, rate of alginate gelation, sequence of CAC gelation, initial cell number, and poly-l-lysine coating conditions. Stability in device microstructure and net cell proliferative effect of CAC-based ECT systems allowed the devices to remain stable and functional for prolonged periods of time in vitro and in the eyes of healthy or dystrophic Royal College of Surgeons rats with inherited retinal degeneration. Encapsulated cell viability and daily GDNF secretion have shown to be closely related. These devices are well-tolerated in vivo without host cells attachment or cell protrusion. Sustained GDNF delivery in dystrophic rats resulted in a dose-dependent photoreceptor and electroretinogram functional rescue 28 days post-implantation. Our pilot data showed that CAC platform maintained its microstructure and supported the growth of proliferative HEK293 cells or non-proliferative immortalized human retinal pigment epithelial cells, ARPE-19, without protrusion 6 months post-implantation. AP-coating improved the encapsulation power, shell microstructure, cell growth kinetics and daily GDNF secretion from CAC systems over 28 days of culture. Preliminary in vivo studies suggested that AP-CAC and CAC devices achieved comparable photoreceptor rescue effect in dystrophic rats.

Tet-on Casp8 HEK293 cells demonstrated good viability under non-induced conditions and effective termination upon Doxycycline (Dox) treatment. Cell death was mediated by pro-Casp8 overexpression and caspase-mediated apoptotic pathway after 72 hours of 100pg-8μg/ml Dox treatment. Comparably, most encapsulated Tet-on cells were terminated after 24 hours of 2ng-2μg/ml Dox treatment and no viable cells were detected at 72 hours. Implanted ECT devices were able to respond to Dox. Most cells in retrieved devices underwent apoptosis after 48 hours of 1mg/ml Dox delivered in sweetened drinking water.

This study characterized the properties and performance of externally controllable CAC-based ocular ECT devices. These devices offer great flexibility in system design and can potentially be applied to many posterior eye disorders.