Ciliary neurotrophic factor (CNTF) is one of the most studied neuroprotective agents with acknowledged potential in treating diseases of the posterior eye segment. Although its efficacy and mechanisms of action in the retina have been studied extensively, it is still not comprehensively understood which retinal cells mediate the therapeutic effects of CNTF. As with therapeutic proteins in general, it is poorly elucidated whether exogenous CNTF administered into the vitreous can enter and distribute into the retina and hence reach potentially responsive target cells. Here, we have characterized our purified recombinant human CNTF (rhCNTF), studied the protein’s in vitro bioactivity in a cell-based assay, and evaluated the thermodynamic and oligomeric status of the protein during storage. Biological activity of rhCNTF was further evaluated in vivo in an animal model of retinal degeneration. The retinal penetration and distribution of rhCNTF after 24 h was studied utilizing two ex vivo retina models. Based on our characterization findings, our rhCNTF is correctly folded and biologically active. Moreover, based on initial screening and subsequent follow-up, we identified two buffers in which rhCNTF retains its stability during storage. Whereas rhCNTF did not show photoreceptor preservative effect or improve the function of photoreceptors in vivo, this could possibly be due to the used disease model or the short duration of action with a single intravitreal injection of rhCNTF. On the other hand, the lack of in vivo efficacy was shown to not be due to distribution limitations; permeation into the retina was observed in both retinal explant models as in 24 h rhCNTF penetrated the inner limiting membrane, and being mostly observed in the ganglion cell layer, distributed to different layers of the neural retina. As rhCNTF can reach deeper retinal layers, in general, having direct effects on resident CNTF-responsive target cells is plausible.

The aim of this study was to develop an injectable hydrogel delivery system for sustained ocular delivery of dexamethasone. To this end, a self-healing hydrogel consisting of a thermosensitive ABA triblock copolymer was designed. The drug was covalently linked to the polymer by copolymerization of methacrylated dexamethasone with N-isopropylacrylamide (NIPAM) and N-acryloxysuccinimide (NAS) through reversible addition–fragmentation chain transfer (RAFT) polymerization, using poly(ethylene glycol) (PEG) functionalized at both ends with a chain transfer agent (CTA). Hydrogel formation was achieved by mixing aqueous solutions of the formed thermosensitive polymer (with a cloud point of 23 °C) with cystamine at 37 °C, to result in covalent cross-linking due to the reaction of the N-hydroxysuccimide (NHS) functionality of the polymer and the primary amines of cystamine. Rheological analysis showed both thermogelation and covalent cross-linking at 37 °C, as well as the self-healing properties of the formed network, which was attributed to the presence of disulfide bonds in the cystamine cross-links, making the system injectable. The release of dexamethasone from the hydrogel occurred through ester hydrolysis following first-order kinetics in an aqueous medium at pH 7.4 over 430 days at 37 °C. Based on simulations, administration of 100 mg of hydrogel would be sufficient for maintaining therapeutic levels of dexamethasone in the vitreous for at least 500 days. Importantly, dexamethasone was released from the hydrogel in its native form as determined by LC–MS analysis. Cytocompatibility studies showed that at clinically relevant concentrations, both the polymer and the cross-linker were well tolerated by adult retinal pigment epithelium (ARPE-19) cells. Moreover, the hydrogel did not show any toxicity to ARPE-19 cells. The injectability of the hydrogel, together with the long-lasting release of dexamethasone and good cytocompatibility with a retinal cell line, makes this delivery system an attractive candidate for treatment of ocular inflammatory diseases.