# Neurorotrophic Factors in Glaucoma and Innovative Delivery Systems
## 3 Advances in Drug and Gene Delivery Systems for Neuroprotection in Glaucoma
### Neurotrophic Factors Delivery in Glaucoma  
The use of NTF-loaded biodegradable microspheres has been tested in preclinical setting for the treatment of ocular diseases including glaucoma [108; 109; 110; 111]. [PERSON] et al. demonstrated a significant increase in long-term RGC survival in a glaucoma animal model (DBA/2J mice) by intravitreally transplanting GDNF- loaded PLGA microspheres. In this study, they demonstrated that the release kinetics was nonlinear, with an initial burst of 59% registered within the first two days followed by a 30-day plateau stage in which approximately 1-2 ng/mg was released. Subsequently, a sustained delivery of 15 ng/mg was observed over the final 40 days, until a cumulative release 35.4 ng/mg was reached at 71 days. The average diameter of microspheres was approximately 10 \(\upmu\)m. Different injection time points were established in order to design four treatment protocols, while maintaining an interval of two months between each injection. Mice receiving early treatment with microsphere-delivered GDNF showed up to 3.5 times greater RGC density than untreated mice at 15 months survival [112]. The same authors performed a dosage analysis of microsphere-released NTs in the vitreous over eight weeks of IOP elevation in a rat model of glaucoma, demonstrating that 10% GDNF microspheres promoted a significant higher percentage of survival of RGCs and axons (61.58%) when compared with 2% GDNF microspheres (38.56%), blank microspheres (35.25%), and phosphate buffered saline (PBS) alone (33.12%). Similar significant results were achieved in terms of retinal inner plexiform layer (IPL) thickness preservation and glial cell inhibition in the retina and optic nerve. Moreover, an associated reduction in optic nerve head excavation was reported [111]. In view of the above, [PERSON] et al. evaluated the neuroprotective effect of GDNF-loaded PLGA microspheres in a pig model of acute retinal ischemia induced by controlled low ocular perfusion pressure. Treatment was intravitreally administered at post-ischemic Day 3 and resulted in significant increase in RGC survival at 42 days following ischemic insult with associated functional improvement, as evaluated by multifocal electroretinography (mfERG) studies [113]. [PERSON] et al. described the effects of gamma-irradiation on proteins microencapsulated according to a novel method based on S/O/W (solid-in-oil-in-water) emulsion process using PLGA microsphere combined with antioxidants [114; 115]. Recombinant human GDNF was encapsulated conforming to three technological approaches aimed at preserving protein biological activity after microencapsulation and sterilization: water-in-oil-in-water (W/O/W), solid-in-oil-in-water included in PLGA microspheres (S/O/W) without oil additive (S/O/W 1), and S/O/W in PLGA in combination with Vitamin E in the inner phase (S/O/W 2) [114]. The microspheres prepared (mean particle size 19 \(\upmu\)m; 25.4 ng GDNF/mg MSs) were able to slowly release bioactive GDNF in vitro for at least 19 weeks. The results demonstrate that the protein released from the S/O/W 2 microspheres was the most biologically active, with significant increased survival in RGCs and axons than those released from either W/O/W or S/O/W 1 [115]. The introduced changes included: (i) increased protein stability as GDNF was not pre-treated before encapsulation and unfolding-refolding phenomena at the organic, i.e., aqueous interface were avoided; (ii) the inclusion of antioxidant vitamin E in the internal phase to promote additional protein protection; and (iii) release modulation [114; 115]. The same group later evaluated the intravitreal pharmacokinetics of GDNF after a single GDNF/vitamin E-loaded microspheres (4%_w/v_; 74.85 ng GDNF) IVT injection (50 \(\mu\)L) in rabbits over an extended period of 24 weeks. Rabbits were selected due to their bigger vitreous cavity in comparison to rats (1.27 mL vs. 20 \(\mu\)L).