# Pharmacokinetics of Pullulan-Dexamethasone Conjugates in Retinal Drug Delivery
## 4 Discussion
The implants release all the drug payload in the eye and thereafter a 'ghost matrix' remains in the eye. Interplay of delivery system retention, drug release and ocular pharmacokinetics has not been discussed in the literature. The presented simulation models provide useful tools for drug developers who optimize intravitreal drug delivery systems towards target product profiles. _Retinal permeation._ The inner limiting membrane (ILM) is a mechanical and electrostatic barrier that limits access of nanoparticles into the retina. The ILM includes a negatively charged network of collagen and glycosaminoglycans [66]. Retinal delivery of materials with the molecular weights \(\leq\) 100 kDa are not limited by ILM [67]. However, the electric charge of the materials can make a difference: FITC-dextrans of 20 kDa and 500 kDa permeated through ILM, but cationic poly-L-lysine (20 kDa) did not [68]. Recently, liposomes (\(\approx\)50 nm) with negative charge and PEG-coating were shown to permeate across bovine ILM that is considered a close model for human ILM [44]. However, larger liposomes of about 100 nm in diameter were not able to cross the ILM barrier in bovine retina. In this study, pullulan conjugates (particle size 200-400 nm) were permeating only to the ganglion cell layer of the bovine ex vivo retina, but not further into the retina. Ganglion cells are an important drug target, especially in the treatment of glaucomatous retinal degeneration [69]. Limited ILM permeation is also an obstacle in the field of retinal gene therapy with viral vectors and, for this reason, retinal gene therapy is performed using subretinal injections [70]. Better understanding of the ILM and other contributing factors will help in development of intravitreal treatments of retinal diseases. In various retinal disorders and upon ageing, ILM may become thinner and presents pores thereby become leakier to the nanoparticles [60]. Retinal permeation of pullulan was also investigated with organotypic mouse retinal explant that can be maintained as biologically active even for one month [71]. This model is useful in mechanistic pharmacological studies [1; 2; 72; 73; 74; 75]. ILM in mouse retina is thinner and leakier than in the bigger eyes, potentially giving optimistic views on retinal permeation of particles [44]. However, this study showed qualitatively distinct retinal distribution: pullulan conjugates did not cross ILM, but they were taken up by the Muller cells that may not be as active in the isolated bovine retina. The pullulan-dexamethasone nanoparticles showed protective effects in the retina, suggesting drug release from the conjugates in the retina. Delivery of pullulan conjugates to Muller cells can be explained on the location and properties of these cells. Muller cells are glial cells capable of phagocytosis cell bodies,fragments of retinal cells and particles [76; 77; 78]. The cell bodies of Muller glia are located in the inner nuclear layer with its two stem processes extending in opposite directions and spanning the entire retina. Therefore, it is likely that intravitreal particles could be phagocytosed by Muller cells and carried into the inner retinal layers. In this study, the cultured retinal explants from wild-type mice were exposed to pullulan-based particles from the ganglion cell layer side on the retina. Colocalization with glutamine synthase antibody (Muller glia marker) confirmed particle localization in Muller cells and the particles were visualized in radial sections (red punctas, Figure 12) through all retinal layers in a linear distribution similar to the Muller cells extension (from the ganglion cell layer until reaching the distal part of the outer nuclear layer (Figure 12, and 3D visualization in Supplementary Materials). This indicates that the pullulan-based particles were phagocytized by the Muller cells, thereby overcoming the barrier of inner limiting membrane. Muller glial cells are a major type of macroglial cells in the neural retina [79; 80; 81; 82; 83].