# Pharmacokinetics of Pullulan-Dexamethasone Conjugates in Retinal Drug Delivery
## 4 Discussion  
_Elimination kinetics._ The intravitreal pharmacokinetics and safety of pullulan-dexamethasone conjugates were investigated in detail. The results indicate that the conjugates are safe after intravitreal injections to the rodent eyes. Furthermore, they show prolonged retention in the rabbit eyes and retinal distribution via [PERSON] glia cells in mouse eyes. Non-modified pullulan behaves as a random coil of individual polymeric chains with diameters of few nanometers [13; 33]. In this study, we used conjugates of pullulan with hydrophobic compound DEX and BDP. Pullulan conjugate with BDP was used since it allows direct and non-invasive monitoring of the particle kinetics after intravitreal injections. A roughly two-fold size difference was seen between pullulan-DEX and BDP-pullulan-DEX, but this should not cause kinetic differences since even several fold changes in liposome diameter (from <50 nm to >200 nm) did not change diffusivity in the vitreous [56]. These conjugates self-assemble to nanoparticles that showed similar retention times (half  
Figure 14: Images of retinal sections from two-months old mice after intravitreal injection (1 \(\upmu\)L) of 5 mg/mL Cy3-pullulan-DEX (cyanine3, red). Müller glial cells were labelled with an antibody against glutamine synthetase (green). Nuclei were stained with DAPI (blue). RG: overlay of red and green channels for Müller glial cell and nanoparticle colocalization. RGB: overlay of red, green and blue channels. Bar size: 20 \(\upmu\)m.  
lives \(\approx\)2.5-6.0 days) in the rabbit vitreous as macromolecules (e.g., antibodies and FITC-dextrans) [15], but shorter than the retention of some polymeric micelles and polymersomes (half-lives \(\approx\)9-31 days) [12]. Based on particle size, longer half-lives would be expected for pullulan conjugates, but the pullulan conjugates are structurally different systems than polymeric micelles and polymersomes. Furthermore, pullulan conjugates showed even distribution in the rat vitreous already at one day after injection, suggesting fast diffusion of the pullulan conjugates in the rat vitreous. The reasons for the described kinetic profile of pullulan conjugates are not known, but could involve biological interactions or particle disassembly. Disassembly of the particles to individual polymer conjugates might explain the results, but particle exposure to homogenized vitreous did not indicate particle disassembly in our previous study [26]. In vivo studies of nanoparticle disassembly and other mechanisms in the vitreous are methodologically challenging and the molecular mechanisms of pullulan-DEX elimination remain open for the time being. In order to elucidate the elimination route of pullulan conjugates from the vitreal cavity, we performed kinetic simulations by assuming that all polymer is eliminated to the anterior chamber at the experimental elimination rate from the vitreous and further eliminated from the anterior chamber at the rate of aqueous humor outflow. The simulated particle concentrations in the aqueous humor were close to the experimental values, indicating that practically all pullulan conjugates are eliminated from the vitreous via anterior route. Moreover, the Maurice plot (\(\mathrm{C_{v}/C_{a}}\) vs. vitreal half-life) derived estimates of anterior elimination suggested that the major fraction (at least 82.7%) of intravitreally injected pullulan conjugate is eliminated via anterior route [49]. An anterior elimination route has been shown earlier for soluble macromolecules (e.g., antibodies) [57; 58], but we have shown here for the first time that this route dominates the vitreal elimination of nanoparticles. According to the observed kinetics of pullulan-DEX in rats and rabbits, the vitreal elimination half-life was longer in the rabbits. This is in line with our previous data with FITC-dextrans in rats and rabbits [15]. The current data shows that similar species difference is seen also with nanoparticles.