# Pharmacokinetics of Pullulan-Dexamethasone Conjugates in Retinal Drug Delivery
## 1 Introduction  
Intravitreal injection is the most important mode of drug administration in the treatment of retinal diseases. In particular, anti-inflammatory drugs (e.g., corticosteroids) [1, 2, 3] and anti-neovascular inhibitors of vascular endothelial growth factor (VEGF) (antibodies, Fab-fragments, soluble receptors and aptamers) are widely used in clinics [4, 5, 6, 7, 8]. In general, intravitreal injections are safe, but frequent injections may result in reduced patient compliance and some rare, but serious, adverse effects (e.g., infection and retinal detachment) [5, 9]. Implants with prolonged ocular residence and controlled release have been developed to prolong injection intervals of dexamethasone (e.g., Ozurdex) [1, 3, 9]. On the other hand, delivery systems for trafficking therapeutics to the retinal cells (e.g., intracellularly actingpeptides, proteins and nucleic acids) are needed. Therefore, nanoparticles and peptide conjugates have been recently investigated for retinal delivery of dexamethasone and nucleic acids [10; 11; 12].  
Modified polysaccharides are promising candidates for the development of ocular drug delivery systems [13]. For example, pullulan [14], dextran [15], hyaluronic acid [16] and chitosan [17] have been investigated in this respect. Many polysaccharides can be chemically functionalized for optimized drug delivery [13; 18] and they can be formulated as macroscopic implants [19; 20], gels [21] and nanosized formulations [22]. In most studies, drugs have been physically encapsulated into the polysaccharide formulations, such as polymeric micelles or nanoparticles [13; 23; 24; 25], whereas chemical covalent conjugation technologies have not been applied for ocular in vivo drug delivery with polysaccharides. Recently, the chemical conjugation through hydrazone bond was used to generate peptide-dexamethasone and pullulan-dexamethasone conjugates that were investigated in vitro [10; 26]. In contrast to the ocular field, polymeric drug conjugates have been widely investigated to provide site-specificity and extended drug release in some other medical indications [27; 28; 29; 30; 31; 32].  
We have been investigating drug carriers based on pullulan, a fungal extracellular polysaccharide produced by _Aureobsodium pullulans_[33; 34]. Inexpensive and conveniently modified pullulan is considered to be a biocompatible polymer. Previously, pullulan has been used as a backbone in the synthesis of bioconjugates for drug delivery to the liver and pancreas [14; 27; 33; 35; 36; 37]. Conjugation of hydrophobic drugs to pullulan results in self-assembled colloids with drug molecules oriented to the core of the nanoparticles [27; 28; 33; 35; 36].  
In this study, we investigated dexamethasone conjugates of pullulan that were obtained with recently published synthetic procedures [26]. Dexamethasone was conjugated to pullulan through a hydrazone bond that is expected to control drug release under the acidic intracellular compartments (endosomes or lysosomes). We investigated ocular safety, retinal distribution and ocular pharmacokinetics of fluorescently labelled pullulan-dexamethasone after intravitreal injections into mouse, rat and rabbit eyes. Retinal distribution of the conjugates was also investigated using ex vivo retinas of mice and cows.