Preparation And Pharmacokinetics Characterization Of Dexamethasone Loaded PLGA Nanoparticles In Rabbit Eyes

Purpose Glucosteroid treatment has already been widely used in ophthalmic clinic and proved to be effective for ocular proliferative diseases such as retinal and choroidal angiogenesis, and inflammatory diseases such as uveitis and retinal vasculitis. However, the application of glucosteroid is limited by its systemic side effects and the various barriers of the eye. Generally, intravitreal injection is supposed to be more direct and effective than oral administration or intravenous injection. But drug concentration decreases fast in vitreous cavity after a single injection while multi-injections would greatly increase the risk of complications such as intraocular hemorrhage, cataract, and retinal detachment, etc. Nanoparticles is a kind of sustained drug delivery system basing on biodegradable and biocompatible polymer that could produce prolonged and stable drug concentration in local intraocular site.In this study: ?Dexamethasone acetate loaded PLGA nanoparticles will be prepared, its mean particle size and size distribution will be described and morphological study will be performed. The efficiency of dexamethasone entrapped in nanoparticles and the drug release rate in vitro will also be examined. (2) Then DA-PLGA nanoparticles will be intravitrealy injected in rabbit eyes and the pharmacokineticscharacterization of DA in various ocular tissues and plasma will be performed by HPLC, in order to investigate the feasibility of intraocular application of drug loaded nanoparticles.Method â‘  Freeze-dried DA-PLGA nanoparticles containing 20% (NP20) or 50% (NP50) DA was prepared by emulsification/solvent evaporation method with slight modifications. The particles was weighed to determine the efficiency of production and entrapment. The morphologic study and surface characterization of the nanoparticles was performed through scanning electron microscope (SEM) and transmission electron microscope(TEM). Nanoparticle size and its distribution were assessed by photon correlation spectroscopy. In vitro drug release from the nanoparticles was performed in sodium phosphate buffer saline (PBS, pH7.4, 154mM) at 37℃ utilizing double-chamber diffusion cells on a shaker stand.(2) After being sterilized by γ-radiation, NP20 and NP50 were suspended in normal saline at the concentration of 20g· L^-1. Rabbits were divided into two groups and each group received intravitreal injection of 0.1 mL NP20 or NP50 suspension respectively. Fundus observation and photography of the rabbits were performed at the 1, 7, 14, 21th day after injection, drug concentration in various ocular tissues and plasma was also measured by HPLC at each time point. The preparation that could maintain higher and longer drug concentration in local site would be selected for future study. After intravitreal injection of the selected DA-PLGA nanoparticles suspension (20g·L^-1,0.1mL) in rabbit eyes, clinical investigation including fundus observation and photography, IOP measurement, and ocular B mode ultrasound were performed at the 1,3,7, 14, 21, 28, 35, 42, 50, 80th day. At each time point, three samples of each ocular tissue, involving cornea, aqueous humor, lens, iris, vitreous, and chorioretina were taken and extracted for HPLC analyse. The control group that treated with DA followed exactly the same process.Results â‘  Emulsification/solvent evaporation method with slight modifications we used produced NP20 and NP50 with production efficiency of 55.31% and 73.05% respectively. The DA entrapment efficiency of NP20 was theoretically 18.2% and actually 19.2%, and those of NP50 was 45.5% and 45.3%. Transmission electron micrography and scanningelectron micrography showed that the DA-PLGA nanoparticles had spherical shape. The effective particles size of NP50 was determined to be 232.5nm, the mean size to be 263.6nm, and the polydispersity index to be 0.19 by photon correlation spectroscopy. In vitro, the release profile of DA from NP20 has a triphasic pattern lasting for about 28 days: an initial burst, a lag phase with relatively high release rate and another lag phase with very low release rate. While NP50 has a quadriphasic release pattern lasting for at least 48 days: an initial burst, a lag phase with relatively high release rate, another burst and a descending phase with declining release rate.(2) Within the 21 days after intravitreal injection of NP20 and NP50 suspension in rabbit eyes, the drug concentration in cornea and aqueous humor was below the detectable limit (10μg ·L^-1) of HPLC assay, and the Cmax in plasma was 0.24mg·L^-1 The drug concentration in chorioretina ranged 0.11-0.42 mg^L-1 in NP₂0 group and 0.38-0.88 mg·L-1 in NP₅0 group, while that in vitreous ranged 0.82²6.52mg·L^-1 and 1.78 85.72mg·L^-1 respectively. Clinical observation had no discovery of abnormality. It seemed that NP50 had superior drug release traits to NP₂0 and thus the former was selected for pharmacokinetics characterization study. NP50 kept releasing DA for 50 days in rabbit eyes with a triphasic pattern involving an initial burst, a lag phase and a declining phase. As for vitreous and chorioretina, the second phase persisted for about 1 month. During the proceeding, the DA concentration in cornea, aqueous humor and lens were all below the detectable limit, and that in iris was detected only on the the 1st day with extremely low value of 0.08±0.02 μg·g^-1. In vitreous, the Cmax was 8.48±1.19 mg·L^-1, and the mean DA concentration of the second phase was 3.85 mg·L^-1, which is 5.5 times of that of plasma (0.7mg·L^-1) with significant statistical difference (P<0.001). Compared with control group that treated with DA, the drug concentration in vitreous (P=0.86), chorioretina(P=0.74) and plasma(P=0.156) showed no significant diffrence, while calculation of areas] under the curve revealed that the bioavailability of DA in test group was far higher. Clinical investigation showed that the nanoparticles were well tolerable and quite safe for intraocular treatment.Conclusion In vitro, DA-PLGA nanoparticles notablely prolonged DA release, the release time and pattern varies according to the drug...