| Purpose: Retinal drug delivery is a challenging area in the field of ophthalmic drug delivery. Drug delivery to retinal tissue and vitreous via systemic administration is constrained due to the presence of a blood-retinal barrier (BRB) which regulates permeation of substances from blood to the retina, so intravitreal injection has become the routine to treat the posterior nodule disease of eye. Although intravitreal administration overcomes this barrier, retains the effective drug concentration in vitreous cavity and avoids the drug systemic side effects, it is associated with several other problems, for example, drug concentration decreases fast after a single injection while multi-injections must be repeated to maintain the drug level within a therapeutic range, with the risks of vitreous hemorrhage, retinal detachment or endophthalmitis, and so on. Advances in the ocular drug delivery systems research are expected to provide new tools for the treatment of the posterior segment diseases with the new therapeutic modalities, which should provide prolonged action, less invasive administration, higher efficacy, and improved safety. An ideal drug delivery system for the retina and vitreous humor has not yet been found, despite extensive research.Controlled delivery systems via targeted drug delivery of a predetermined dose over a sustained period have been used to overcome the shortcomings of conventional dosage forms. Nanoparticulate technology is advocated as an ophthalmic drug delivery approach that may enhance dosage form acceptability while providing sustained release in the ocular milieu. Thus, sustained-release technologies are being proposed, and the benefits of using colloidal carriers in intravitreal injections are currently under investigation for posterior drug delivery. Poly(N-isopropylacrylamide)- polyethylene oxide(PNIPAAm-PEO)is a new kind of synthetical polymerizer,whose PNIPAAm plays the important role in the framework and has been widely examined as a smart drug delivery material due to its unique phase separation behavior upon external temperature changes. In this study: (1) To assess the potential of PNIPAAm-PEO nanoparticles as ocular sustained drug delivery carrier by investigating the retinal toxicity of intravitreal injection and also their interaction with the ocular tissue in vivo. (2) Then norvancomycin loaded PNIPAAm-PEO nanoparticles will be intravitrealy in normal rabbit eyes and the intraocular pharmacoklnetics characterization of norvancomycin in various ocular tissues and plasma will be performed by HPLC, in order to investigate the feasibility of intraocular application of drug loaded nanoparticles.Methods: (1) After being sterilized byγ-radiation, PNIPAAm-PEO nanoparticles were suspended in normal saline at the different concentration of 1mg/0.1ml,2mg/0.1 ml,3mg/0.1ml and 4mg/0.1ml. New Zealand rabbits were randomly into 5 groups. Rabbits in group 1, 2, 3, 4 received intravitreal injection of 0.1ml PNIPAAm-PEO nanoparticles at different dosage respectively, which are 1mg/0.1ml,2mg/0.1 ml,3mg/0.1ml,4mg/0.1ml. Rabbits in group 5 as control received intravitreal injection of sterilized normal saline. The eyes were examined at the 1,2,3,7,14th day after intravitreal injections by slit-lamp microscope, ophthalmoscopy, light microscope and some also by transmission electronic microscope. To all eyes, ERG were recorded. While these different concentration diluent were also used as eye dropping locally in order to observe the stimulation of ocular tissue in rabbits.(2) After being sterilized byγ-radiation, NV-PNIPAAm-PEO nanoparticles were suspended in normal saline at the concentration of 2mg/0.1ml. Twenty-one New Zealand rabbits (42 eyes) received intravitreal injection of 0.1ml NV-PNIPAAm-PEO nanoparticles,then were divided into 7 groups of 3 animals each. After intravitreal injection, clinical investigation including funds observation and photography,ocular B mode ultrasound were performed at the l,2,3,7,4,2l,28th day . At each time point,three samples of plasma and each ocular tissue,involving cornea,aqueous humor,vitreous and chorioretina were taken and extracted for HPLC analyse.Results: (1) The rabbit ocular surface showed no signs of inflammation or alteration after different dosage PNIPAAm-PEO exposure compared with the control. No evidence of toxicity was found in rabbits` eyes after intravitreal injections of the PNIPAAm-PEO nanoparticles of 1mg/0.1ml and 2mg/0.1ml, but intravitreal injections of the 3mg/0.1ml and 4mg/0.1ml induced toxic effects on the retinas,which could make the b-wave of ERG diminished, and maily shew extensive vacuolar degeneration in histopathology. In 3 mg group lightly changes in retina were observed by light microscope after 14 days, but electron microscope changes were more observable. However, obvious changes of all of examinations were found in 4 mg group in whole 14 days, when the decline rates of b-wave of ERG were above 30%.(2) Within the experimental duration, norvancomycin concentration in the cornea was below the limit of detection of the HPLC assay,and the Cmax in plasma was 0.26μg/mL . Nanoparticles containing 22 % norvancomycin produced drug concentration ranging 0.08～2.16μg/mL in the aqueous humor, and 0.11～2.54μg/g in the chorioretina, while that in vitreous ranging 5.65~436.69μg/mL respectively. norvancomycin -PNIPAAm-PEO nanoparticles kept releasing norvancomycin for 28 days in vitreous and chorioretina of rabbit eyes with a diphase pattern involving an initial burst and a declining phase. As for aqueous humor and plasma the second phase persisted for less than 21 days. The drug concentrations in vitreous body were higher than minimal inhibitory concentration (MIC) of the most gram-positive bacteria. The therapeutic drug concentration could be reached and last for 21 days in vitreous. Cilinical observation had no discovery of abnormality and showed good biocompatibility of the norvancomycin-PNIPAAm-PEO nanoparicles.Conclusion: (1) The PNIPAAm-PEO nanoparticles were well tolerated by the ocular surface tissues. They could reduce retina toxicity at the dosage of more than 3mg/0.1ml in vitreous. So these facts add further support for the potential use of the PNIPAAm-PEO nanoparticles for ocular drug delivery, and ocular safety problems should be paid more attention to while the large dose intravitreal injections. (2) In vivo, PNIPAAm-PEO nanoparticles containing 22 %norvancomycin notablely prolonged norvancomycin release, and exhibited the effective dosage maintaining 21days. Norvancomycin-PNIPAAm-PEO nanoparicles showed satisfactory biocompatibility and pharmacokinetics characters that superior to norvancomycin after cilinical intravitreal injections. Norvancomycin was concentrated in vitreous and choroiretina, and the vitreous norvancomycin concentration was higher than the plasma norvancomycin concentration. Thses results indicated that the PNIPAAm-PEO nanoparicles had quite potential for ocular drug delievery systems.
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