Kompella Uday B, Sundaram Sneha, Raghava Swita, Escobar Edith R
Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198-5840, USA.
Mol Vis. 2006 Oct 17;12:1185-98.
To determine whether topical ocular delivery of <100 nm nanoparticles can be enhanced by coating their exterior with peptide or protein ligands for cell surface receptors.
A novel ex vivo bovine eye model was validated for its integrity up to 60 min. Using this model, the uptake of 20 nm polystyrene nanoparticles (administered as a single 50 mul drop) before and after surface conjugation with deslorelin, a luteinizing hormone-releasing hormone (LHRH) agonist, or transferrin was determined at 5 and 60 min in individual layers of cornea and aqueous humor. Selected studies were done in the absence of corneal epithelium in the ex vivo model or using excised cornea and conjunctiva. LHRH and transferrin receptor mRNA and protein expression in corneal epithelium and conjunctiva were determined by real-time PCR and western blot, respectively.
Corneal histology, ZO-1 immunostain pattern, and mannitol permeability were similar in controls and at the end of the ex vivo study. Corneal epithelial nanoparticle uptake in the absence of surface modification was 1.1-1.6% at 5 min and remained at about this level even at 60 min. Removal of the corneal epithelium resulted in about 22% particle uptake in the corneal stroma at 5 and 60 min compared to about 0.5% in the presence of epithelium, indicating the barrier nature of corneal epithelium. Deslorelin and transferrin conjugation enhanced corneal epithelial uptake of nanoparticles by 3- and 4.5 fold at 5 min and by 4.5- and 3.8 fold at 60 min, respectively. The total corneal uptake in 5 min is approximately 2.4, 9, and 16% with plain, deslorelin-functionalized, and transferrin-functionalized nanoparticles. In all groups, the nanoparticle uptake per unit tissue weight was in the order: corneal epithelium>stroma>endothelium with levels in the aqueous humor being undetectable. In excised cornea and conjunctiva studies, nanoparticle transport and uptake was elevated for both deslorelin and transferrin conjugated nanoparticles. Expression of LHRH and transferrin receptors was observed in corneal epithelium as well as conjunctiva.
The ex vivo bovine eye model is a useful tool in understanding disposition of nanoparticles after topical delivery. The corneal epithelium is a significant barrier for topical nanoparticle delivery to the anterior segment. Surface modification of nanoparticles by conjugating an LHRH agonist or transferrin is a useful approach to provide rapid, efficient delivery of intact nanoparticles into and/or across cornea and conjunctiva.
确定通过用细胞表面受体的肽或蛋白质配体包被其外部，是否可以增强小于100纳米纳米颗粒的眼部局部递送。
一种新型的离体牛眼模型经证实其完整性可达60分钟。使用该模型，在与促黄体激素释放激素（LHRH）激动剂地洛瑞林或转铁蛋白进行表面偶联之前和之后，于5分钟和60分钟时在角膜和房水的各层中测定20纳米聚苯乙烯纳米颗粒（以单次50微升滴眼剂给药）的摄取量。在离体模型中无角膜上皮的情况下或使用切除的角膜和结膜进行了选定的研究。分别通过实时PCR和蛋白质印迹法测定角膜上皮和结膜中LHRH和转铁蛋白受体的mRNA和蛋白质表达。
对照组和离体研究结束时，角膜组织学、ZO-1免疫染色模式和甘露醇通透性相似。在无表面修饰的情况下，角膜上皮纳米颗粒摄取在5分钟时为1.1%-1.6%，即使在60分钟时仍保持在该水平左右。去除角膜上皮后，在5分钟和60分钟时角膜基质中的颗粒摄取约为22%，而在有上皮存在时约为0.5%，这表明角膜上皮具有屏障性质。地洛瑞林和转铁蛋白偶联分别在5分钟时将纳米颗粒的角膜上皮摄取提高了3倍和4.5倍，在60分钟时提高了4.5倍和3.8倍。在5分钟时，普通、地洛瑞林功能化和转铁蛋白功能化纳米颗粒的角膜总摄取量分别约为2.4%、9%和16%。在所有组中，每单位组织重量的纳米颗粒摄取顺序为：角膜上皮>基质>内皮细胞。房水中的水平无法检测到。在切除的角膜和结膜研究中，地洛瑞林和转铁蛋白偶联的纳米颗粒的纳米颗粒转运和摄取均升高。在角膜上皮以及结膜中均观察到LHRH和转铁蛋白受体的表达。
离体牛眼模型是了解局部给药后纳米颗粒处置情况的有用工具。角膜上皮是纳米颗粒局部递送至眼前节的重要屏障。通过偶联LHRH激动剂或转铁蛋白对纳米颗粒进行表面修饰是一种将完整纳米颗粒快速、有效地递送至角膜和结膜内及/或穿过角膜和结膜的有用方法。
