Laboratory of Bioengineering and Physical Sciences, National Institute of Biomedical Imaging and Bioengineering, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-5766, USA.
J Ocul Pharmacol Ther. 2010 Feb;26(1):55-64. doi: 10.1089/jop.2009.0059.
An effective treatment modality for posterior eye diseases would provide prolonged delivery of therapeutic agents, including macromolecules, to eye tissues using a safe and minimally invasive method. The goal of this study was to assess the ability of a thermosetting gel to deliver a fluorescently labeled protein, Alexa 647 ovalbumin, to the choroid and retina of rats following a single subconjunctival injection of the gel. Additional experiments were performed to compare in vitro to in vivo ovalbumin release rates from the gel.
The ovalbumin content of the eye tissues was monitored by spectrophotometric assays of tissue extracts of Alexa 647 ovalbumin from dissected sclera, choroid, and retina at time points ranging from 2 h to 14 days. At the same time points, fluorescence microscopy images of tissue samples were also obtained. Measurement of intact ovalbumin was verified by LDS-PAGE analysis of the tissue extract solutions. In vitro release of Alexa 488 ovalbumin into 37 degrees C PBS solutions from ovalbumin-loaded gel pellets was also monitored over time by spectrophotometric assay. In vivo ovalbumin release rates were determined by measurement of residual ovalbumin extracted from gel pellets removed from rat eyes at various time intervals.
Our results indicate that ovalbumin concentrations can be maintained at measurable levels in the sclera, choroid, and retina of rats for up to 14 days using the thermosetting gel delivery system. The concentration of ovalbumin exhibited a gradient that decreased from sclera to choroid and to retina. The in vitro release rate profiles were similar to the in vivo release profiles.
Our findings suggest that the thermosetting gel system may be a feasible method for safe and convenient sustained delivery of proteins to choroidal and retinal tissue in the posterior segments of the eye.
对于后眼部疾病，有效的治疗方式是采用安全微创的方法，将治疗剂（包括大分子）递送至眼组织，实现长时间的药物输送。本研究的目的是评估热固性凝胶在单次结膜下注射凝胶后，将荧光标记的蛋白质（Alexa 647 卵清蛋白）递送至大鼠脉络膜和视网膜的能力。此外，还进行了比较凝胶体外和体内卵清蛋白释放率的实验。
通过分光光度法对从分离的巩膜、脉络膜和视网膜组织提取物中 Alexa 647 卵清蛋白的含量进行监测，以监测眼组织中的卵清蛋白含量。时间点范围从 2 小时到 14 天不等。同时，还获得了组织样本的荧光显微镜图像。通过组织提取物溶液的 LDS-PAGE 分析，验证了完整卵清蛋白的测量。通过分光光度法监测卵清蛋白负载的凝胶小球在 37°C PBS 溶液中的体外释放情况，还监测了不同时间点的卵清蛋白释放情况。通过测量从大鼠眼内取出的凝胶小球中提取的残留卵清蛋白，确定了体内卵清蛋白的释放率。
我们的结果表明，使用热固性凝胶递药系统，卵清蛋白浓度可在大鼠的巩膜、脉络膜和视网膜中维持长达 14 天的可测量水平。卵清蛋白浓度呈现从巩膜到脉络膜再到视网膜逐渐降低的梯度。体外释放率曲线与体内释放率曲线相似。
我们的研究结果表明，热固性凝胶系统可能是一种安全方便的后眼部脉络膜和视网膜组织中蛋白质持续输送的可行方法。
