Nanomedicine and Drug Delivery Laboratory, Department of Pharmaceutical Sciences, University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045, United States.
Mol Pharm. 2013 Aug 5;10(8):2858-67. doi: 10.1021/mp300716t. Epub 2013 Jul 8.
A light-activated polycaprolactone dimethacrylate (PCM) and hydroxyethyl methacrylate (HEMA) based gel network was developed to sustain the release of stable, active bevacizumab (an anti-VEGF antibody used to treat choroidal neovascularization) and used to assess sustained ex vivo delivery in rabbit eyes and in vivo delivery in rat eyes following in situ gel formation in the suprachoroidal space. PCM was synthesized from polycaprolactone diol (PCD) and evaluated using NMR spectroscopy. PCM was used to cross-link HEMA in the presence of 365 nm UV light and 2,2-dimethoxy-2-phenylacetophenone (DMPA) as a photoinitiator. Bevacizumab was entrapped in the gel using three different cross-linking durations of 3, 7, and 10 min. In vitro release of bevacizumab in PBS pH 7.4 at 37 °C during a 4 month study was quantified using a VEGF-binding based ELISA. The stability of released bevacizumab was monitored by size exclusion chromatography (SEC) and circular dichroism. Alexa Fluor 488 dye conjugated bevacizumab mixed with polymers was injected suprachoroidally in rabbit eyes to study the effect of different cross-linking durations on the spread of the dye conjugated bevacizumab. In vivo delivery was assessed in Sprague-Dawley (SD) rats by injecting Alexa Fluor 488 dye conjugated bevacizumab mixed with polymers followed by cross-linking for 10 min. Spread in the rabbit eyes and in vivo delivery in rat eyes was monitored noninvasively using a fundus camera and Fluorotron Master. The formation of PCM was confirmed by the disappearance of hydroxyl peak in NMR spectra. A cross-linking duration of 10 min resulted in a burst release of 21% of bevacizumab. Other cross-linking durations had ≥62% burst release. Bevacizumab release from 10 min cross-linked gel was sustained for ∼4 months. Release samples contained ≥96.1% of bevacizumab in the monomeric form as observed in SEC chromatograms. Circular dichroism confirmed that secondary β-sheet structure of bevacizumab was maintained after release from the gel. As the cross-linking duration was increased to 10 min, the gel/antibody was better confined at the injection site in excised rabbit eye suprachoroidal space. Delivery of Alexa Fluor 488 dye conjugated bevacizumab was sustained for at least 60 days in the suprachoroidal space of SD rats. PCM and HEMA gel sustained bevacizumab release for 4 months and maintained the stability and VEGF-binding activity of bevacizumab. Therefore, light-activated PCM and HEMA gel is suitable for in situ gel formation and sustained protein delivery in the suprachoroidal space.
一种光激活的聚己内酯二甲基丙烯酸酯（PCM）和羟乙基甲基丙烯酸酯（HEMA）基凝胶网络被开发出来，以维持稳定、活性的贝伐单抗（一种用于治疗脉络膜新生血管的抗 VEGF 抗体）的释放，并用于评估在兔眼的体外持续释放以及在原位凝胶形成后在大鼠眼的体内释放。PCM 是由聚己内酯二醇（PCD）合成的，并通过 NMR 光谱进行评估。PCM 用于在 365nm UV 光和 2,2-二甲氧基-2-苯乙酮（DMPA）存在下交联 HEMA，作为光引发剂。贝伐单抗在 3、7 和 10 分钟的三种不同交联时间内被包埋在凝胶中。在 4 个月的研究中，在 37°C 的 PBS pH 7.4 中使用基于 VEGF 结合的 ELISA 定量测定贝伐单抗的体外释放。通过尺寸排阻色谱（SEC）和圆二色性监测释放的贝伐单抗的稳定性。将与聚合物混合的 Alexa Fluor 488 染料缀合的贝伐单抗注射到兔眼的脉络膜上，以研究不同交联时间对缀合的贝伐单抗染料扩散的影响。通过注射与聚合物混合的 Alexa Fluor 488 染料缀合的贝伐单抗，然后交联 10 分钟，在 Sprague-Dawley（SD）大鼠中评估体内递送。通过使用眼底相机和 Fluorotron Master 非侵入性地监测兔眼的扩散和大鼠眼的体内递送。NMR 光谱中羟基峰的消失证实了 PCM 的形成。交联时间为 10 分钟导致 21%的贝伐单抗突释。其他交联时间的突释率≥62%。10 分钟交联凝胶的贝伐单抗释放持续了大约 4 个月。释放样品中 SEC 色谱图观察到至少 96.1%的贝伐单抗以单体形式存在。圆二色性证实，贝伐单抗从凝胶释放后，其二级β-折叠结构得以保持。随着交联时间增加到 10 分钟，在切除的兔眼脉络膜上的眼外空间中，凝胶/抗体更好地局限在注射部位。SD 大鼠脉络膜上的 Alexa Fluor 488 染料缀合的贝伐单抗的递送至少持续 60 天。PCM 和 HEMA 凝胶持续释放贝伐单抗 4 个月，并保持贝伐单抗的稳定性和 VEGF 结合活性。因此，光激活的 PCM 和 HEMA 凝胶适合于在脉络膜上形成原位凝胶和持续的蛋白质递送。
