Elsaid Naba, Jackson Timothy L, Elsaid Zeeneh, Alqathama Aljawharah, Somavarapu Satyanarayana
University of Hertfordshire , Hatfield, United Kingdom.
King's College London , London, United Kingdom.
Mol Pharm. 2016 Sep 6;13(9):2923-40. doi: 10.1021/acs.molpharmaceut.6b00335. Epub 2016 Jun 22.
Age-related macular degeneration (AMD) is the leading cause of certified vision loss worldwide. The standard treatment for neovascular AMD involves repeated intravitreal injections of therapeutic proteins directed against vascular endothelial growth factor, such as ranibizumab. Biodegradable polymers, such as poly(lactic-co-glycolic acid) (PLGA), form delivery vehicles which can be used to treat posterior segment eye diseases, but suffer from poor protein loading and release. This work describes a "system-within-system", PLGA microparticles incorporating chitosan-based nanoparticles, for improved loading and sustained intravitreal delivery of ranibizumab. Chitosan-N-acetyl-l-cysteine (CNAC) was synthesized and its synthesis confirmed using FT-IR and (1)H NMR. Chitosan-based nanoparticles composed of CNAC, CNAC/tripolyphosphate (CNAC/TPP), chitosan, chitosan/TPP (chit/TPP), or chit/TPP-hyaluronic acid (chit/TPP-HA) were incorporated in PLGA microparticles using a modified w/o/w double emulsion method. Nanoparticles and final nanoparticles-within-microparticles were characterized for their protein-nanoparticle interaction, size, zeta potential, morphology, protein loading, stability, in vitro release, in vivo antiangiogenic activity, and effects on cell viability. The prepared nanoparticles were 17-350 nm in size and had zeta potentials of -1.4 to +12 mV. Microscopic imaging revealed spherical nanoparticles on the surface of PLGA microparticles for preparations containing chit/TPP, CNAC, and CNAC/TPP. Ranibizumab entrapment efficiency in the preparations varied between 13 and 69% and was highest for the PLGA microparticles containing CNAC nanoparticles. This preparation also showed the slowest release with no initial burst release compared to all other preparations. Incorporation of TPP to this formulation increased the rate of protein release and reduced entrapment efficiency. PLGA microparticles containing chit/TPP-HA showed the fastest and near-complete release of ranibizumab. All of the prepared empty particles showed no effect on cell viability up to a concentration of 12.5 mg/mL. Ranibizumab released from all preparations maintained its structural integrity and in vitro activity. The chit/TPP-HA preparation enhanced antiangiogenic activity and may provide a potential biocompatible platform for enhanced antiangiogenic activity in combination with ranibizumab. In conclusion, the PLGA microparticles containing CNAC nanoparticles showed significantly improved ranibizumab loading and release profile. This novel drug delivery system may have potential for improved intravitreal delivery of therapeutic proteins, thereby reducing the frequency, risk, and cost of burdensome intravitreal injections.
年龄相关性黄斑变性（AMD）是全球经认证的视力丧失的主要原因。新生血管性AMD的标准治疗方法包括反复玻璃体内注射针对血管内皮生长因子的治疗性蛋白质，如雷珠单抗。可生物降解的聚合物，如聚乳酸-乙醇酸共聚物（PLGA），形成可用于治疗眼后段疾病的递送载体，但存在蛋白质负载和释放不佳的问题。这项工作描述了一种“系统内系统”，即包含基于壳聚糖的纳米颗粒的PLGA微粒，用于改善雷珠单抗的负载和持续玻璃体内递送。合成了壳聚糖-N-乙酰-L-半胱氨酸（CNAC），并使用傅里叶变换红外光谱（FT-IR）和核磁共振氢谱（¹H NMR）对其合成进行了确认。使用改良的水包油包水（w/o/w）双乳液法将由CNAC、CNAC/三聚磷酸钠（CNAC/TPP）、壳聚糖、壳聚糖/TPP（chit/TPP）或壳聚糖/TPP-透明质酸（chit/TPP-HA）组成的基于壳聚糖的纳米颗粒掺入PLGA微粒中。对纳米颗粒和最终的微粒内纳米颗粒进行了蛋白质-纳米颗粒相互作用、尺寸、zeta电位、形态、蛋白质负载、稳定性、体外释放、体内抗血管生成活性以及对细胞活力影响的表征。所制备的纳米颗粒尺寸为17 - 350 nm，zeta电位为-1.4至+12 mV。显微镜成像显示，对于含有chit/TPP、CNAC和CNAC/TPP的制剂，PLGA微粒表面有球形纳米颗粒。制剂中雷珠单抗的包封率在13%至69%之间变化，对于含有CNAC纳米颗粒的PLGA微粒包封率最高。与所有其他制剂相比，该制剂还显示出最慢的释放速度且无初始突释。向该制剂中加入TPP会增加蛋白质释放速率并降低包封率。含有壳聚糖/TPP-透明质酸的PLGA微粒显示出雷珠单抗最快且近乎完全的释放。所有制备的空白颗粒在浓度高达12.5 mg/mL时对细胞活力均无影响。从所有制剂中释放的雷珠单抗保持其结构完整性和体外活性。壳聚糖/TPP-透明质酸制剂增强了抗血管生成活性，并可能为与雷珠单抗联合增强抗血管生成活性提供一个潜在的生物相容性平台。总之，含有CNAC纳米颗粒的PLGA微粒显示出显著改善的雷珠单抗负载和释放特性。这种新型药物递送系统可能具有改善治疗性蛋白质玻璃体内递送的潜力，从而降低繁重的玻璃体内注射的频率、风险和成本。
