USC Roski Eye Institute, USC Ginsburg Institute for Biomedical Therapeutics, Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033, USA.
Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Science Center, Oklahoma City, OK 73104, USA.
Cells. 2022 Aug 18;11(16):2573. doi: 10.3390/cells11162573.
To assess the transretinal penetration of intravitreally injected retinal multicell-derived exosomes and to develop exosome-based active targeting of choroidal neovascularization (CNV) by bioengineering with ASL, which is composed of a membrane Anchor (BODIPY), Spacer (PEG), and targeting Ligands (cyclic RGD peptide).
Retinal multicell-derived exosomes were recovered from a whole mouse retina using differential ultracentrifugation. Their size, number, and morphology were characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Exosome markers were confirmed using an exosome detection antibody array. Intravitreal injection of fluorescent (PKH-26)-labeled or engineered ASL exosomes (1 × 10 exosomes/μL) were given to the wild-type mouse or laser-induced CNV mouse model. Retinal uptake of exosomes was assessed by in vivo retinal imaging microscopy and histological staining with DAPI, GSA, and anti-integrin for retinal sections or choroid/RPE flat mounts. Active targeting of CNV was assessed by comparing retinal uptake between areas with and without CNV and by colocalization analysis of ASL exosomes with integrin within CNV. Staining with anti-F4/80, anti-ICAM-1, and anti-GFAP antibodies on retinal sections were performed to identify intracellular uptake of exosomes and immediate reactive retinal gliosis after exosome treatment.
An average of 2.1 × 10 particles/mL with a peak size of 140 nm exosomes were recovered. Rapid retinal penetration of intravitreally injected exosomes was confirmed by retinal imaging microscopy at 3 and 24 h post-injection. Intravitreally delivered PKH-26-labeled exosomes reached inner and outer retinal layers including IPL, INL, OPL, and ONL at 1 and 7 days post-injection. Intravitreally injected ASL exosomes were predominantly delivered to the area of CNV including ONL, RPE, and choroid in laser-induced CNV mouse models with 89.5% of colocalization with integrin . Part of exosomes was also taken intracellularly to vascular endothelial cells and macrophages. After intravitreal injection, neither naive exosomes nor ASL exosomes induced immediate reactive gliosis.
Intravitreally delivered retinal multicell-derived exosomes have good retinal penetration, and ASL modification of exosomes actively targets CNV with no immediate reactive gliosis. ASL exosomes have a great potential to serve as an intraocular drug delivery vehicle, allowing an active targeting strategy.
通过生物工程用 ASL 评估玻璃体内注射的视网膜多细胞衍生的外体的视网膜穿透，并通过 ASL 开发针对脉络膜新生血管（CNV）的外体主动靶向，ASL 由膜锚（BODIPY）、间隔物（PEG）和靶向配体（环状 RGD 肽）组成。
使用差速超速离心从全鼠视网膜中回收视网膜多细胞衍生的外体。使用纳米颗粒跟踪分析（NTA）和透射电子显微镜（TEM）对其大小、数量和形态进行表征。使用外体检测抗体阵列确认外体标记物。将荧光（PKH-26）标记或工程化 ASL 外体（1×10 个外体/μL）玻璃体注射到野生型小鼠或激光诱导的 CNV 小鼠模型中。通过体内视网膜成像显微镜和 DAPI、GSA 和抗整合素的视网膜切片或脉络膜/RPE 平片组织染色评估外体的视网膜摄取。通过比较有 CNV 和无 CNV 区域之间的视网膜摄取以及 ASL 外体与 CNV 内整合素的共定位分析来评估 CNV 的主动靶向。对视网膜切片上的抗 F4/80、抗 ICAM-1 和抗 GFAP 抗体进行染色，以鉴定外体处理后外体的细胞内摄取和即刻反应性视网膜神经胶质增生。
平均回收 2.1×10 个/mL 粒径峰值为 140nm 的外体。视网膜成像显微镜证实，玻璃体注射后 3 和 24 小时内，外体迅速穿透视网膜。玻璃体注射的 PKH-26 标记的外体在 1 和 7 天注射后到达内、外视网膜层，包括 IPL、INL、OPL 和 ONL。在激光诱导的 CNV 小鼠模型中，玻璃体注射的 ASL 外体主要递送至 CNV 区域，包括 ONL、RPE 和脉络膜，与整合素的共定位率为 89.5%。部分外体也被细胞内摄取到血管内皮细胞和巨噬细胞中。玻璃体注射后，未修饰的外体和 ASL 外体均未引起即刻反应性神经胶质增生。
玻璃体内递送至视网膜的多细胞衍生外体具有良好的视网膜穿透性，外体的 ASL 修饰可主动靶向 CNV，且无即刻反应性神经胶质增生。ASL 外体很有潜力作为眼内药物递送载体，允许主动靶向策略。
