Institute of Medical Science, University of Toronto, Toronto, Ontario M5G 1L7, Canada.
Princess Margaret Cancer Center, University Health Network, Toronto, Ontario M5G 1L7, Canada.
ACS Nano. 2023 Mar 14;17(5):4688-4703. doi: 10.1021/acsnano.2c10936. Epub 2023 Feb 28.
Lipid nanoparticles (LNPs) have achieved clinical success in delivering small interfering RNAs (siRNAs) for targeted gene therapy. However, endosomal escape of siRNA into the cytosol remains a fundamental challenge for LNPs. Herein, we report a strategy termed light-activated siRNA endosomal release (LASER) to address this challenge. We established a porphyrin-LNP by incorporating porphyrin-lipids into the clinically approved Onpattro formulation. The porphyrin-LNP maintained the physical properties of an LNP and generated reactive oxygen species (ROS) when irradiated with near-infrared (NIR) light. Using confocal microscopy, we revealed that porphyrin-lipids within the LNP translocate to endosomal membranes during endocytosis. The translocated porphyrin-lipids generated ROS under light irradiation and enabled LASER through endosomal membranes disruption as observed through GAL-9 recruitment and transmission electron microscopy (TEM). By establishing a quantitative confocal imaging method, we confirmed that porphyrin-LNPs can increase siRNA endosomal escape efficiency by up to 2-fold via LASER and further enhance luciferase target knockdown by 4-fold more in luciferase-transfected prostate cancer cells. Finally, we formulated porphyrin-LNPs encapsulated with gold nanoparticles (GNP) and visualized the LASER effect within prostate tumors via TEM, confirming the light-activated endosomal membrane disruption and subsequent GNP release into cytosols . Overall, porphyrin-LNPs and the LASER approach enhanced siRNA endosomal escape and significantly improved knockdown efficacy. We believe the versatility of this technology could be applied to various LNP-based RNA therapeutics.
脂质纳米颗粒（LNPs）在递送小干扰 RNA（siRNA）用于靶向基因治疗方面已取得临床成功。然而，siRNA 从内涵体逃逸到细胞质仍然是 LNPs 的一个基本挑战。在此，我们报告了一种称为光激活 siRNA 内涵体释放（LASER）的策略来解决这一挑战。我们通过将卟啉脂质纳入临床批准的 Onpattro 制剂中，建立了一种卟啉-LNP。该卟啉-LNP 保持了 LNP 的物理性质，并且在近红外（NIR）光照射下产生活性氧（ROS）。通过共聚焦显微镜，我们揭示了在内涵体摄取过程中，LNP 内的卟啉脂质向内涵体膜转移。在光照下，转运的卟啉脂质产生 ROS，并通过内涵体膜破裂实现 LASER，这可以通过 GAL-9 募集和透射电子显微镜（TEM）观察到。通过建立定量共聚焦成像方法，我们证实卟啉-LNP 可以通过 LASER 将 siRNA 内涵体逃逸效率提高多达 2 倍，并在转染荧光素酶的前列腺癌细胞中进一步将荧光素酶靶标敲低提高 4 倍。最后，我们将卟啉-LNP 与金纳米颗粒（GNP）封装，并通过 TEM 可视化前列腺肿瘤内的 LASER 效应，证实了光激活内涵体膜破裂以及随后 GNP 释放到细胞质中。总体而言，卟啉-LNP 和 LASER 方法增强了 siRNA 内涵体逃逸，并显著提高了敲低效果。我们相信这项技术的多功能性可以应用于各种基于 LNP 的 RNA 治疗。
