卟啉脂质纳米颗粒的光激活 siRNA 内体释放 (LASER),ACS Nano

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卟啉脂质纳米颗粒的光激活 siRNA 内体释放 (LASER)
ACS Nano ( IF 15.8 ) Pub Date : 2023-02-28 , DOI: 10.1021/acsnano.2c10936
Yulin Mo _{1,

2} , Miffy H Y Cheng ₃ , Andrew D'Elia ₂ , Katie Doran ₂ , Lili Ding ₂ , Juan Chen ₂ , Pieter R Cullis ₃ , Gang Zheng _{1,

2,

4}

Affiliation

脂质纳米颗粒 (LNP) 在递送用于靶向基因治疗的小干扰 RNA (siRNA) 方面取得了临床成功。然而，siRNA 的内体逃逸到胞质溶胶中仍然是 LNP 的一个基本挑战。在此，我们报告了一种称为光激活 siRNA 内体释放 (LASER) 的策略来应对这一挑战。我们通过将卟啉脂质掺入临床批准的 Onpattro 配方中，建立了卟啉-LNP。卟啉-LNP 保持了 LNP 的物理特性，并在用近红外 (NIR) 光照射时产生活性氧 (ROS)。使用共聚焦显微镜，我们发现 LNP 内的卟啉脂质在内吞作用期间易位至内体膜。如通过 GAL-9 募集和透射电子显微镜 (TEM) 观察到的，易位的卟啉脂质在光照射下产生 ROS，并通过内体膜破坏启用激光。通过建立定量共聚焦成像方法，我们证实卟啉-LNPs 可以通过激光将 siRNA 内体逃逸效率提高多达 2 倍，并在荧光素酶转染的前列腺癌细胞中进一步增强荧光素酶靶标敲低 4 倍以上。最后，我们配制了用金纳米粒子 (GNP) 封装的卟啉-LNP，并通过 TEM 观察了前列腺肿瘤内的激光效应，证实了光激活的内体膜破裂和随后的 GNP 释放到胞质溶胶中通过建立定量共聚焦成像方法，我们证实卟啉-LNPs 可以通过激光将 siRNA 内体逃逸效率提高多达 2 倍，并在荧光素酶转染的前列腺癌细胞中进一步增强荧光素酶靶标敲低 4 倍以上。最后，我们配制了用金纳米粒子 (GNP) 封装的卟啉-LNP，并通过 TEM 观察了前列腺肿瘤内的激光效应，证实了光激活的内体膜破裂和随后的 GNP 释放到胞质溶胶中通过建立定量共聚焦成像方法，我们证实卟啉-LNPs 可以通过激光将 siRNA 内体逃逸效率提高多达 2 倍，并在荧光素酶转染的前列腺癌细胞中进一步增强荧光素酶靶标敲低 4 倍以上。最后，我们配制了用金纳米粒子 (GNP) 封装的卟啉-LNP，并通过 TEM 观察了前列腺肿瘤内的激光效应，证实了光激活的内体膜破裂和随后的 GNP 释放到胞质溶胶中体内。总体而言，卟啉-LNP 和激光方法增强了 siRNA 内体逃逸并显着提高了敲低效果。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 疗法。

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Light-Activated siRNA Endosomal Release (LASER) by Porphyrin Lipid Nanoparticles

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 in vivo. 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.

更新日期：2023-02-28

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