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

Abstract

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.

Activatable NIR‐II Photothermal Lipid Nanoparticles for Improved Messenger RNA Delivery

“…In acidic environment, ionizable lipids (SM-102 and Cy-lipid in our system) are protonated to become positively charged, resulting in extra electrostatic repulsive force between these charged ionizable lipids and lead to the unstable structure, then the lipids may undergo rearrangement to fuse large sized assembly. [17] Most importantly, when the solution was irradiated by 1064 nm laser, spontaneous morphology change occurred and large sized aggregations were formed with particle size increased up to 4426.8 nm, which was consistent with TEM imaging findings (Figure 2h). These results clearly demonstrated that photothermally promoted NIR-II LNP morphology change is highly dependent on both acidic environment and NIR-II 1064 nm laser irradiation, which provided a prerequisite for remotely NIR-II light-triggered mRNA endosome escape.…”

Colloidal Quantum Dots as an Emerging Vast Platform and Versatile Sensitizer for Singlet Molecular Oxygen Generation

“…The porphyrin lipids enabled LNPs to generate 1 O 2 upon light irradiation which disrupted the endosomal membrane, therby triggering siRNA endosomal escape to cytosol. 83 1.1.3. Biomaterial Modifications.…”

“…In the system, the therapeutic agents are conjugated/chemically linked with a light-sensitive photosensitizer. Upon exposure to light, the photosensitizers produce 1 O 2 which triggers the release of therapeutic agents at a site of interest and a desired time. − For example, Mo et al developed singlet oxygen-responsive light-activated siRNA endosomal release (LASER) using porphyrin lipid nanoparticle (LNP) encapsulated siRNA to achieve an enhanced siRNA endosomal escape and significantly improved knockdown efficacy. The porphyrin lipids enabled LNPs to generate 1 O 2 upon light irradiation which disrupted the endosomal membrane, therby triggering siRNA endosomal escape to cytosol …”

Nanoparticles Internalization through HIP-55-Dependent Clathrin Endocytosis Pathway

“…There are two main patterns for extracellular particles to enter cells: membrane fusion and endocytosis. Most nanoparticles enter cells through endocytic pathways, including lipid nanoparticles, 17 solid lipid nanoparticles, 18 polymeric nanoparticles, 19 and most of inorganic nanoparticles. 12,20,21 Many nanoparticles, such as quantum dots (QDs), 22−24 gold nanoparticles, 25 and ultrastable organic dye nanoparticles, 26 are internalized into cells through clathrin-mediated endocytosis (CME).…”

“…There are two main patterns for extracellular particles to enter cells: membrane fusion and endocytosis. Most nanoparticles enter cells through endocytic pathways, including lipid nanoparticles, solid lipid nanoparticles, polymeric nanoparticles, and most of inorganic nanoparticles. ,, Many nanoparticles, such as quantum dots (QDs), − gold nanoparticles, and ultrastable organic dye nanoparticles, are internalized into cells through clathrin-mediated endocytosis (CME). For example, polystyrene nanoparticles can enter cervical epithelial (HeLa), lung epithelial (A549), brain astrocytoma, and macrophage (J774A.1) through the CME pathway. , Structurally, the size range of clathrin-coated vesicles is 60–120 nm, which is exactly suitable for most nanoparticles to be endocytosed.…”