Image-based analysis of lipid nanoparticle–mediated siRNA delivery, intracellular trafficking and endosomal escape

Abstract

Delivery of short interfering RNAs (siRNAs) remains a key challenge in the development of RNA interference (RNAi) therapeutics. A better understanding of the mechanisms of siRNA cellular uptake, intracellular transport and endosomal release could critically contribute to the improvement of delivery methods. Here we monitored the uptake of lipid nanoparticles (LNPs) loaded with traceable siRNAs in different cell types in vitro and in mouse liver by quantitative fluorescence imaging and electron microscopy. We found that LNPs enter cells by both constitutive and inducible pathways in a cell type-specific manner using clathrin-mediated endocytosis as well as macropinocytosis. By directly detecting colloidal-gold particles conjugated to siRNAs, we estimated that escape of siRNAs from endosomes into the cytosol occurs at low efficiency (1-2%) and only during a limited window of time when the LNPs reside in a specific compartment sharing early and late endosomal characteristics. Our results provide insights into LNP-mediated siRNA delivery that can guide development of the next generation of delivery systems for RNAi therapeutics.

Rod‐Shaped Active Drug Particles Enable Efficient and Safe Gene Delivery

“…Unlike other cationic vectors, these vectors induced little toxicity to cells, as well as normal organs (heart, live, kidney, lung, and spleen), thereby suggesting their better biocompatibility. Conventional vectors render gene escape from lysosomes to cytoplasm of target cells via a mechanism of “proton sponge effect,”26 yet this escape efficiency is extremely low at only 1–2% 12. Moreover, this process results in rupture of the lysosome membrane and, in turn, induces cytotoxicity 27.…”

“…Additionally, these vector/gene complexes tend to accumulate in lysosomes followed by internalization, and subsequently the obtained gene escapes from lysosomes by taking advantage of the “proton sponge effect” into the cytosol of target cells, where they associate with the target gene to exert their silencing function 11. Unfortunately, the escape efficiency of genes from lysosomes into the cytosol is extremely low at only 1–2%, and it only occurs in a short limited time‐period when the vectors are located in a specific compartment that shares both early and late endosomal characteristics 12. Critically, during this trafficking process, significant lysosomal degradation of the gene by digestive enzymes and acidic conditions with a pH less than 4.5 occurs in lysosomes,13 thereby greatly diminishing the therapeutic efficacy.…”

Light‐Patterned RNA Interference of 3D‐Cultured Human Embryonic Stem Cells

“…We found that the siRNA protected by HGNs remain intact at neutral pH for at least a month, whereas overnight exposure to GSH at 0.01 × 10 −3 m releases half of the loading ( Figure S8, Supporting Information). However, the siRNA-HGN constructs accumulate in the endosome [38] and endosomal GSH levels are quite low. [39] Furthermore, release of siRNA from the endosome requires laser exposure, as shown by the unchanged GFP expression in both hESC and HeLa cells 6 d post siRNA coated HGN internalization ( Figure 4 and Figure S7 (Supporting Information)).…”

High-content analysis for drug delivery and nanoparticle applications

“…HCA was used to monitor lipid-based NPs loaded with traceable siRNAs in cell lines and in mouse liver cells i to quantify siRNA uptake and intracellular trafficking [81]. The lipid NPs comprised ionizable lipid/ distearoyl phosphatidyl choline/cholesterol/,PEG-DMG in ratios of 50:10:38.5:1.5 and entrapped antiGFP siRNA labeled with Alexa Fluor 647.…”

“…To date, no such genome-wide characterization of NP uptake has been reported, but small-scale studies are now adding further depth to the pharmacological studies. For example, in the HCA study of the uptake of lipid NPs outlined above [81], siRNAs were used to deplete cells of fundamental endocytic machinery, and showed that lipid NPs primarily endocytose by either clathrin-mediation or macropinocytosis. The parallel study using a limited number of siRNAs also found that macropinocytosis (as judged by depletion of Cdc42 and Rac1) was important for the internalization of this particle type [82].…”