Caveolae-Mediated Endocytosis Drives Robust siRNA Delivery of Polymeric Nanoparticles to Macrophages

Abstract

Cytosolic delivery of small interfering RNA (siRNA) remains challenging, and a profound understanding of the cellular uptake and intracellular processing of siRNA delivery systems could greatly improve the development of siRNA-based therapeutics. Here, we show that caveolaemediated endocytosis (CvME) accounts for the robust siRNA delivery of mannose-modified trimethyl chitosan-cysteine/ tripolyphosphate nanoparticles (MTC/TPP NPs) to macrophages by circumventing lysosomes. We show that the Golgi complex and ER are key organelles required for the efficient delivery of siRNA to macrophages in which the siRNA accumulation positively correlates with its silencing efficiency (r = 0.94). We also identify syntaxin6 and Niemann−Pick type C1 (NPC1) as indispensable regulators for MTC/TPP NPs-delivered siRNA into macrophages both in vitro and in vivo. Syntaxin6 and NPC1 knockout substantially decrease the cellular uptake and gene silencing of the siRNA delivered in MTC/ TPP NPs in macrophages, which result in poor therapeutic outcomes for mice bearing acute hepatic injury. Our results suggest that highly efficient siRNA delivery can be achieved via CvME, which would give ideas for designing optimal delivery vectors to facilitate the clinical translation of siRNA drugs.

In Cellulo and In Vivo Comparison of Cholesterol, Beta-Sitosterol and Dioleylphosphatidylethanolamine for Lipid Nanoparticle Formulation of mRNA

“…We further investigated the intracellular trafficking of Cy3-labeled mRNA using confocal microscopy and cells expressing chimera fluorescent proteins to label critical organelles: EGFP-Rab5, EGFP-Rab7, and EGFP-Rab11 to label early endosomes, late endosomes, and recycling endosomes, respectively ( Figure 3 A–C and Figure S2 ) [ 16 , 55 , 56 , 57 ]. Endosomal trafficking was studied 4 h after transfection as in the original report comparing cholesterol and β-sitosterol LNPs [ 21 ].…”

Microfluidic-Based Cationic Cholesterol Lipid siRNA Delivery Nanosystem: Highly Efficient In Vitro Gene Silencing and the Intracellular Behavior

“…Since the complexity of physical–chemical nanocomplexes and diversity of transfected cell types, the “preferred endocytosis pathway” remains controversial. Recent research disclosed that the caveolae-mediated endocytosis route could allow gene/drug-loaded nanoparticles to bypass the lysosome and smoothly reach the cytoplasm, e.g., Song et al found that caveolae-mediated endocytosis was conductive to the robust siRNA delivery of mannose-modified trimethyl chitosan-cysteine/tripolyphosphate nanoparticles through the subcellular organelle of the Golgi-complex and endoplasmic reticulum [ 42 ]. Qiu et al designed endoplasmic reticulum (ER) membrane-decorated siRNA nanoparticles to effectively transport siRNA through the endosome–Golgi–ER pathway to avoid lysosomal degradation [ 43 ].…”

A Unique Core–Shell Structured, Glycol Chitosan-Based Nanoparticle Achieves Cancer-Selective Gene Delivery with Reduced Off-Target Effects

“…The rapid clinical deployment of cationic lipids for delivering SAR-CoV-19 mRNA during the pandemic underscores the utility of such reagents. Recent studies have employed polymer-based NPs, pH-sensitive copolymers, or targeting ligand conjugates, such as ethylaminoconjugated GC (chitosan) [74], poly(amidoamine)-conjugated CS [75], poly(lactide-coglycolide)-graft-PEI [76], PEG-conjugated CS [77], and trimethyl CS-cysteine conjugates [78]. Despite the reasonable therapeutic effects, however, the efficiency of the cytosolic delivery of those agents needs further attention as accumulation in the cytosol is essential for theranostic applications.…”