Super-resolution Imaging of Proton Sponge-Triggered Rupture of Endosomes and Cytosolic Release of Small Interfering RNA

Abstract

The intracellular delivery of nucleic acids and proteins remains a key challenge in the development of biologic therapeutics. In gene therapy, the inefficient delivery of small interfering RNA (siRNA) to the cytosol by lipoplexes or polyplexes is often ascribed to the entrapment and degradation of siRNA payload in the endosomal compartments. A possible mechanism by which polyplexes rupture the endosomal membrane and release their nucleic acid cargo is commonly defined as the "proton sponge effect". This is an osmosis-driven process triggered by the proton buffering capacities of polyplexes. Herein, we investigate the molecular basis of the "proton sponge effect" through direct visualization of the siRNA trafficking process, including analysis of individual polyplexes and endosomes, using stochastic optical reconstruction microscopy. We probed the sequential siRNA trafficking steps through single molecule superresolution analysis of subcellular structures, polyplexes, and silencing RNA molecules.Specifically, individual intact polyplexes released in the cytosol upon rupture of the endosomes, the damaged endosomal vesicles, and the disassembly of the polyplexes in the cytosol were examined. We found that the architecture of the polyplex and the rigidity of the cationic polymer chains are crucial parameters that control the mechanism of endosomal escape driven by the proton sponge effect. We provide evidence that in highly branched and rigid cationic polymers, such as glycogen or polyethylenimine, immobilized on silica nanoparticles, the proton sponge effect is effective in inducing osmotic swelling and rupture of endosomes.KEYWORDS: endosomal escape, polyplexes, proton sponge effect, STORM, glycogenThe complexation of nucleic acids with cationic lipid and polymers to form lipoplexes and polyplexes, respectively, is widely used for intracellular transfection of DNA, siRNA, microRNAs, and lately CRISPR-Cas9. 1,2 To efficiently deliver their cargo and promote alteration 3 of gene expression and transcription, these vectors must overcome multiple biological barriers, including substantial cellular uptake, endosomal escape, and intracellular targeting devoid of offtarget effects. The endolysosomal escape 3-7 and endocytic recycling 8 processes are reported to be the major bottlenecks encountered in the intracellular release of nucleic acid payloads. In particular, the translocation of intact and functional RNA double strands from late endosomes to the cytosol prior to their degradation in the lysosome compartments is usually considered the ratelimiting step in siRNA-mediated knockdown of protein expression (RNAi). 3,6 It has been estimated that the escape of siRNAs from endosomes to the cytosol occurs at a very low efficiency (0.01-2%). 5,7 The delivery of siRNAs, mediated by lipoplexes and polyplexes, has been investigated by time-lapse confocal and electron microscopy. [3][4][5][6] This qualitative and quantitative image-based analysis of lipid and polymer-based nanocarriers, in fixed and live cells, have revealed dif...

Tracking the DNA complexation state of pBAE polyplexes in cells with super resolution microscopy

“…After internalization, polyplexes are known to be trafficked through the endolysosomal pathway with different possible fates, such as lysosomes, Golgi or recycled vesicles. 41,42 Lastly, we observe polyplexes mostly in the perinuclear region (t [12][13][14][15][16][17][18][19][20][21][22][23][24] ), the fate of the non-recycled polyplexes. This localization has been proposed to be the previous step to nuclear entry.…”

“…2c). However, at later time points, we could only observe discrete pDNA not covered by the polymer (t [12][13][14][15][16][17][18][19][20][21][22][23][24] ), making it evident that the pDNA release has occurred after a few hours of internalization. Moreover, we observed a reduction in the amount of the free polymer that, as mentioned previously, enters rapidly in the cell.…”

“…Super-resolution microscopy, initially developed to study biological structures, has recently proved to be a powerful tool to study nanoparticles in vitro and in cells. [17][18][19] Recently, we have proven the applicability of 2-colour super-resolution microscopy for polyplex stoichiometry quantification. 20 In our approach, direct stochastic optical reconstruction microscopy (dSTORM) 21,22 is used to obtain high-resolution images of polyplexes where their structure is clearly resolved.…”

Glycogen as a Building Block for Advanced Biological Materials

“…Mitani et al have shown that oral administration of ESG suppresses dextran sulfate sodium‐ and 2,4,6‐trinitrobenzenesulfonic acid‐induced colitis and oxidative stress in mice, suggesting that ESG could prevent inflammatory bowel disease. Interestingly, as natural glycogen is typically only present in the cytoplasm, there is limited data on the uptake pathway of exogenous glycogen nanoparticles into cells . A recent study by Ida‐Yonemochi et al showed that uptake of ESG into MC3T3‐E1 cells was accompanied with an increase in expression of the glucose transporter 1 (GLUT‐1) protein, and when this protein was inhibited, the ESG particles mainly aggregated on the cell surface, with some particles internalized by endocytosis (Figure G–I).…”

“…The structures of polysaccharides vary considerably but span from linear to highly branched polymers with molecular weights ranging from thousands to millions, composed of mono‐ or disaccharides, or short‐chain oligomers bound together by glycosidic linkages . There is a rich history of research on the use of polysaccharides in a range of therapeutic applications including: i) soft tissue engineering, where the materials can be designed to mimic the mechanical properties of the extracellular matrix in the tissue; ii) as drug, protein, or gene delivery systems, where polysaccharides have a large number of reactive groups that allow for tunable properties such as pH‐responsiveness; and iii) as nanoprobes for cellular and tissue imaging . Of the available polysaccharides, the most studied biopolymers for use as therapeutic materials are arguably derivatives or composites of cellulose, chitosan, hyaluronic acid, and/or alginate .…”