A Novel Mechanism Is Involved in Cationic Lipid-Mediated Functional siRNA Delivery

Lu, James; Langer, Róbert; Chen, Jianzhu

doi:10.1021/mp900023v

Cited by 201 publications

(215 citation statements)

References 47 publications

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“…We hypothesize that following cellular uptake only a small fraction of the siRNA molecules complexed to the nanogels reaches the cell cytoplasm and becomes available for incorporation into the RNAi pathway [22]. Indeed, also other reports in literature suggest that only a minor fraction of the siRNA transported across the cellular membrane by nonviral carriers is eventually responsible for the RNAi effect [29][30][31][32].…”

Section: Resultsmentioning

confidence: 70%

“…nanogels) and a lipid vesicle type carrier. Liposomal carriers most likely give rise to an immediate release of the loaded siRNA in the cytosol due to fusion with the endosomal membrane and even with the plasmamembrane [30]. Opposite to our nanogels, which remain trapped inside intracellular vesicles, liposomes do not create an intracellular depot of siRNA, thus making these transfection agents refractive towards a PCI controlled siRNA release.…”

Section: Resultsmentioning

confidence: 89%

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Prolonged gene silencing by combining siRNA nanogels and photochemical internalization

Raemdonck

Naeye

Høgset

et al. 2010

Journal of Controlled Release

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Small interfering RNAs (siRNAs) show potential for the treatment of a wide variety of pathologies with a known genetic origin through sequence-specific gene silencing. However, siRNAs do not have favorable drug-like properties and need to be packaged into nanoscopic carriers that are designed to guide the siRNA to the cytoplasm of the target cell. In this report biodegradable cationic dextran nanogels are used to deliver siRNA across the intracellular barriers. For the majority of non-viral siRNA carriers studied so far, endosomal confinement is identified as the most prominent hurdle, limiting the full gene silencing potential. Thus, there is a major interest in methods that are able to enhance endosomal escape of siRNA to improve its intracellular bioavailability. Photochemical internalization (PCI) is a method that employs amphiphilic photosensitizers to destabilize endosomal vesicles. We show that applying PCI at a later time-point post-transfection significantly prolonged the knockdown of the target protein only in case the siRNA was carried by nanogels and not when a liposomal carrier was used. Combining siRNA nanogels and PCI creates new possibilities to prolong gene silencing by using intracellular vesicles as depots for siRNA and applying PCI at the time when maintaining the RNAi effect becomes critical. Graphical abstractCombining siRNA nanogels and photochemical internalization (PCI) creates new possibilities to prolong gene silencing by using intracellular vesicles as depots and applying PCI at the time when maintaining the RNAi effect becomes critical.

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Section: Resultsmentioning

confidence: 70%

Section: Resultsmentioning

confidence: 89%

Prolonged gene silencing by combining siRNA nanogels and photochemical internalization

Raemdonck

Naeye

Høgset

et al. 2010

Journal of Controlled Release

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“…Cationic lipids represent one of the most well-studied classes of synthetic materials for siRNA delivery. To date, the most advanced examples of these materials demonstrate the ability to bind and condense siRNA into nanocomplexes through electrostatic interactions and to deliver the payload across the cellular membrane into the cytoplasm of target cells (16,17).…”

mentioning

confidence: 99%

Lipid-like materials for low-dose, in vivo gene silencing

Love

Mahon

Levins

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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817

803

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Significant effort has been applied to discover and develop vehicles which can guide small interfering RNAs (siRNA) through the many barriers guarding the interior of target cells. While studies have demonstrated the potential of gene silencing in vivo, improvements in delivery efficacy are required to fulfill the broadest potential of RNA interference therapeutics. Through the combinatorial synthesis and screening of a different class of materials, a formulation has been identified that enables siRNA-directed liver gene silencing in mice at doses below 0.01 mg/kg. This formulation was also shown to specifically inhibit expression of five hepatic genes simultaneously, after a single injection. The potential of this formulation was further validated in nonhuman primates, where high levels of knockdown of the clinically relevant gene transthyretin was observed at doses as low as 0.03 mg/kg. To our knowledge, this formulation facilitates gene silencing at orders-of-magnitude lower doses than required by any previously described siRNA liver delivery system.

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“…This shows that the free fraction exerts its influence separately from the siRNA-containing core polyplexes, and the higher uptake by N/P2 than N/P3 bPEI 25kDa polyplexes suggests that the free fraction plays a role unrelated to uptake. This could be by facilitating endosomal escape or by facilitating alternate routes of polyplex uptake such as the caveolar pathway as suggested by Pack et al [19] or direct membrane fusion as proposed for lipoplex uptake [47].…”

Section: Discussionmentioning

confidence: 99%

Elucidating the role of free polycations in gene knockdown by siRNA polyplexes

et al. 2016

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Future improvements of non-viral vectors for siRNA delivery require better understanding of intracellular processing and vector interactions with target cells. Here, we have compared the siRNA delivery properties of a lipid derivative of bPEI 1.8kDa (DOPE-PEI) with branched polyethyleneimine (bPEI) with average molecular weights of 1.8kDa (bPEI 1.8kDa) and 25 kDa (bPEI 25kDa). We find mechanistic differences between the DOPE-PEI conjugate and bPEI regarding siRNA condensation and intracellular processing. bPEI 1.8kDa and bPEI 25kDa have similar properties with respect to condensation capability, but are very different regarding siRNA decondensation, cellular internalization and induction of reporter gene knockdown. Lipid conjugation of bPEI 1.8kDa improves the siRNA delivery properties, but with markedly different 2 formulation requirements and mechanisms of action compared to conventional PEIs. Interestingly, strong knockdown using bPEI 25kDa is dependent on the presence of a free vector fraction which does not increase siRNA uptake. Finally, we have investigated the effect on lysosomal pH induced by these vectors to elucidate the differences in the proton sponge effect between lipid conjugated PEI and conventional PEI: Neither DOPE-PEI nor bPEI 25kDa affected lysosomal pH as a function of time, underlining that the possible proton sponge effect is not associated with changes in lysosomal pH.

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A Novel Mechanism Is Involved in Cationic Lipid-Mediated Functional siRNA Delivery

Cited by 201 publications

References 47 publications

Prolonged gene silencing by combining siRNA nanogels and photochemical internalization

Prolonged gene silencing by combining siRNA nanogels and photochemical internalization

Lipid-like materials for low-dose, in vivo gene silencing

Elucidating the role of free polycations in gene knockdown by siRNA polyplexes

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