Cytosolic delivery remains a major bottleneck for siRNA therapeutics. To facilitate delivery, siRNAs are often enclosed in nanoparticles (NPs). However, upon endocytosis such NPs are mainly trafficked towards lysosomes. To avoid degradation, cytosolic release of siRNA should occur prior to fusion of endosomes with lysosomes, but current endosomal escape strategies remain inefficient. In contrast to this paradigm, we aim to exploit lysosomal accumulation by treating NP-transfected cells with low molecular weight drugs that release the siRNA from the lysosomes into the cytosol. We show that FDA-approved cationic amphiphilic drugs (CADs) significantly improved gene silencing by siRNA-loaded nanogels in cancer cells through simple sequential incubation. CADs induced lysosomal phospholipidosis, leading to transient lysosomal membrane permeabilization and improved siRNA release without cytotoxicity. Of note, the lysosomes could be applied as an intracellular depot for triggered siRNA release by multiple CAD treatments.
Small
nucleic acid (NA) therapeutics, such as small interfering
RNA (siRNA), are generally formulated in nanoparticles (NPs) to overcome
the multiple extra- and intracellular barriers upon in vivo administration. Interaction with target cells typically triggers
endocytosis and sequesters the NPs in endosomes, thus hampering the
pharmacological activity of the encapsulated siRNAs that occurs in
the cytosol. Unfortunately, for most state-of-the-art NPs, endosomal
escape is largely inefficient. As a result, the bulk of the endocytosed
NA drug is rapidly trafficked toward the degradative lysosomes that
are considered as a dead end for siRNA nanomedicines. In contrast
to this paradigm, we recently reported that cationic amphiphilic drugs
(CADs) could strongly promote functional siRNA delivery from the endolysosomal
compartment via transient induction of lysosomal
membrane permeabilization. However, many questions still remain regarding
the broader applicability of such a CAD adjuvant effect on NA delivery.
Here, we report a drug repurposing screen (National Institutes of
Health Clinical Collection) that allowed identification of 56 CAD
adjuvants. We furthermore demonstrate that the CAD adjuvant effect
is dependent on the type of nanocarrier, with NPs that generate an
appropriate pool of decomplexed siRNA in the endolysosomal compartment
being most susceptible to CAD-promoted gene silencing. Finally, the
CAD adjuvant effect was verified on human ovarian cancer cells and
for antisense oligonucleotides. In conclusion, this study strongly
expands our current knowledge on how CADs increase the cytosolic release
of small NAs, providing relevant insights to more rationally combine
CAD adjuvants with NA-loaded NPs for future therapeutic applications.
RNA therapeutics are poised to revolutionize medicine. To unlock the full potential of RNA drugs, safe and efficient (nano)formulations to deliver them inside target cells are required. Endosomal sequestration of nanocarriers represents a major bottleneck in nucleic acid delivery. Gaining more detailed information on the intracellular behavior of RNA nanocarriers is crucial to rationally develop delivery systems with improved therapeutic efficiency. Surfactant protein B (SP-B) is a key component of pulmonary surfactant (PS), essential for mammalian breathing. In contrast to the general belief that PS should be regarded as a barrier for inhaled nanomedicines, we recently discovered the ability of SP-B to promote gene silencing by siRNA-loaded and lipid-coated nanogels. However, the mechanisms governing this process are poorly understood. The major objective of this work was to obtain mechanistic insights in the SP-B mediated cellular delivery of siRNA. To this end, we combined siRNA knockdown experiments, confocal microscopy and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) imaging in an in vitro non-small cell lung carcinoma model with lipid mixing assays on vesicles that mimic the composition of (intra)cellular membranes. Our work highlights a strong correlation between SP-B mediated fusion with anionic endosomal membranes and cytosolic siRNA delivery, a mode-of-action resembling that of certain viruses and virus-derived cell-penetrating peptides. Building on these gained insights, we optimized the SP-B proteolipid composition, which dramatically improved delivery efficiency. Altogether, our work provides a mechanistic understanding of SP-B induced perturbation of intracellular membranes, offering opportunities to fuel rational design of SP-B inspired RNA nanoformulations for inhalation therapy.
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