RNA interference (RNAi) is a valuable method for understanding the gene function and holds great potential for insect pest management. While RNAi is efficient and systemic in coleopteran insects, RNAi is inefficient in lepidopteran insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW),
DNA-inspired nanomaterials for enhanced endosomal escape
To realize RNA interference (RNAi) therapeutics, it is necessary to deliver therapeutic RNAs (such as small interfering RNA or siRNA) into cell cytoplasm. A major challenge of RNAi therapeutics is the endosomal entrapment of the delivered siRNA. In this study, we developed a family of delivery vehicles called Janus base nanopieces (NPs). They are rod-shaped nanoparticles formed by bundles of Janus base nanotubes (JBNTs) with RNA cargoes incorporated inside via charge interactions. JBNTs are formed by noncovalent interactions of small molecules consisting of a base component mimicking DNA bases and an amino acid side chain. NPs presented many advantages over conventional delivery materials. NPs efficiently entered cells via macropinocytosis similar to lipid nanoparticles while presenting much better endosomal escape ability than lipid nanoparticles; NPs escaped from endosomes via a “proton sponge” effect similar to cationic polymers while presenting significant lower cytotoxicity compared to polymers and lipids due to their noncovalent structures and DNA-mimicking chemistry. In a proof-of-concept experiment, we have shown that NPs are promising candidates for antiviral delivery applications, which may be used for conditions such as COVID-19 in the future.
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- Award ID(s):
- 1905785
- NSF-PAR ID:
- 10230809
- Date Published:
- Journal Name:
- Proceedings of the National Academy of Sciences
- Volume:
- 118
- Issue:
- 19
- ISSN:
- 0027-8424
- Page Range / eLocation ID:
- e2104511118
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract Spodoptera frugiperda cells by formulating dsRNA with Cellfectin II (CFII) transfection reagent. The CFII formulated dsRNA was protected from degradation by endonucleases present in Sf9 cells conditioned medium, hemolymph and midgut lumen contents collected from the FAW larvae. Lipid formulated dsRNA also showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing Sf9 cells and tissues to CFII formulated dsRNA caused a significant knockdown of endogenous genes. CFII formulated dsIAP fed to FAW larvae induced knockdown ofiap gene, growth retardation and mortality. Processing of dsRNA into siRNA was detected in Sf9 cells andSpodoptera frugiperda larvae treated with CFII conjugated32P‐UTP labeled dsGFP. Overall, the present study concluded that delivering dsRNA formulated with CFII transfection reagent helps dsRNA escapes from the endosomal accumulation and improved RNAi efficiency in the FAW cells and tissues. -
Cationic liposomes (CLs) are effective carriers of a variety of therapeutics. Their applications as vectors of nucleic acids (NAs), from long DNA and mRNA to short interfering RNA (siRNA), have been pursued for decades to realize the promise of gene therapy, with approvals of the siRNA therapeutic patisiran and two mRNA vaccines against COVID-19 as recent milestones. The long-term goal of developing optimized CL-based NA carriers for a broad range of medical applications requires a comprehensive understanding of the structure of these vectors and their interactions with cell membranes and components that lead to the release and activity of the NAs within the cell. Structure–activity relationships of lipids for CL-based NA and drug delivery must take into account that these lipids act not individually but as components of an assembly of many molecules. This review summarizes our current understanding of how the choice of the constituting lipids governs the structure of their CL–NA self-assemblies, which constitute distinct liquid crystalline phases, and the relation of these structures to their efficacy for delivery. In addition, we review progress toward CL–NA nanoparticles for targeted NA delivery in vivo and close with an outlook on CL-based carriers of hydrophobic drugs, which may eventually lead to combination therapies with NAs and drugs for cancer and other diseases.more » « less
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Systemic, non-viral siRNA delivery for cancer treatment is mainly achieved via condensation by cationic materials ( e.g. , lipids and cationic polymers), which nevertheless, suffers from poor serum stability, non-specific tissue interaction, and unsatisfactory membrane activity against efficient in vivo gene knockdown. Here, we report the design of a metastable, cancer-targeting siRNA delivery system based on two functional polymers, PVBLG-8, a cationic, helical cell-penetrating polypeptide, and poly( l -glutamic acid) (PLG), an anionic random-coiled polypeptide. PVBLG-8 with rigid, linear structure showed weak siRNA condensation capability, and PLG with flexible chains was incorporated as a stabilizer which provided sufficient molecular entanglement with PVBLG-8 to encapsulate the siRNA within the polymeric network. The obtained PVBLG-8/siRNA/PLG nanoparticles (PSP NPs) with positive charges were sequentially coated with additional amount of PLG, which reversed the surface charge from positive to negative to yield the metastable PVBLG-8/siRNA/PLG@PLG (PSPP) NPs. The PSPP NPs featured desired serum stability during circulation to enhance tumor accumulation via the enhanced permeability and retention (EPR) effect. Upon acidification in the tumor extracellular microenvironment and intracellular endosomes, the partial protonation of PLG on PSPP NPs surface would lead to dissociation of PLG coating from NPs, exposure of the highly membrane-active PVBLG-8, and surface charge reversal from negative to positive, which subsequently promoted tumor penetration, selective cancer cell internalization, and efficient endolysosomal escape. When siRNA against epidermal growth factor receptor (EGFR) was encapsulated, the PSPP NPs showed excellent tumor penetration capability, tumor cell uptake level, EGFR silencing efficiency, and tumor growth inhibition efficacy in U-87 MG glioblastoma tumor spheroids in vitro and in xenograft tumor-bearing mice in vivo , outperforming the PSP NPs and several commercial reagents such as Lipofectamine 2000 and poly( l -lysine) (PLL). This study therefore demonstrates a facile and unique design approach of metastable and charge reversal NPs, which overcomes multiple biological barriers against systemic siRNA delivery toward anti-cancer treatment.more » « less
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null (Ed.)As an overarching immune mechanism, RNA interference (RNAi) displays pathogen specificity and memory via different pathways. The small interfering RNA (siRNA) pathway is the primary antiviral defense mechanism against RNA viruses of insects and plays a lesser role in defense against DNA viruses. Reflecting the pivotal role of the siRNA pathway in virus selection, different virus families have independently evolved unique strategies to counter this host response, including protein-mediated, decoy RNA–based, and microRNA-based strategies. In this review, we outline the interplay between insect viruses and the different pathways of the RNAi antiviral response; describe practical application of these interactions for improved expression systems and for pest and disease management; and highlight research avenues for advancement of the field.more » « less
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Abstract RNA delivery into deep tissues with dense extracellular matrix (ECM) has been challenging. For example, cartilage is a major barrier for RNA and drug delivery due to its avascular structure, low cell density and strong negative surface charge. Cartilage ECM is comprised of collagens, proteoglycans, and various other noncollagneous proteins with a spacing of 20nm. Conventional nanoparticles are usually spherical with a diameter larger than 50-60nm (after cargo loading). Therefore, they presented limited success for RNA delivery into cartilage. Here, we developed Janus base nanotubes (JBNTs, self-assembled nanotubes inspired from DNA base pairs) to assemble with small RNAs to form nano-rod delivery vehicles (termed as “Nanopieces”). Nanopieces have a diameter of ∼20nm (smallest delivery vehicles after cargo loading) and a length of ∼100nm. They present a novel breakthrough in ECM penetration due to the reduced size and adjustable characteristics to encourage ECM and intracellular penetration.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1073/pnas.2104511118
