Targeted si-RNA with liposomes and exosomes (extracellular vesicles): How to unlock the potential

Purpose: RNA interference (RNAi) therapeutics have shown potential for treating genetic, viral, and cancerous diseases, but the main bottleneck remains delivery of functional RNA molecules into the cell cytoplasm. The review focuses on liposomes and other lipid carriers, and on exosomes/extracellular vesicles (EVs), as approaches to overcome siRNA delivery barriers, and proposes integrating these two vesicular systems to unlock RNAi therapeutic potential.

Hypothesis: As a review, there is no single experimental hypothesis. Central thesis: if liposomes/lipid carriers are engineered for stable, high siRNA loading and efficient endosomal escape, and exosomes/EVs are exploited for their organotropism and biocompatibility, then integrating the advantages of both systems may overcome current barriers and unlock the therapeutic potential of RNAi. Cationic lipids can complex siRNA and promote endosomal escape, but toxicity and serum instability limit them; exosomes offer natural targeting but have low loading capacity and rapid clearance.

Aims: Introduce the theoretical basis of RNAi therapeutics and the main barriers/challenges for siRNA delivery. - Provide an overview of different delivery systems/carriers used to overcome siRNA delivery issues. - Focus on liposomes and other lipid carriers—neutral liposomes, cationic liposomes/lipoplexes, solid lipid nanoparticles (SLNs), stable nucleic acid lipid particles (SNALPs), lipopolyplexes/MENDs, and membrane-core nanoparticles (MCNPs)—and on exosomes/EVs. - Propose integrating liposomes and exosomes as a potential solution to realize RNAi therapy.

Delivery system: Platform: Lipid carriers and extracellular vesicles. Categories include neutral liposomes, cationic liposomes/lipoplexes, SNALPs, lipopolyplexes/MENDs, MCNPs, SLNs, targeted liposomes, PEGylated liposomes, cuboplexes, and exosomes/EVs. - Payloads: siRNA, miRNA, shRNA, antisense oligonucleotides; exosomes naturally carry interfering RNAs. - Key lipids: Cationic lipids such as DOTMA, DOTAP, DOGS, DC-Chol, DLinDMA, DLin-MC3-DMA, DLin-KC2-DMA, DODAP, DODMA; helper lipids DOPE, cholesterol, DSPC, DOPC; PEGylated lipids; anionic lipids such as DMPG. - Targeting ligands: folate, transferrin, integrins, N-acetyl-galactosamine (GalNAc/NAG), rabies virus glycoprotein (RVG) peptide, anisamide, aptamers, antibodies (anti-CD4, anti-CD20, scFv), and RGD peptides. - Chemical modifications: 2′-fluorination, 2′-oxymethylation, 2′-amination, locked nucleic acids (LNAs), phosphorodiamidate morpholino oligomers (PMOs), peptide nucleic acids (PNAs), and multivalent GalNAc-siRNA conjugates. - Exosome loading methods: electroporation, chemical-based transfection, transfection of exosome-producing cells, and cell activation.

Approach: Review/synthesis of preclinical and clinical studies. No new primary experimental data. In vitro and in vivo models include cancer cells, hepatocytes, neurons, fibrosarcoma, glioblastoma, breast cancer, pancreatic cancer, and viral infection models. Clinical trial data include lipid-based siRNA formulations such as DCR-MYC, TKM-080301, ND-L02-s0201, PRO-040201, Atu027, siRNA-EphA2-DOPC, ALN-PCS02, and patisiran (ALN-TTR02). Exosome-related clinical studies include Antisense102 (NCT02507583), EXOSOMES (NCT02310451), and plant exosomes for curcumin delivery (NCT01294072). Not a systematic review.

Key methods: RNAi mechanism and RISC loading; physicochemical barriers of siRNA (nuclease degradation, renal clearance, RES uptake, low membrane permeability, endosomal/lysosomal degradation); complexation and endosomal escape mechanisms; targeting ligand design; exosome isolation and characterization; exosome loading by electroporation, chemical transfection, producer-cell transfection, and cell activation; clinical trial database searches; gene silencing, tumor growth inhibition, survival, and toxicity endpoints.

Key results: Naked siRNA has a plasma half-life of <20 min, small size <10 nm, and is rapidly cleared by glomerular filtration and RES uptake. - GalNAc-siRNA conjugates bind the asialoglycoprotein receptor (ASGR) at nanomolar levels, target hepatocytes, and have entered clinical evaluation; one GalNAc-siRNA is in Phase 3 for transthyretin-mediated amyloidotic cardiomyopathy, with others in Phase 1/2 for hemophilia A/B, antitrypsin deficiency, and porphyria. - SNALP siRNA formulations effectively deliver siRNA to hepatocytes with no or minimal toxicity; ALN-TTR02 (patisiran) reached Phase III, the most advanced stage for an LNP-based therapy. - VP35-targeting siRNA-LNP treatment resulted in up to 100% survival of non-human primates infected with Sudan Ebola virus. - A PEGylated liposomal siRNA formulation with OA-R8 transferrin targeting showed 61.7% tumor inhibition, hepatocyte and tumor uptake in HepG2-bearing nude mice, and no immunogenicity, renal, or hepatic toxicity. - Exosome electroporation loaded approximately 25% of total siRNA; electroporation-loaded exosomes showed superior transfection compared with commercial transfection reagents. However, electroporation can cause siRNA aggregation and overestimate loading. - ClinicalTrials.gov search for “siRNA” returned 45 studies: 24 completed, 12 active, 5 terminated, 1 withdrawn, and 3 unknown. Of these, 9 involved siRNA and lipid; 11 systemic studies used lipid formulations, 2 of which were targeted. - DCR-MYC clinical development was discontinued; an ApoB-SNALP trial was terminated due to immunotoxicity; ALN-PCS02 showed no serious side effects or toxicities. - Exosome-delivered GAPDH and BACE1 siRNA reduced mRNA expression in neurons; exosome-delivered RAD51/RAD52 siRNA decreased fibrosarcoma cell viability; exosomal miR-146b inhibited cancer growth in a glioma model.

Interpretation: Lipid-based delivery systems are the most explored and promising non-viral siRNA carriers, and SNALP siRNA formulations are a sound approach for liver-targeted delivery. However, non-hepatic delivery remains the major challenge, and actively targeted liposomal formulations have not yet been realized clinically. Exosomes/EVs offer excellent organotropism, biocompatibility, and possible lysosomal avoidance, but suffer from low drug loading, rapid clearance, and manufacturing limitations. The authors argue that liposomes and exosomes are highly complementary: liposome engineering can provide high, reproducible siRNA loading, while exosome biology can provide targeting insights. Integrating the two may unlock the therapeutic potential of RNAi.

Limitations: This is a review, not a primary study. No “ideal system” for siRNA delivery has yet been identified. Cationic lipid carriers face toxicity, serum instability, rapid clearance, and PEG-related trade-offs. Active targeting of liposomes has not been successfully translated; no actively targeted liposomal drug is on the market. Exosomes have low drug loading, rapid blood clearance in some cases, heterogeneous composition, and unresolved manufacturing/scale-up issues. Clinical translation is mostly limited to liver targets, and many studies remain preclinical. Long-term toxicity in humans, especially for cationic lipid systems, remains to be addressed.