Macromolecular Design Principles for Polymer-Lipid Hybrid Nanoparticles in RNA Delivery.

Tunable polymers for polymer–lipid hybrid nanoparticles facilitate controlled organ-selective RNA delivery

Electrostatically‐Stabilized PEG‐Free Lipid Nanoparticles for Systemic Nucleic Acid Delivery

Lipid nanoparticles (LNPs) have enabled the clinical translation of various nucleic acid cargos, including messenger RNA. Traditional LNP formulations contain a small proportion of lipids conjugated to poly(ethylene glycol), or PEG, which provides advantages of size control, stability, and extended circulation. However, PEGylation of both LNPs and other nano‐carriers raises challenges to efficient gene delivery, including endogenous production of anti‐PEG antibodies, accelerated clearance of multiple doses of LNPs, and reduced cellular uptake and endosomal escape. Here, we design and characterize a fully PEG‐free LNP platform, stabilized alternatively through the electrostatic adsorption of charged polymeric coatings that leverage principles of layer‐by‐layer self‐assembly. A library of carboxylated polyanions confers stability to non‐PEGylated (nonPEG) LNPs under biological stresses. nonPEG layered LNPs (LLNPs) significantly improve transfection of cancer and immune cells in vitro and generate favorable transfection in vivo via multiple routes of administration. In particular, nonPEG LLNPs reduce hepatic transfection by an order of magnitude, a major challenge for targeted LNP gene delivery, while maintaining similar circulation and transfection in other organs. Ultimately, this platform offers a highly modular, tunable approach to incorporate various bioactive moieties to achieve PEG‐free extrahepatic gene delivery.

Extracellular Vesicle–Lipid Hybrid Systems for RNA Delivery in Cancer: Structural Classification, Functional Delivery, and Translational Challenges

RNA therapeutics offer considerable potential for cancer treatment. Their therapeutic application, however, remains limited by rapid degradation, inefficient cellular uptake, and restricted intracellular release. Extracellular vesicles (EVs) are cell-derived membrane vesicles that have been exploited as promising vehicles for drug delivery due to their high biocompatibility and low immunogenicity, whereas liposomes and lipid nanoparticles provide tunable lipid composition and efficient loading of exogenous nucleic acids. Combining these carriers has led to EV–lipid hybrid systems designed to integrate their complementary properties. This review summarizes recent advances in EV–lipid hybrids for cancer therapy and organizes the reported systems according to their structural architecture and preparation. EV–liposome fusion hybrids, EV–lipid nanoparticle hybrids, and EV membrane-integrated lipid nanocarriers are discussed in relation to their RNA-loading strategies and representative therapeutic designs. The review also examines the key processes involved in functional RNA delivery and summarizes representative applications across different cancer types. Challenges associated with safety evaluation are also discussed, together with future directions for clinical translation. Overall, EV–lipid hybrids represent a promising strategy for RNA-based cancer therapy.

Stabilizing Anionic mRNA Lipid Nanoparticles by Cleavable Crosslinking of Cholesterol.

Lipid nanoparticles (LNPs) are the leading platform for mRNA delivery, with their in vivo performance governed by lipid composition and colloidal stability. While anionic helper lipids can bias LNP expression toward the spleen, weak RNA-lipid interactions during purification often induce nanoparticle rearrangement and reduced activity. These stability limitations effectively narrow the accessible formulation design and screening space, leaving large regions of anionic compositional space underexplored. Here, we extend our cleavable crosslinking strategy to stabilize anionic LNPs without replacing the primary lipid constituents of the parent LNP formulation. By tuning the lengths of the cholesterol-derived acid-cleavable crosslinker and PEG-diamine, we achieved balanced structural stability. The optimized crosslinked formulation exhibited a significant increase in splenic mRNA expression at 12 h compared to the uncrosslinked LNPs. Notably, 33.2% of CD45+ tdTomato+ cells in the spleen were identified as T cells. Mechanistic analyses suggest that controlled mRNA release and altered intracellular processing contribute to the improved transfection efficiency. Together, these findings define a tunable crosslinking window that expands the accessible design landscape for tissue- and cell-specific mRNA delivery.

Phospholipid-Mimetic Amphiphilic Polyion Complexes Enable Intracellular Oligonucleotide Delivery under Charge-Neutral Conditions.

Here, we report phospholipid-mimetic cationic copolymers that facilitate intracellular oligonucleotide delivery at a net charge-neutral polymer/DNA mixing ratio. Conventional nucleic acid delivery nanocarriers are mainly internalized through endocytosis, which often leads to endosomal sequestration and nucleic acid degradation, thereby reducing delivery efficiency and therapeutic efficacy. To address these issues, we developed a cationic phospholipid-mimetic random copolymer designed to physically form polyion complexes with anionic oligomeric DNA. Instead of simply maximizing cationic charge, our approach utilizes the membrane-interactive interfacial amphiphilicity of copolymer/oligonucleotide polyion complexes alongside hydrophobic monomer unit-mediated interactions to regulate interactions with cellular membranes. Consequently, at a net charge-neutral mixing ratio of the cationic polymer and anionic DNA, the polyion complexes showed reduced LysoTracker colocalization, uptake that was partially retained under ATP-depleted and endocytosis-inhibited conditions, and exhibited minimal cytotoxicity and negligible plasma membrane damage in vitro. Functionally, this system enabled antisense oligonucleotide delivery, reducing Bcl-2 mRNA expression by 57% relative to untreated cells in HepG2 cells. These findings suggest that interfacial regulation through phospholipid-mimetic amphiphilic polyion complexes can facilitate intracellular oligonucleotide delivery under net charge-neutral conditions, with uptake showing reduced sensitivity to suppression of classical energy-dependent endocytosis while maintaining negligible plasma membrane damage.

Rational Design of Pyrrole-Based Ionizable Lipids for mRNA Delivery.

Ionizable lipids are key components of lipid nanoparticles for mRNA delivery. Inspired by the DLin-KC2-DMA ionizable lipid, we developed a series of pyrrole-based ionizable lipids to evaluate the feasibility of incorporating a rigid heterocyclic scaffold into ionizable lipid design. Systematic variation of headgroup architecture revealed that positioning the tertiary amine outside the pyrrole core substantially improved RNA encapsulation and transfection activity and identified lipid 6 as the lead structure within this series. Lipid nanoparticles formulated with lipid 6, administered via intramuscular injection, outperformed other pyrrole analogs and achieved lymph node transfection comparable to structural reference DLin-KC2-DMA. Mechanistic studies combining Förster resonance energy transfer assays and multiscale molecular dynamics simulations suggested that the improved delivery was associated with enhanced lipid-RNA electrostatic interactions and favorable membrane fusion behavior. Overall, this work establishes pyrrole as a feasible scaffold for ionizable lipid development and expands the chemical design space of mRNA delivery materials.