Aug 2026· Biomaterials Science· 0 citations· 33 references
Medicine
TL;DR
Tuning polycation chain length with core π-π interactions is key to unlocking highly efficient mRNA delivery in micelles, and achieves 2-fold higher protein expression in vivo compared to shorter or longer chain variants.
Abstract
Messenger RNA (mRNA)-based therapeutics hold great promise by enabling in situ expression of therapeutic proteins. The success of these therapies in vivo critically depends on the design of safe and efficient delivery systems. Polymeric micelles formed by block catiomers can encapsulate mRNA, protect it from degradation, and facilitate intracellular delivery. Polycation chain length has been proposed as a design parameter of micelles, yet how it governs stabilizing interactions to define mRNA delivery performance remains unestablished. Here, we systematically investigate this relationship using a series of poly(ethylene glycol)-poly(glycidyl-phenylalanate) (PEG-PG(Phe)) block copolymers with varying PG(Phe) chain lengths. These polymers interact with mRNA via polyion complexation and π-π stacking. Our findings revealed that the block catiomers having PG(Phe) segments with 90 units formed micelles with superior stability and enhanced endosomal escape, while micelles with shorter segments were rapidly dissociated and micelles with longer blocks showed impaired PEG shielding. Moreover, the polymer with a PG(Phe) block of 90 units achieved 2-fold higher protein expression in vivo compared to shorter or longer chain variants. These findings reveal that tuning polycation chain length with core π-π interactions is key to unlocking highly efficient mRNA delivery.
The clinical translation of small interfering RNA (siRNA) therapeutics critically depends on delivery systems capable of protecting nucleic acids, enabling efficient cellular uptake, and promoting cytosolic release. Liquid crystalline nanoparticles (LCNs) are promising carriers due to tunable internal nanostructure and biocompatibility, yet how composition controls membrane affinity and biological performance remains limited. Here, we compared two cationic reverse-hexagonal LCNs formulated from monoolein, oleic acid, poly(allylamine hydrochloride) and either poloxamer 407 (P407) or poloxamer 188 (P188). Both LCN-P407 and LCN-P188 shared reverse-hexagonal organization, mean diameters of 150–185 nm, low polydispersity (0.09–0.18), positive zeta potentials (10–20 mV), and protection of siRNA from RNase A. Langmuir isotherms and Brewster angle microscopy revealed pronounced adsorption and expansion of DPPC monolayers, with LCN-P188 inducing stronger perturbations, consistent with its higher cytotoxicity (∼20% increase in cell death) relative to LCN-P407. Crucially, siRNA delivered by LCN-P407 showed significantly greater uptake (> 1.7-fold) than that administered by LCN-P188. Functionally, as a proof of concept, we showed that LCN-P407-siTNFα induced robust gene silencing in LPS-stimulated macrophages, reducing TNFα secretion by 1.2–3.5-fold depending on particle concentration and incubation time. LCN-P188-siTNFα produced only delayed and modest reductions (1.3–1.6-fold). In addition, in dermatomized porcine skin, LCN-P407 also exhibited superior cutaneous penetration, delivering siRNA efficiently into the viable epidermis and dermis. Collectively, these results reveal a direct relationship between LCN composition, membrane affinity, and functional performance, providing mechanistic insight for the rational design of LCN-based carriers in RNA therapeutics.
Ana Vitória Pupo Silvestrini, Márcia Carvalho de Abreu Fantini, Ana Paula Ramos et al.· International Journal of Pha...· 0 citations
Polyion complex (PIC) vesicles are attractive carriers for mRNA delivery, yet their assembly is often compromised by charged cargos that disrupt the electrostatic interactions required for vesicle formation. Here, we report triblock polyampholyte vesicles (TBPVs) that encode preferential polymer-polymer interactions, enabling robust vesicle assembly independent of cargo-mediated disruption. Constructed from poly(ethylene glycol)-b-poly(L-lysine)-b-poly(aspartic acid) copolymers, TBPVs encapsulate both free mRNA and pre-condensed mRNA polyplexes while preserving vesicle integrity. Notably, the TBPVs encapsulating pre-condensed mRNA polyplexes demonstrated significant protein expression both in vitro and in vivo. Disulfide crosslinking of the vesicular membrane further confers stability under physiological conditions, while providing redox responsiveness for intracellular disassembly and cargo release. Systemically administered SS-TBPVs loaded with mRNA polyplexes enable detectable hepatic transgene expression in vivo. These findings support triblock polyampholyte PIC vesicles as a versatile and cargo-tolerant platform for polyplex-based nucleic acid delivery.
Guanghao Hu, Takayoshi Watanabe, Pengwen Chen et al.· Colloids and Surfaces B: Bio...· 0 citations
Clinical translation of messenger RNA (mRNA) therapeutics is often hampered by the poor storage stability of lipid-based delivery vectors. Many lipopolyplex (LPP) delivery systems struggle to achieve efficient nucleic acid release through the membrane fusion pathway. Herein, we developed a novel ternary lipopolyplex (LPP) platform of lipid/HBPL/mRNA LPPs by incorporating hyperbranched poly-L-lysine (HBPL) as a functional polymeric core. The highly branched architecture of HBPL enabled a unique balance between high mRNA encapsulation efficiency, efficient intracellular release, and exceptional colloidal stability, thereby overcoming a key limitation of conventional lipid nanoparticles. This platform primarily promoted cellular uptake through a membrane fusion mechanism in vitro, thereby achieving highly efficient cytoplasmic delivery, while the HBPL core enabled superior mRNA release compared to its linear ε-Polylysine (ε-PLL, hereafter referred to as PLL)-based counterpart. These attributes collectively yielded high transfection efficiency across multiple cell lines, significantly enhanced dendritic cell (DC) maturation, and superior biocompatibility over a commercial transfection reagent. Efficient mRNA delivery to the lungs and spleen was also confirmed in vivo, with protein expression sustained even after 1 month of storage. By successfully integrating long-term stability with high delivery efficiency, this HBPL-based LPP platform represents a highly promising candidate for advancing mRNA therapeutics and vaccines.
Huidi Meng, Bingjie Fu, Min Liang et al.· Advanced Healthcare Material...· 0 citations
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.
Yunhe Su, Joseph Choy, Xiang Liu et al.· ACS Applied Materials and In...· 0 citations
The development of nonviral RNA delivery has been shaped by the chemistry of cationic materials and their capacity to organize nucleic acids through multivalent electrostatic interactions. Before ionizable lipid nanoparticle (LNP) formulations became central to clinically translated RNA therapeutics, nonviral nucleic acid delivery relied largely on cationic liposomes, cationic polymers, and polymer-lipid complexes that condensed anionic DNA or RNA into nanoscale assemblies. These systems established principles that continue to guide RNA nanomedicine, including electrostatic complexation, colloidal assembly, endosomal trafficking, charge-associated toxicity, degradable carrier design, and intracellular cargo release. Over the past decade, the clinical success of mRNA vaccines has consolidated small-molecule ionizable lipid LNPs as a major formulation platform, owing to their ability to combine efficient RNA encapsulation with improved tolerability, manufacturability, and in vivo expression. This Perspective revisits cationic polymer-lipid nanoparticles not as historical precursors to ionizable lipid LNPs, but as a macromolecular materials space that can clarify and extend current RNA delivery design. We discuss these systems within a structure-assembly-biointerface framework, emphasizing how polymer architecture, charge distribution, degradability, topology, and polymer-lipid interfacial organization govern RNA packaging, nanoparticle formation, intracellular release, tissue-selective interactions, and biological identity.
Enhancing the endosomal escape of nucleic acids remains a critical bottleneck in improving their therapeutic efficacy. Inspired by advancements in lipid nanoparticles, we describe a chemical approach to deliver multi-tailed DNA-surfactant conjugates (DSCs) into cells using nucleic acid nanocapsules (NANs). NANs are enzyme degradable, crosslinked DNA micelles that exhibit enhanced membrane destabilizing properties. Previously, we reported the successful delivery of therapeutic oligonucleotides to cells using single-tailed DSCs delivered by esterase-cleavable NANs. This work features the rational design of a set of asymmetrically synthesized hydrophobic micelle crosslinkers using a synthetic approach that incorporates either one or two alkyl chains and an esterase-labile group into a nonionic surfactant flanked by clickable azides. These crosslinkers are covalently incorporated into the NAN construct, and upon their enzymatic degradation within the cell, multi-tailed DSCs are released. We show that these DSCs have superior transfection capabilities over single-tailed DSCs. Additionally, we show that these multi-tailed DSCs improve DNAzyme-mediated gene silencing and enhance mRNA delivery in vitro, owing to their enhanced membrane disruption capabilities reflected in both cell studies and molecular dynamics simulations. Importantly, our chemical approach enables the formation of multi-tailed surfactants in situ, post micelle assembly. This allows an oligonucleotide nanomaterial delivery platform to act as a "pro-drug" where the active therapeutic and the potential for its cytosolic delivery are the product of the nanoformulation's degradation.
Joseph Gorecki, Ina F de la Fuente, Patrick M. Corrigan et al.· ACS Applied Materials and In...· 0 citations