Aug 2026· Advanced Healthcare Materials· Vol 15, pp.
e71571
· 0 citations· 41 references
Medicine
TL;DR
A novel ternary lipopolyplex (LPP) platform of lipid/HBPL/mRNA LPPs by incorporating hyperbranched poly-L-lysine (HBPL) as a functional polymeric core that yields high transfection efficiency across multiple cell lines, significantly enhanced dendritic cell (DC) maturation, and superior biocompatibility over a commercial transfection reagent.
Abstract
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.
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
Lipid nanoparticles (LNPs) are effective carriers for messenger ribonucleic acid (mRNA) delivery in vaccines; however, their reliance on extreme cold-chain storage limits global manufacturing and distribution. Conventional LNPs are formed by rapidly mixing four lipids with mRNA through electrostatic interactions between cationic ionizable lipids and negatively charged nucleic acids, facilitating nucleation and precipitation of mRNA-loaded LNPs. However, this binding also accelerates mRNA degradation, requiring stringent cold storage which limits widespread vaccine deployment. To overcome this limitation, we introduce a post-loading strategy in which empty LNPs (eLNPs) are first fabricated and RNA is subsequently loaded at a later stage. Using scalable confined impinging jet (CIJ) mixers, we optimized pH, buffer composition, lipid concentration, and ethanol content to produce colloidally stable eLNPs. Controlled adjustment of ethanol content and pH enabled efficient incorporation of four distinct RNA payloads while maintaining loaded LNP diameters below 100 nm. Post-loaded LNPs demonstrated mRNA delivery efficiencies in HeLa cells comparable to those of conventionally co-precipitated LNPs. Consistent size distributions and zeta potentials further confirmed comparable surface properties. Structural characterization by x-ray and neutron scattering revealed similar internal architectures for post-loaded and co-precipitated LNPs without compromising RNA loading efficiency. Together, these results demonstrate equivalent cellular delivery performance between the two formulations. This post-loading approach enables decentralized assembly of mRNA LNPs at the point of administration, with both eLNPs and mRNA stored under mild refrigeration, thereby improving vaccine accessibility. Moreover, eLNPs function as modular laboratory reagents, facilitating the translation of mRNA research toward clinical applications.
N. Bizmark, David F. Amelemah, Satya K. Nayagam et al.· bioRxiv· 0 citations
This work presents a metabolically targeted, bioorthogonal-activated delivery strategy to address the selectivity and efficiency limitations of current mRNA medicines, providing a promising platform for precision oncology.
Mingzhe Zhang, Chunhong Wang, Xiaohan Xu et al.· Journal of the American Chem...· 0 citations
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.
Yuki Nakashima, Pengwen Chen, Guanghao Hu et al.· Biomaterials Science· 0 citations
Polyethylenimine (PEI) is a general-purpose polycationic polymer which has received much attention as a non-viral vector for therapeutic nucleic acid delivery in medicine. The high level of amine groups enables electrostatic complexation with negatively charged genetic material, leading to the formation of polyplex and higher cellular uptake. Once internalized, PEI can facilitate endosomal escape because of its buffering capacity and or its ability to destabilize cellular membranes, which promotes efficient delivery of DNA and RNA. Recent use of PEI-based systems for gene delivery (DNA and small interfering RNA) and other gene therapy and neurological applications are emphasized. The transfection efficiency is high, but clinical translation is hindered by its cytotoxicity and low biodegradability and systemic stability. Current strategies, namely molecular modification, crosslinking, surface shielding and structural optimization of PEI-based gene-delivery system are then explored for enhancing the safety and therapeutic promise of this platform. This review focuses on major forms of PEI, molecular structure, physicochemical properties influence gene-delivery performance and applications on non-viral gene delivery.
Unknown authors· Advanced International Journ...· 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
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