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The membrane-fusion-based internalization without lysosomal entrapment is advantageous for intracellular delivery over endocytosis. However, protein corona formed on the membrane-fusogenic liposome surface converts its membrane-fusion performance to lysosome-dependent endocytosis, causing poorer delivery efficiency in biological conditions. Herein, we develop an antifouling membrane-fusogenic liposome for effective intracellular delivery in vivo. Leveraging specific lipid composition at an optimized ratio, such antifouling membrane-fusogenic liposome facilitates fusion capacity even in protein-rich conditions, attributed to the copious zwitterionic phosphorylcholine groups for protein-adsorption resistance. Consequently, the antifouling membrane-fusogenic liposome demonstrates robust membrane-fusion-mediated delivery in the medium with up to 38% fetal bovine serum, outclassing two traditional membrane-fusogenic liposomes effective at 4% and 6% concentrations. When injected into mice, antifouling membrane-fusogenic liposomes can keep their membrane-fusion-transportation behaviors, thereby achieving efficient luciferase transfection and enhancing gene-editing-mediated viral inhibition. This study provides a promising tool for effective intracellular delivery under complex physiological environments, enlightening future nanomedicine design., (© 2024. The Author(s).)

Membrane fusion, a fundamental biological process of the fusion of the membrane composition between cells, is vital for cell-cell communication and cargo transport between living cells. This fusion interaction achieves the transportation of the inner content to the cellular cytosol as well as the simultaneous blending of foreign substances with the cell membrane. Inspired by this biological process, emerging membrane-fusogenic particles have been developed, opening a new area for bioengineering and biomedical applications. Especially, membrane-fusion-mediated transfer of inner cargoes can bypass endosomal entrapment to maximize the transportation efficiency, emerging as a unique cytoplasmic delivery platform distinct from those depending on conventional endocytosis-based pathways. In addition, the membrane fusion enables cell surface modification through lipid diffusion and mixing, providing a tool for direct cell membrane engineering. In this review, we focus on the development of membrane-fusogenic particles and their up-to-date progress. We briefly introduce the concept of membrane fusion, elaborate inspiring strategies of membrane-fusogenic particles, and highlight the recent advances and the promising applications of membrane-fusogenic particles as a next-generation bioengineering tool. In the end, we conclude with the present challenges and opportunities, providing insights in the future research of membrane-fusogenic particles.

Clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9)-based therapies are attractive to achieve precise and targeted disruption of the hepatitis B virus (HBV) genome for HBV infection treatment. However, unspecific in vivo distribution of the CRISPR/Cas9 vector after systemic administration not only impedes CRISPR/Cas9 expression in the liver, but also causes undesired gene editing in off-site organs, decreasing the therapeutic efficacy and leading to potential side effects. Herein, we designed a hepatocyte-confined CRISPR/Cas9-based nanocleaver (HepCCCleaver) to realize precise and efficient cleavage of HBV DNA, specifically in hepatocytes, for safe and effective HBV treatment. This HepCCCleaver was formed by encapsulating the hepatocyte-specific CRISPR/Cas9 plasmid into a galactose-modified membrane-fusogenic carrier. Our galactose-modified membrane-fusogenic carrier allowed the HepCCCleaver to specifically accumulate in the liver and enter hepatocytes through membrane fusion, bypassing lysosome entrapment to avoid degradation for superior delivery efficiency. The hepatocyte-specific CRISPR/Cas9 plasmid released in the cytoplasm could switch on the gene expression only in hepatocytes, ensuring precise cleavage on viral DNA in HBV-infected but not other types of cells. By integrating the liver-targeting membrane-fusogenic delivery system with hepatocyte-specific CRISPR/Cas9 plasmid, this HepCCCleaver could precisely and efficiently confine over 95% events of viral DNA gene editing in hepatocytes. This HepCCCleaver is a universal gene editing platform enabling safe and efficient disease treatment, specifically in the cell type of interest. [Display omitted] • A hepatocyte-specific chimeric promoter is constructed into a hepatocyte-specifically-activated CRISPR/Cas9 plasmid. • HepCCCleaver can perform membrane-fusion-mediated delivery to achieve efficient gene transfection. • HepCCCleaver can confine CRISPR/Cas9 gene editing in hepatocyte, achieving safe and effective HBV treatment. [ABSTRACT FROM AUTHOR]

Targeting the activated epidermal growth factor receptor (EGFR) via clustered regularly interspaced short palindromic repeat (CRISPR) technology is appealing to overcome the drug resistance of hepatocellular carcinoma (HCC) towards tyrosine kinase inhibitor (TKI) therapy. However, combining these two distinct drugs using traditional liposomes results in a suboptimal synergistic anti-HCC effect due to the limited CRISPR/Cas9 delivery efficiency caused by lysosomal entrapment after endocytosis. Herein, we developed a liver-targeting gene-hybridizing-TKI fusogenic liposome (LIGHTFUL) that can achieve high CRISPR/Cas9 expression to reverse the EGFR-mediated drug resistance for enhanced TKI-based HCC therapy efficiently. Coated with a galactose-modified membrane-fusogenic lipid layer, LIGHTFUL reached the targeting liver site to fuse with HCC tumor cells, directly and efficiently transporting interior CDK5- and PLK1-targeting CRISPR/Cas9 plasmids (pXG333-CPs) into the HCC cell cytoplasm and then the cell nucleus for efficient expression. Such membrane-fusion-mediated pXG333-CP delivery resulted in effective downregulation of both CDK5 and PLK1, sufficiently inactivating EGFR to improve the anti-HCC effects of the co-delivered TKI, lenvatinib. This membrane-fusion-participant codelivery strategy optimized the synergetic effect of CRISPR/Cas9 and TKI combinational therapy as indicated by the 0.35 combination index in vitro and the dramatic reduction of subcutaneous and orthotopic TKI-insensitive HCC tumor growth in mice. Therefore, the established LIGHTFUL provides a unique co-delivery platform to combine gene editing and TKI therapies for enhanced synergetic therapy. [Display omitted] [ABSTRACT FROM AUTHOR]