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While anionic helper lipids can bias LNP expression toward the spleen, weak RNA\u2013lipid 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.<\/jats:p>","DOI":"10.1021\/acsami.6c06888","type":"journal-article","created":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T17:07:33Z","timestamp":1784221653000},"page":"40246-40259","source":"Crossref","is-referenced-by-count":0,"title":["Stabilizing Anionic mRNA Lipid Nanoparticles by Cleavable Crosslinking of Cholesterol"],"prefix":"10.1021","volume":"18","author":[{"given":"Yunhe","family":"Su","sequence":"first","affiliation":[{"name":"Johns Hopkins University , , , ,","place":["Baltimore, Maryland, United States, 21218"]},{"name":"Johns Hopkins University School of Medicine , , , ,","place":["Baltimore, Maryland, United States, 21218"]},{"name":"Johns Hopkins University School of Medicine , , , ,","place":["Baltimore, Maryland, United States, 21231"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joseph","family":"Choy","sequence":"additional","affiliation":[{"name":"Johns Hopkins University , , , ,","place":["Baltimore, Maryland, United States, 21218"]},{"name":"Johns Hopkins University , , , ,","place":["Baltimore, Maryland, United States, 21218"]},{"name":"Johns Hopkins University School of Medicine , , , ,","place":["Baltimore, Maryland, United States, 21231"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiang","family":"Liu","sequence":"additional","affiliation":[{"name":"Johns Hopkins University , , , ,","place":["Baltimore, Maryland, United States, 21218"]},{"name":"Johns Hopkins University , , , ,","place":["Baltimore, Maryland, United States, 21218"]},{"name":"Johns Hopkins University School of Medicine , , , ,","place":["Baltimore, Maryland, United States, 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21231"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christine","family":"Wei","sequence":"additional","affiliation":[{"name":"Johns Hopkins University , , , ,","place":["Baltimore, Maryland, United States, 21218"]},{"name":"Johns Hopkins University School of Medicine , , , ,","place":["Baltimore, Maryland, United States, 21218"]},{"name":"Johns Hopkins University School of Medicine , , , ,","place":["Baltimore, Maryland, United States, 21231"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kailei D.","family":"Goodier","sequence":"additional","affiliation":[{"name":"Johns Hopkins University , , , ,","place":["Baltimore, Maryland, United States, 21218"]},{"name":"Johns Hopkins University , , , ,","place":["Baltimore, Maryland, United States, 21218"]},{"name":"Johns Hopkins University School of Medicine , , , ,","place":["Baltimore, Maryland, United States, 21231"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Di","family":"Yu","sequence":"additional","affiliation":[{"name":"Johns Hopkins University , , , ,","place":["Baltimore, Maryland, United States, 21218"]},{"name":"Johns Hopkins University School of Medicine , , , ,","place":["Baltimore, Maryland, United States, 21218"]},{"name":"Johns Hopkins University School of Medicine , , , ,","place":["Baltimore, Maryland, United States, 21231"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leonardo","family":"Cheng","sequence":"additional","affiliation":[{"name":"Johns Hopkins University , , , ,","place":["Baltimore, Maryland, United States, 21218"]},{"name":"Johns Hopkins University School of Medicine , , , ,","place":["Baltimore, Maryland, United States, 21218"]},{"name":"Johns Hopkins University School of Medicine , , , ,","place":["Baltimore, Maryland, United States, 21231"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Milan","family":"Patel","sequence":"additional","affiliation":[{"name":"Johns Hopkins University , , , ,","place":["Baltimore, Maryland, United States, 21218"]},{"name":"Johns Hopkins University School of Medicine , , , ,","place":["Baltimore, Maryland, United States, 21218"]},{"name":"Johns Hopkins University School of Medicine , , , ,","place":["Baltimore, Maryland, United States, 21231"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoya","family":"Lu","sequence":"additional","affiliation":[{"name":"Johns Hopkins University , , , ,","place":["Baltimore, Maryland, United States, 21218"]},{"name":"Johns Hopkins University , , , ,","place":["Baltimore, Maryland, United States, 21218"]},{"name":"Johns Hopkins University School of Medicine , , , ,","place":["Baltimore, Maryland, United States, 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