Lipid nanoparticle (LNP)-mRNA complexes are transforming medicine. However, the medical applications of LNPs are limited by their low endosomal disruption rates, high toxicity and long tissue persistence times. LNPs that rapidly hydrolyse in endosomes (RD-LNPs) could solve the problems limiting LNP-based therapeutics and dramatically expand their applications but have been challenging to synthesize. Here we present an acid-degradable linker termed 'azido-acetal' that hydrolyses in endosomes within minutes and enables the production of RD-LNPs. Acid-degradable lipids composed of polyethylene glycol lipids, anionic lipids and cationic lipids were synthesized with the azido-acetal linker and used to generate RD-LNPs, which significantly improved the performance of LNP-mRNA complexes in vitro and in vivo. Collectively, RD-LNPs delivered mRNA more efficiently to the liver, lung, spleen and brains of mice and to haematopoietic stem and progenitor cells in vitro than conventional LNPs. These experiments demonstrate that engineering LNP hydrolysis rates in vivo has great potential for expanding the medical applications of LNPs.

Department of Bioengineering and Cancer Center at Illinois University of Illinois Urbana Champaign Urbana IL USA

Department of Bioengineering and Innovative Genomics Institute University of California Berkeley CA USA

Department of Bioengineering School of Medicine Stanford University Stanford CA USA

Department of Cellular and Integrative Physiology University of Texas Health Science Center at San Antonio San Antonio TX USA

Department of Pathophysiology Faculty of Medicine in Pilsen Charles University Plzen Czech Republic

Department of Science and Environment Roskilde University Roskilde Denmark

Department of Surgery Department of Biomedical Engineering and Institute for Pediatric Regenerative Medicine Shriners Children's University of California Davis Sacramento CA USA

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Tenchov, R., Sasso, J. M. & Zhou, Q. A. PEGylated lipid nanoparticle formulations: immunological safety and efficiency perspective. Bioconjug. Chem. 34, 941–960 (2023). PubMed DOI PMC

Müller, S. S. et al. Biodegradable hyperbranched polyether–lipids with in-chain pH-sensitive linkages. Polym. Chem. 7, 6257–6268 (2016). DOI

Kedmi, R., Ben-Arie, N. & Peer, D. The systemic toxicity of positively charged lipid nanoparticles and the role of Toll-like receptor 4 in immune activation. Biomaterials 31, 6867–6875 (2010). PubMed DOI

Huotari, J. & Helenius, A. Endosome maturation. EMBO J. 30, 3481–3500 (2011). PubMed DOI PMC

Gao, W., Chan, J. M. & Farokhzad, O. C. pH-Responsive nanoparticles for drug delivery. Mol. Pharm. 7, 1913–1920 (2010). PubMed DOI PMC

Zhuo, S. et al. pH-sensitive biomaterials for drug delivery. Molecules 25, 5649 (2020). PubMed DOI PMC

Shin, J., Shum, P. & Thompson, D. H. Acid-triggered release via dePEGylation of DOPE liposomes containing acid-labile vinyl ether PEG-lipids. J. Control. Release 91, 187–200 (2003). PubMed DOI

Guo, X. & Szoka, F. C. Steric stabilization of fusogenic liposomes by a low-pH sensitive PEG-diortho ester–lipid conjugate. Bioconjug. Chem. 12, 291–300 (2000). DOI

Jayaraman, M. et al. Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angew. Chem. Int. Ed. 51, 8529–8533 (2012). DOI

Zhang, Y., Sun, C., Wang, C., Jankovic, K. E. & Dong, Y. Lipids and lipid derivatives for RNA delivery. Chem. Rev. 121, 12181–12277 (2021). PubMed DOI PMC

Roise, J. J. et al. Acid-sensitive surfactants enhance the delivery of nucleic acids. Mol. Pharm. 19, 67–79 (2022). PubMed DOI

Yang, X. et al. Making smart drugs smarter: the importance of linker chemistry in targeted drug delivery. Med. Res. Rev. 40, 2682–2713 (2020). PubMed DOI PMC

Liu, B. & Thayumanavan, S. S. Substituent effects on the pH sensitivity of acetals and ketals and their correlation with encapsulation stability in polymeric nanogels. J. Am. Chem. Soc. 139, 2306–2317 (2017). PubMed DOI PMC

Hansch, C., Leo, A. & Taft, R. W. A survey of Hammett substituent constants and resonance and field parameters. Chem. Rev. 91, 165–195 (1991). DOI

Takahata, Y. & Chong, D. P. Estimation of Hammett sigma constants of substituted benzenes through accurate density-functional calculation of core–electron binding energy shifts. Int. J. Quantum Chem. 103, 509–515 (2005). DOI

Waggoner, L. E., Miyasaki, K. F. & Kwon, E. J. Analysis of PEG-lipid anchor length on lipid nanoparticle pharmacokinetics and activity in a mouse model of traumatic brain injury. Biomater. Sci. 11, 4238–4253 (2023). PubMed DOI PMC

Kim, J. et al. Engineering lipid nanoparticles for enhanced intracellular delivery of mRNA through inhalation. ACS Nano 16, 14792–14806 (2022). PubMed DOI PMC

Lokugamage, M. P. et al. Optimization of lipid nanoparticles for the delivery of nebulized therapeutic mRNA to the lungs. Nat. Biomed. Eng. 5, 1059–1068 (2021). PubMed DOI PMC

Coelho, F., Salonen, L. M. & Silva, B. F. B. Hemiacetal-linked pH-sensitive PEG-lipids for non-viral gene delivery. N. J. Chem. 46, 15414–15422 (2022). DOI

Fang, Y. et al. Cleavable PEGylation: a strategy for overcoming the “PEG dilemma” in efficient drug delivery. Drug Deliv. 24, 22–32 (2017). PubMed DOI PMC

Kozma, G. T., Shimizu, T., Ishida, T. & Szebeni, J. Anti-PEG antibodies: properties, formation, testing and role in adverse immune reactions to PEGylated nano-biopharmaceuticals. Adv. Drug Deliv. Rev. 154-155, 163–175 (2020). PubMed DOI

Wang, H. et al. Polyethylene glycol (PEG)-associated immune responses triggered by clinically relevant lipid nanoparticles in rats. NPJ Vaccines 8, 169 (2023). PubMed DOI PMC

Yanez Arteta, M. et al. Successful reprogramming of cellular protein production through mRNA delivered by functionalized lipid nanoparticles. Proc. Natl Acad. Sci. USA 115, E3351–E3360 (2018). PubMed DOI PMC

Kilchrist, K. V. et al. Gal8 visualization of endosome disruption predicts carrier-mediated biologic drug intracellular bioavailability. ACS Nano 13, 1136–1152 (2019). PubMed PMC

Schmiderer, L. et al. Efficient and non-toxic biomolecule delivery to primary human hematopoietic stem cells using nanostraws. Proc. Natl Acad. Sci. USA 117, 21267–21273 (2020). PubMed DOI PMC

Levetzow, G. V. et al. Nucleofection, an efficient non-viral method to transfer genes into human hematopoietic stem and progenitor cells. Stem Cells Dev. 15, 278–285 (2006). DOI

Vhora, I., Lalani, R., Bhatt, P., Patil, S. & Misra, A. Lipid-nucleic acid nanoparticles of novel ionizable lipids for systemic BMP-9 gene delivery to bone-marrow mesenchymal stem cells for osteoinduction. Int. J. Pharm. 563, 324–336 (2019). PubMed DOI

Shi, D., Toyonaga, S. & Anderson, D. G. In vivo RNA delivery to hematopoietic stem and progenitor cells via targeted lipid nanoparticles. Nano Lett. 23, 2938–2944 (2023). PubMed DOI PMC

Kumar, V. et al. Shielding of lipid nanoparticles for siRNA delivery: impact on physicochemical properties, cytokine induction, and efficacy. Mol. Ther. Nucleic Acids 3, e210 (2014). PubMed DOI PMC

Sakurai, Y. et al. Efficient siRNA delivery by lipid nanoparticles modified with a non-standard macrocyclic peptide for EpCAM-targeting. Mol. Pharm. 14, 3290–3298 (2017). PubMed DOI

Chander, N., Basha, G., Yan Cheng, M. H., Witzigmann, D. & Cullis, P. R. Lipid nanoparticle mRNA systems containing high levels of sphingomyelin engender higher protein expression in hepatic and extra-hepatic tissues. Mol. Ther. Methods Clin. Dev. 30, 235–245 (2023). PubMed DOI PMC

Ruoslahti, E. Brain extracellular matrix. Glycobiology 6, 489–492 (1996). PubMed DOI

Dankovich, T. M. et al. Extracellular matrix remodeling through endocytosis and resurfacing of Tenascin-R. Nat. Commun. 12, 7129 (2021). PubMed DOI PMC

Cheng, Q. et al. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR–Cas gene editing. Nat. Nanotechnol. 15, 313–320 (2020). PubMed DOI PMC

LoPresti, S. T., Arral, M. L., Chaudhary, N. & Whitehead, K. A. The replacement of helper lipids with charged alternatives in lipid nanoparticles facilitates targeted mRNA delivery to the spleen and lungs. J. Control. Release 345, 819–831 (2022). PubMed DOI PMC

Frohlich, E. The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles. Int. J. Nanomed. 7, 5577–5591 (2012). DOI

Hu, M., Zhou, N., Cai, W. & Xu, H. Lysosomal solute and water transport. J. Cell Biol. 221, e202109133 (2022). PubMed DOI PMC

Zahid, M. U., Ma, L., Lim, S. J. & Smith, A. M. Single quantum dot tracking reveals the impact of nanoparticle surface on intracellular state. Nat. Commun. 9, 1830 (2018). PubMed DOI PMC

Pei, Y. et al. Synthesis and bioactivity of readily hydrolysable novel cationic lipids for potential lung delivery application of mRNAs. Chem. Phys. Lipids 243, 105178 (2022). PubMed DOI PMC

Qiu, M. et al. Lung-selective mRNA delivery of synthetic lipid nanoparticles for the treatment of pulmonary lymphangioleiomyomatosis. Proc. Natl Acad. Sci. USA 119, e2116271119 (2022). PubMed DOI PMC

Li, Q. et al. Engineering caveolae-targeted lipid nanoparticles to deliver mRNA to the lungs. ACS Chem. Biol. 15, 830–836 (2020). PubMed DOI

Landesman-Milo, D. & Peer, D. Toxicity profiling of several common RNAi-based nanomedicines: a comparative study. Drug Deliv. Transl. Res. 4, 96–103 (2014). PubMed DOI

Sanders, L. M. & Zeisel, S. H. Choline: dietary requirements and role in brain development. Nutr. Today 42, 181–186 (2007). PubMed DOI PMC

Gibellini, F. & Smith, T. K. The Kennedy pathway—de novo synthesis of phosphatidylethanolamine and phosphatidylcholine. IUBMB Life 62, 414–428 (2010). PubMed DOI

Raghu, G., Nyberg, F. & Morgan, G. The epidemiology of interstitial lung disease and its association with lung cancer. Br. J. Cancer 91, S3–S10 (2004). PubMed DOI PMC

McAleer, J. P. & Kolls, J. K. Directing traffic IL‐17 and IL‐22 coordinate pulmonary immune defense. Immunol. Rev. 260, 129–144 (2014). PubMed DOI PMC

Muhl, H. et al. IL-22 in tissue-protective therapy. Br. J. Pharmacol. 169, 761–771 (2013). PubMed DOI PMC

Mizoguchi, A. et al. Clinical importance of IL-22 cascade in IBD. J. Gastroenterol. 53, 465–474 (2018). PubMed DOI

Hwang, S., Feng, D. & Gao, B. Interleukin-22 acts as a mitochondrial protector. Theranostics 10, 7836–7840 (2020). PubMed DOI PMC