Ionizable lipids as a promising platform for creating mRNA vaccines

Objectives. Gene therapy involves the administration of various types of therapeutic nucleic acids into the organism, in order to treat severe hereditary diseases, as well as cancer. Furthermore, the COVID-19 pandemic demonstrated the possibility of rapid development and the effectiveness of both DNA and mRNA vaccines for the prevention of viral diseases. Numerous studies in the field of gene therapy have revealed that in most cases successful delivery of nucleic acids requires a special delivery system which protects nucleic acids from the effects of external and internal biological factors. Among the various types of such tools, non-viral delivery systems have proven to be the most versatile and safe ones. In the case of mRNA delivery, such systems are usually called mRNA vaccines, consisting of cationic or ionizable lipids. The purpose of this review is to justify the choice of the optimal structure of lipid components of mRNA vaccines and highlight the current prospects for their clinical use.

Results. In this review, we have considered the evolution of lipid structures, from cationic to ionizable, as the main components of mRNA delivery systems. Furthermore, the study demonstrated the necessity to use other types of lipids in mRNA vaccines. It also presents a review of clinical trials of mRNA vaccines against viral and oncological diseases, and provides recommendations for the design of the optimal structure of both cationic and ionizable lipids.

Conclusions. The most promising lipids for the development of mRNA vaccines are ionizable. They do not have a permanent positive charge which reduces their cytotoxicity and undesirable binding to components of the immune system. In general, mRNA vaccines can be universal and effective means for treating various types of diseases. However, their composition needs to be careful optimized.

1. Puumalainen A.M., Vapalahti M., Agrawal R.S., Kossila M., Laukkanen J., Lehtolainen P., Viita H., Paljärvi L., Vanninen R., Ylä-Herttuala S. β-galactosidase gene transfer to human malignant glioma in vivo using replication-deficient retroviruses and adenoviruses. Human Gene Ther. 1998;9(12):1769–1774. https://doi.org/10.1089/hum.1998.9.12-1769

2. Blaese R.M., Culver K.W., Miller A.D., Carter C.S., Fleisher T., Clerici M., Shearer G., Chang L., Chiang Y., Tolstoshev P., Greenblatt J.J, Rosenberg S.A., Klein H., Berger M., Mullen C.A., Ramsey W.J., Muul L., Morgan R.A., Anderson W.F. T Lymphocyte-Directed Gene Therapy for ADA−SCID: Initial Trial Results After 4 Years. Science. 1995;270(5235):475–480. https://doi.org/10.1126/science.270.5235.475

3. Kulkarni J.A., Witzigmann D., Thomson S.B., Chen S., Leavitt B.R., Cullis P.R., van der Meel R. The current landscape of nucleic acid therapeutics. Nature Nanotechnol. 2021;16: 630–643. https://doi.org/10.1038/s41565-021-00898-0

4. Liu G., Lin Q., Jin S., Gao C. The CRISPR-Cas toolbox and gene editing technologies. Mol. Cell. 2022;82(2):333–347. https://doi.org/10.1016/J.MOLCEL.2021.12.002

5. Liu C., Zhang L., Zhu W., Guo R., Sun H., Chen X., Deng N. Barriers and Strategies of Cationic Liposomes for Cancer Gene Therapy. Mol. Ther. Methods Clin. Dev. 2020;18: 751–764. https://doi.org/10.1016/j.omtm.2020.07.015

6. Goradel N.H., Negahdari B., Ghorghanlu S., Jahangiri S., Arashkia A. Strategies for enhancing intratumoral spread of oncolytic adenoviruses. Pharmacol. Ther. 2020;213:107586. https://doi.org/10.1016/j.pharmthera.2020.107586

7. Volodin T.A., Polikashina P.P., Budanova U.A., Sebyakin Y.L. Symmetrical and asymmetric dimeric cationic amphiphiles based on lipopeptides of irregular structure as potential components of cationic liposomes. Fine Chem. Technol. 2025;20(5):441–453. https://doi.org/10.32362/2410-6593-2025-20-5-441-453

8. Wang C., Pan C., Yong H., Wang F., Bo T., Zhao Y., Ma B., He W., Li M. Emerging non-viral vectors for gene delivery. J. Nanobio- technology. 2023;21:272. https://doi.org/10.1186/s12951-023-02044-5

9. Law S.S.Y., Liou G., Nagai Y., Giménez-Dejoz J., Tateishi A., Tsuchiya K., Kodama Y., Fujigaya T., Numata K. Polymercoated carbon nanotube hybrids with functional peptides for gene delivery into plant mitochondria. Nature Communications. 2022;13:2417. https://doi.org/10.1038/s41467-022-30185-y

10. Lin G., Revia R.A., Zhang M. Inorganic NanomaterialMediated Gene Therapy in Combination with Other Antitumor Treatment Modalities. Adv. Funct. Mater. 2021;31(5):2007096. https://doi.org/10.1002/adfm.202007096

11. Jones I., Roy P. Sputnik V COVID-19 vaccine candidate appears safe and effective. The Lancet. 2021;397(10275): 642–643. https://doi.org/10.1016/S0140-6736(21)00191-4

12. Walsh E.E., Frenck R.W., Falsey A.R., Kitchin N., Absalon J., Gurtman A., Lockhart S., Neuzil K., Mulligan M.J., Bailey R., Swanson K.A., Li P., Koury K., Kalina W., Cooper D., Fontes-Garfias C., Shi P.-Y., Türeci Ö., Tompkins K.R., Lyke K.E., Raabe V., Dormitzer P.R., Jansen K.U., Şahin U., Gruber W.C. Safety and Immunogenicity of Two RNA-Based Covid-19 Vaccine Candidates. New Engl. J. Med. 2020;383(25): 2439–2450. https://doi.org/10.1056/nejmoa2027906

13. Baden L.R., El Sahly H.M., Essink B., Kotloff K., Frey S., Novak R., Diemert D., Spector S.A., Rouphael N., Creech C.B., McGettigan J., Khetan S., Segall N., Solis J., Brosz A., Fierro C., Schwartz H., Neuzil K., Corey L., Gilbert P., Janes H., Follmann D., Marovich M., Mascola J., Polakowski L., Ledgerwood J., Graham B.S., Bennett H., Pajon R., Knightly C., Leav B., Deng W., Zhou H., Han S., Ivarsson M., Miller J., Zaks T. Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. New Engl. J. Med. 2021;384(5):403–416. https://doi.org/10.1056/nejmoa2035389

14. Jin L., Zeng X., Liu M., Deng Y., He N. Current progress in gene delivery technology based on chemical methods and nano-carriers. Theranostics. 2014;4(3):240–255. https://doi.org/10.7150/thno.6914

15. Kranz L.M., Diken M., Haas H., Kreiter S., Loquai C., Reuter K.C., Meng M., Fritz D., Vascotto F., Hefesha H., Grunwitz C., Vormehr M., Hüsemann Y., Selmi A., Kuhn A.N., Buck J., Derhovanessian E., Rae R., Attig S., Diekmann J., Jabulowsky R.A., Heesch S., Hassel J., Langguth P., Grabbe S., Huber C., Türeci Ö., Sahin U. Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy. Nature. 2016;534:396–401. https://doi.org/10.1038/nature18300

16. Sayour E.J., Grippin A., De Leon G., Stover B., Rahman M., Karachi A., Wummer B., Moore G., Castillo-Caro P., Fredenburg K., Sarkisian M.R., Huang J., Deleyrolle L.P., Sahay B., Carrera-Justiz S., Mendez-Gomez H.R., Mitchell D.A. Personalized Tumor RNA Loaded Lipid-Nanoparticles Prime the Systemic and Intratumoral Milieu for Response to Cancer Immunotherapy. Nano Lett. 2018;18(10):6195–6206. https://doi.org/10.1021/acs.nanolett.8b02179

17. Melo M., Porter E., Zhang Y., Silva M., Li N., Dobosh B., Liguori A., Skog P., Landais E., Menis S., Sok D., Nemazee D., Schief W.R., Weiss R., Irvine D.J. Immunogenicity of RNA Replicons Encoding HIV Env Immunogens Designed for SelfAssembly into Nanoparticles. Mol. Therapy. 2019;27(12): 2080–2090. https://doi.org/10.1016/j.ymthe.2019.08.007

18. Krienke C., Kolb L., Diken E., Streuber M., Kirchhoff S., Bukur T., Akilli-Öztürk Ö., Kranz L.M., Berger H., Petschenka J., Diken M., Kreiter S., Yogev N., Waisman A., Karikó K., Türeci Ö., Sahin U. A noninflammatory mRNA vaccine for treatment of experimental autoimmune encephalomyelitis. Science. 2021;371(6525):145–153. https://doi.org/10.1126/science.aay3638

19. Bevers S., Kooijmans S.A.A., Van de Velde E., Evers M.J.W., Seghers S., Gitz-Francois J.J.J.M., van Kronenburg N.C.H., Fens M.H.A.M., Mastrobattista E., Hassler L., Sork H., Lehto T., Ahmed K.E., El Andaloussi S., Fiedler K., Breckpot K., Maes M., Van Hoorick D., Bastogne T., Schiffelers R.M., De Koker S. mRNA-LNP vaccines tuned for systemic immunization induce strong antitumor immunity by engaging splenic immune cells. Mol. Therapy. 2022;30(9):3078–3094. https://doi.org/10.1016/j.ymthe.2022.07.007

20. Anderluzzi G., Lou G., Gallorini S., Brazzoli M., Johnson R., O’Hagan D.T., Baudner B.C., Perrie Y. Investigating the impact of delivery system design on the efficacy of selfamplifying RNA vaccines. Vaccines (Basel). 2020;8:212. https://doi.org/10.3390/vaccines8020212

21. Fedorovskiy A.G., Antropov D.N., Dome A.S., Puchkov P.A., Makarova D.M., Konopleva M.V., Matveeva A.M., Panova E.A., Shmendel E.V., Maslov M.A., Dmitriev S.E., Stepanov G.A., Markov O.V. Novel Efficient Lipid-Based Delivery Systems Enable a Delayed Uptake and Sustained Expression of mRNA in Human Cells and Mouse Tissues. Pharmaceutics. 2024;16(5):684. https://doi.org/10.3390/pharmaceutics16050684

22. Cullis P.R., Hope M.J. Lipid Nanoparticle Systems for Enabling Gene Therapies. Mol. Therapy. 2017;25(7): 1467–1475. https://doi.org/10.1016/j.ymthe.2017.03.013

23. Farbiak L., Cheng Q., Wei T., Álvarez-Benedicto E., Johnson L.T., Lee S., Siegwart D.J. All-In-One DendrimerBased Lipid Nanoparticles Enable Precise HDR-Mediated Gene Editing In Vivo. Adv. Mater. 2021;33(30):e2006619. https://doi.org/10.1002/adma.202006619

24. Heyes J., Palmer L., Bremner K., MacLachlan I. Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids. J. Controll. Release. 2005;107(2):276–287. https://doi.org/10.1016/j.jconrel.2005.06.014

25. Sato Y., Hashiba K., Sasaki K., Maeki M., Tokeshi M., Harashima H. Understanding structure-activity relationships of pH-sensitive cationic lipids facilitates the rational identification of promising lipid nanoparticles for delivering siRNAs in vivo. J. Controll. Release. 2019;295:140–152. https://doi.org/10.1016/j.jconrel.2019.01.001

26. Patel S., Ryals R.C., Weller K.K., Pennesi M.E., Sahay G. Lipid nanoparticles for delivery of messenger RNA to the back of the eye. J. Controll. Release. 2019;303:91–100. https://doi.org/10.1016/J.JCONREL.2019.04.015

27. Gote V., Bolla P.K., Kommineni N., Butreddy A., Nukala P.K., Palakurthi S.S., Khan W. A Comprehensive Review of mRNA Vaccines. Int. J. Mol. Sci. 2023;24:2700. https://doi.org/10.3390/ijms24032700

28. Semple S.C., Akinc A., Chen J., Sandhu A.P., Mui B.L., Cho C.K., Sah DW.Y., Stebbing D., Crosley E.J., Yaworski E., Hafez I.M., Dorkin J.R., Qin J., Lam K., Rajeev K.G., Wong K.F., Jeffs L.B., Nechev L., Eisenhardt M.L., Jayaraman M., Kazem M., Maier M.A., Srinivasulu M., Weinstein M.J., Chen Q., Alvarez R., Barros S.A., De S., Klimuk S.K., Borland T., Kosovrasti V., Cantley W.L., Tam Y.K., Manoharan M., Ciufolini M.A., Tracy M.A., De Fougerolles A., MacLachlan I., Cullis P.R., Madden T.D., Hope M.J. Rational design of cationic lipids for siRNA delivery. Nat. Biotechnol. 2010;28:172–176. https://doi.org/10.1038/nbt.1602

29. McKay P.F., Hu K., Blakney A.K., Samnuan K., Brown J.C., Penn R., Zhou J., Bouton C.R., Rogers P., Polra K., Lin P.J.C., Barbosa C., Tam Y.K., Barclay W.S., Shattock R.J. Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine candidate induces high neutralizing antibody titers in mice. Nat. Commun. 2020;11:3523. https://doi.org/10.1038/S41467-020-17409-9

30. Akinc A., Maier M.A., Manoharan M., Fitzgerald K., Jayaraman M., Barros S., Ansell S., Du X., Hope M.J., Madden T.D., Mui B.L., Semple S.C., Tam Y.K., Ciufolini M., Witzigmann D., Kulkarni J.A., van der Meel R., Cullis P.R. The Onpattro story and the clinical translation of nanomedicines containing nucleic acid-based drugs. Nat. Nanotechnol. 2019;14:1084–1087. https://doi.org/10.1038/s41565-019-0591-y

31. Zhang M., Sun J., Li M., Jin X. Modified mRNA-LNP Vaccines Confer Protection against Experimental DENV-2 Infection in Mice. Mol. Ther. Methods Clin. Dev. 2020;18:702–712. https://doi.org/10.1016/j.omtm.2020.07.013

32. Sabnis S., Kumarasinghe E.S., Salerno T., Mihai C., Ketova T., Senn J.J., Lynn A., Bulychev A., McFadyen I., Chan J., Almarsson Ö., Stanton M.G., Benenato K.E. A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates. Mol. Therapy. 2018;26(6):1509–1519. https://doi.org/10.1016/j.ymthe.2018.03.010

33. Huo H., Cheng X., Xu J., Lin J., Chen N., Lu X. A fluorinated ionizable lipid improves the mRNA delivery efficiency of lipid nanoparticles. J. Mater. Chem. B. 2023;11(19):4171–4180. https://doi.org/10.1039/d3tb00516j

34. Corbett K.S., Edwards D.K., Leist S.R., Abiona O.M., Boyoglu-Barnum S., Gillespie R.A., Himansu S., Schäfer A., Ziwawo C.T., DiPiazza A.T., Dinnon K.H., Elbashir S.M., Shaw C.A., Woods A., Fritch E.J., Martinez D.R., Bock K.W., Minai M., Nagata B.M., Hutchinson G.B., Wu K., Henry C., Bahl K., Garcia-Dominguez D., Ma L.Z., Renzi I., Kong W.P., Schmidt S.D., Wang L., Zhang Y., Phung E., Chang L.A., Loomis R.J., Altaras N.E., Narayanan E., Metkar M., Presnyak V., Liu C., Louder M.K., Shi W., Leung K., Yang E.S., West A., Gully K.L., Stevens L.J., Wang N., Wrapp D., Doria-Rose N.A., Stewart-Jones G., Bennett H., Alvarado G.S., Nason M.C., Ruckwardt T.J., McLellan J.S., Denison M., Chappell J.D., Moore I.N., Morabito K.M., Mascola J.R., Baric R.S., Carfi A., Graham B.S. SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness. Nature. 2020;586:567–571. https://doi.org/10.1038/s41586-020-2622-0

35. Tanaka H., Sakurai Y., Anindita J., Akita H. Development of lipid-like materials for RNA delivery based on intracellular environment-responsive membrane destabilization and spontaneous collapse. Adv. Drug. Deliv. Rev. 2020; 154-155:210–226. https://doi.org/10.1016/j.addr.2020.07.001

36. Buschmann M.D., Carrasco M.J., Alishetty S., Paige M., Alameh M.G., Weissman D. Nanomaterial delivery systems for mRNA vaccines. Vaccines (Basel). 2021;9(1):65. https://doi.org/10.3390/vaccines9010065

37. Hassett K.J., Higgins J., Woods A., Levy B., Xia Y., Hsiao C.J., Acosta E., Almarsson Ö., Moore M.J., Brito L.A. Impact of lipid nanoparticle size on mRNA vaccine immunogenicity. J. Controll. Release. 2021;335:237–246. https://doi.org/10.1016/j.jconrel.2021.05.021

38. Vogel A.B., Kanevsky I., Che Y., Swanson K.A., Muik A., Vormehr M., Kranz L.M., Walzer K.C., Hein S., Güler A., Loschko J., Maddur M.S., Ota-Setlik A., Tompkins K., Cole J., Lui B.G., Ziegenhals T., Plaschke A., Eisel D., Dany S.C., Fesser S., Erbar S., Bates F., Schneider D., Jesionek B., Sänger B., Wallisch A.K., Feuchter Y., Junginger H., Krumm S.A., Heinen A.P., Adams-Quack P., Schlereth J., Schille S., Kröner C., de la Caridad Güimil Garcia R., Hiller T., Fischer L., Sellers R.S., Choudhary S., Gonzalez O., Vascotto F., Gutman M.R., Fontenot J.A., Hall-Ursone S., Brasky K., Griffor M.C., Han S., Su A.A.H., Lees J.A., Nedoma N.L., Mashalidis E.H., Sahasrabudhe P.V., Tan C.Y., Pavliakova D., Singh G., Fontes-Garfias C., Pride M., Scully I.L., Ciolino T., Obregon J., Gazi M., Carrion R., Alfson K.J., Kalina W.V., Kaushal D., Shi P.Y., Klamp T., Rosenbaum C., Kuhn A.N., Türeci Ö., Dormitzer P.R., Jansen K.U., Sahin U. BNT162b vaccines protect rhesus macaques from SARS-CoV-2. Nature. 2021;592:283–289. https://doi.org/10.1038/s41586-021-03275-y

39. Panova E.A., Kleymenov D.A., Shcheblyakov D.V., Bykonia E.N., Mazunina E.P., Dzharullaeva A.S., Zolotar A.N., Derkaev A.A., Esmagambetov I.B., Sorokin I.I., Usachev E.V., Noskov A.N., Ivanov I.A., Zatsepin T.S., Dmitriev S.E., Gushchin V.A., Naroditsky B.S., Logunov D.Y., Gintsburg A.L. Single-domain antibody delivery using an mRNA platform protects against lethal doses of botulinum neurotoxin A. Front. Immunol. 2023;14:1098302. https://doi.org/10.3389/fimmu.2023.1098302

40. Mazunina E.P., Gushchin V.A., Bykonia E.N., Kleymenov D.A., Siniavin A.E., Kozlova S.R., Mukasheva E.A., Shidlovskaya E.V., Kuznetsova N.A., Usachev E.V., Zlobin V.I., Burtseva E.I., Ivanov R.A., Logunov D.Y., Gintsburg A.L. Immunogenicity and Efficacy of Combined mRNA Vaccine Against Influenza and SARS-CoV-2 in Mice Animal Models. Vaccines. 2024;12(11): 1206. https://doi.org/10.3390/VACCINES12111206

41. Mazunina E.P., Gushchin V.A., Kleymenov D.A., Siniavin A.E., Burtseva E.I., Shmarov M.M., Mukasheva E.A., Bykonia E.N., Kozlova S.R, Evgrafova E.A., Zolotar A.N., Shidlovskaya E.V., Kirillova E.S., Krepkaia A.S., Usachev E.V., Kuznetsova N.A., Ivanov I.A., Dmitriev S.E., Ivanov R.A., Logunov D.Y., Gintsburg A.L. Trivalent mRNA vaccine-candidate against seasonal flu with cross-specific humoral immune response. Front. Immunol. 2024;15:1381508. https://doi.org/10.3389/fimmu.2024.1381508

42. Miao L., Lin J., Huang Y., Li L., Delcassian D., Ge Y., Shi Y., Anderson D.G. Synergistic lipid compositions for albumin receptor mediated delivery of mRNA to the liver. Nat. Commun. 2020;11:2424. https://doi.org/10.1038/s41467-020-16248-y

43. Miao L., Zhang Y., Huang L. mRNA vaccine for cancer immunotherapy. Mol. Cancer. 2021;20:41. https://doi.org/10.1186/s12943-021-01335-5

44. Lokugamage M.P., Vanover D., Beyersdorf J., Hatit M.Z.C., Rotolo L., Echeverri E.S., Peck HE., Ni H., Yoon J.K., Kim Y.T., Santangelo P.J., Dahlman J.E. Optimization of lipid nanoparticles for the delivery of nebulized therapeutic mRNA to the lungs. Nat. Biomed. Eng. 2021;5:1059–1068. https://doi.org/10.1038/s41551-021-00786-x

45. Ogata F.T., Branco V., Vale F.F., Coppo L. Glutaredoxin: Discovery, redox defense and much more. Redox Biol. 2021;43: 101975. https://doi.org/10.1016/J.REDOX.2021.101975

46. Begas P., Liedgens L., Moseler A., Meyer A.J., Deponte M. Glutaredoxin catalysis requires two distinct glutathione interaction sites. Nat. Commun. 2017;8:14835. https://doi.org/10.1038/ncomms14835

47. Gallogly M.M., Starke D.W., Mieyal J.J. Mechanistic and kinetic details of catalysis of thiol-disulfide exchange by glutaredoxins and potential mechanisms of regulation. Antioxid Redox Signal. 2009;11(5):1059–1081. https://doi.org/10.1089/ars.2008.2291

48. Holmgren A. Thioredoxin. Annu. Rev. Biochem. 1985;54: 237–271. https://doi.org/10.1146/annurev.bi.54.070185.001321

49. Powis G., Montfort W.R. Properties and biological activities of thioredoxins. Annu. Rev. Pharmacol. Toxicol. 2001;41: 261–295. https://doi.org/10.1146/annurev.pharmtox.41.1.261

50. Luster A.D., Weinshank R.L., Feinman R., Ravetch J.V. Molecular and biochemical characterization of a novel gamma-interferoninducible protein. J. Biol. Chem. 1988;263(24):12036–12043. https://doi.org/10.1016/S0021-9258(18)37889-X

51. Arunachalam B., Phan U.T., Geuze H.J., Cresswell P. Enzymatic reduction of disulfide bonds in lysosomes: Characterization of a Gamma-interferon-inducible lysosomal thiol reductase (GILT). Proc. Natl. Acad. Sci. USA. 2000;97(2):745–750. https://doi.org/10.1073/pnas.97.2.745

52. Shirazi R.S., Ewert K.K., Leal C., Majzoub R.N., Bouxsein N.F., Safinya C.R. Synthesis and characterization of degradable multivalent cationic lipids with disulfide-bond spacers for gene delivery. Biochim. Biophys. Acta Biomembr. 2011;1808(9): 2156–2166. https://doi.org/10.1016/j.bbamem.2011.04.020

53. Tanaka H., Takahashi T., Konishi M., Takata N., Gomi M., Shirane D., Miyama R., Hagiwara S., Yamasaki Y., Sakurai Y., Ueda K., Higashi K., Moribe K., Shinsho E., Nishida R., Fukuzawa K., Yonemochi E., Okuwaki K., Mochizuki Y., Nakai Y., Tange K., Yoshioka H., Tamagawa S., Akita H. Self-Degradable Lipid-Like Materials Based on “Hydrolysis accelerated by the intraParticle Enrichment of Reactant (HyPER)” for Messenger RNA Delivery. Adv. Funct. Mater. 2020;30(34):1910575. https://doi.org/10.1002/adfm.201910575

54. Akita H., Ishiba R., Togashi R., Tange K., Nakai Y., Hatakeyama H., Harashima H. A neutral lipid envelope-type nanoparticle composed of a pH-activated and vitamin E-scaffold lipid-like material as a platform for a gene carrier targeting renal cell carcinoma. J. Controll. Release. 2015;200:97–105. https://doi.org/10.1016/j.jconrel.2014.12.029

55. Miyabe H., Hyodo M., Nakamura T., Sato Y., Hayakawa Y., Harashima H. A new adjuvant delivery system “cyclic di-GMP/YSK05 liposome” for cancer immunotherapy. J. Controll. Release. 2014;184:20–27. https://doi.org/10.1016/j.jconrel.2014.04.004

56. Shobaki N., Sato Y., Harashima H. Mixing lipids to manipulate the ionization status of lipid nanoparticles for specific tissue targeting. Int. J. Nanomedicine. 2018;13:8395–8410. https://doi.org/10.2147/IJN.S188016

57. Warashina S., Nakamura T., Sato Y., Fujiwara Y., Hyodo M., Hatakeyama H., Harashima H. A lipid nanoparticle for the efficient delivery of siRNA to dendritic cells. J. Controll. Release. 2016;225:183–191. https://doi.org/10.1016/j.jconrel.2016.01.042

58. Shobaki N., Sato Y., Suzuki Y., Okabe N., Harashima H. Manipulating the function of tumor-associated macrophages by siRNA-loaded lipid nanoparticles for cancer immunotherapy. J. Controll. Release. 2020;325:235–248. https://doi.org/10.1016/j.jconrel.2020.07.001

59. Hashiba K., Taguchi M., Sakamoto S., Otsu A., Maeda Y., Ebe H., Okazaki A., Harashima H., Sato Y. Overcoming thermostability challenges in mRNA–lipid nanoparticle systems with piperidinebased ionizable lipids. Commun. Biol. 2024;7:556. https://doi.org/10.1038/s42003-024-06235-0

60. Akinc A., Goldberg M., Qin J., Dorkin J.R., Gamba-Vitalo C., Maier M., Jayaprakash K.N., Jayaraman M., Rajeev K.G., Manoharan M., Koteliansky V., Röhl I., Leshchiner E.S., Langer R., Anderson D.G. Development of lipidoid-siRNA formulations for systemic delivery to the liver. Mol. Therapy. 2009;17(5):872–879. https://doi.org/10.1038/mt.2009.36

61. Akinc A., Zumbuehl A., Goldberg M., Leshchiner E.S., Busini V., Hossain N., Bacallado S.A., Nguyen D.N., Fuller J., Alvarez R., Borodovsky A., Borland T., Constien R., De Fougerolles A., Dorkin J.R., Jayaprakash K.N., Jayaraman M., John M., Koteliansky V., Manoharan M., Nechev L., Qin J., Racie T., Raitcheva D., Rajeev K.G., Sah D.W.Y., Soutschek J., Toudjarska I., Vornlocher H.P., Zimmermann T.S., Langer R., Anderson D.G. A combinatorial library of lipid-like materials for delivery of RNAi therapeutics. Nat. Biotechnol. 2008;26:561–569. https://doi.org/10.1038/nbt1402

62. Love K.T., Mahon K.P., Levins C.G., Whitehead K.A., Querbes W., Dorkin J.R., Qin J., Cantley W., Qin L.L., Racie T., Frank-Kamenetsky M., Yip K.N., Alvarez R., Sah D.W.Y., De Fougerolles A., Fitzgerald K., Koteliansky V., Akinc A., Langer R., Anderson D.G. Lipid-like materials for low-dose, in vivo gene silencing. Proc. Natl. Acad. Sci. USA. 2010;107(5): 1864–1869. https://doi.org/10.1073/pnas.0910603106

63. Turnbull I.C., Eltoukhy A.A., Fish K.M., Nonnenmacher M., Ishikawa K., Chen J., Hajjar R.J., Anderson D.G., Costa K.D. Myocardial delivery of lipidoid nanoparticle carrying modRNA induces rapid and transient expression. Mol. Therapy. 2016;24(1):66–75. https://doi.org/10.1038/mt.2015.193

64. Jiang C., Mei M., Li B., Zhu X., Zu W., Tian Y., Wang Q., Guo Y., Dong Y., Tan X. A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo. Cell Res. 2017;27:440–443. https://doi.org/10.1038/cr.2017.16

65. Liu S., Cheng Q., Wei T., Yu X., Johnson L.T., Farbiak L., Siegwart D.J. Membrane-destabilizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR–Cas gene editing. Nat. Mater. 2021;20:701–710. https://doi.org/10.1038/s41563-020-00886-0

66. Li B., Luo X., Deng B., Wang J., McComb D.W., Shi Y., Gaensler K.M.L., Tan X., Dunn A.L., Kerlin B.A., Dong Y. An Orthogonal Array Optimization of Lipid-like Nanoparticles for mRNA Delivery in Vivo. Nano Lett. 2015;15(12): 8099–8107. https://doi.org/10.1021/acs.nanolett.5b03528

67. Zeng C., Hou X., Yan J., Zhang C., Li W., Zhao W., Du S., Dong Y. Leveraging mRNA Sequences and Nanoparticles to Deliver SARS-CoV-2 Antigens In Vivo. Adv. Mater. 2020;32(40):е2004452. https://doi.org/10.1002/adma.202004452

68. Gindy M.E., Feuston B., Glass A., Arrington L., Haas R.M., Schariter J., Stirdivant S.M. Stabilization of Ostwald ripening in low molecular weight amino lipid nanoparticles for systemic delivery of siRNA Therapeutics. Mol. Pharm. 2014;11(11):4143–4153. https://doi.org/10.1021/mp500367k

69. Suzuki Y., Hyodo K., Tanaka Y., Ishihara H. siRNA-lipid nanoparticles with long-term storage stability facilitate potent gene-silencing in vivo. J. Controll. Release. 2015;220:44–50. https://doi.org/10.1016/J.JCONREL.2015.10.024

70. Suzuki Y., Hyodo K., Suzuki T., Tanaka Y., Kikuchi H., Ishihara H. Biodegradable lipid nanoparticles induce a prolonged RNA interference-mediated protein knockdown and show rapid hepatic clearance in mice and nonhuman primates. Int. J. Pharm. 2017;519(1-2):34–43. https://doi.org/10.1016/j.ijpharm.2017.01.016

71. Lokugamage M.P., Sago C.D., Gan Z., Krupczak B.R., Dahlman J.E. Constrained Nanoparticles Deliver siRNA and sgRNA to T Cells In Vivo without Targeting Ligands. Adv. Mater. 2019;31(41):е1902251. https://doi.org/10.1002/adma.201902251

72. Ni H., Hatit M.Z.C., Zhao K., Loughrey D., Lokugamage M.P., Peck H.E., Del Cid A., Muralidharan A., Kim Y.T., Santangelo P.J., Dahlman J.E. Piperazinederived lipid nanoparticles deliver mRNA to immune cells in vivo. Nat. Commun. 2022;13(1):4766. https://doi.org/10.1038/s41467-022-32281-5

73. Miao L., Li L., Huang Y., Delcassian D., Chahal J., Han J., Shi Y., Sadtler K., Gao W., Lin J., Doloff J.C., Langer R., Anderson D.G. Delivery of mRNA vaccines with heterocyclic lipids increases anti-tumor efficacy by STING-mediated immune cell activation. Nat. Biotechnol. 2019;37:1174–1185. https://doi.org/10.1038/s41587-019-0247-3

74. Hou X., Zhang X., Zhao W., Zeng C., Deng B., McComb D.W., Du S., Zhang C., Li W., Dong Y. Vitamin lipid nanoparticles enable adoptive macrophage transfer for the treatment of multidrug-resistant bacterial sepsis. Nat. Nanotechnol. 2020;15(1):41–46. https://doi.org/10.1038/s41565-019-0600-1

75. Ho W., Gao M., Li F., Li Z., Zhang X.Q., Xu X. NextGeneration Vaccines: Nanoparticle-Mediated DNA and mRNA Delivery. Adv. Healthc. Mater. 2021;10(8):e2001812. https://doi.org/10.1002/adhm.202001812

76. Knezevic I., Liu M.A., Peden K., Zhou T., Kang H.N. Development of mRNA Vaccines: Scientific and Regulatory Issues. Vaccines (Basel). 2021;9(2):81. https://doi.org/10.3390/VACCINES9020081

77. Bauman J., Burris H., Clarke J., Patel M., Cho D., Gutierrez M., Julian R., Scott A., Cohen P., Frederick J., Robert-Tissot C., Zhou H., Mody K., Keating K., Meehan R., Gainor J. 798 Safety, tolerability, and immunogenicity of mRNA-4157 in combination with pembrolizumab in subjects with unresectable solid tumors (KEYNOTE-603): an update. J. ImmunoTher. Cancer. 2020;8(Suppl.3):0798. https://doi.org/10.1136/JITC-2020-SITC2020.0798.

78. Soens M., Ananworanich J., Hicks B., Lucas K.J., Cardona J., Sher L., Livermore G., Schaefers K., Henry C., Choi A., Avanesov A., Chen R., Du E., Pucci A., Das R., Miller J., Nachbagauer R. A phase 3 randomized safety and immunogenicity trial of mRNA-1010 seasonal influenza vaccine in adults. Vaccine. 2025;50:126847. https://doi.org/10.1016/J.VACCINE.2025.126847

79. Shaw C., Panther L., August A., Zaks T., Smolenov I., Bart S., Watson M. Safety and immunogenicity of a mRNA-based chikungunya vaccine in a phase 1 doseranging trial. Int. J. Infectious Dis. 2019;79(Suppl. 1):17. https://doi.org/10.1016/j.ijid.2018.11.058

80. Feldman R.A., Fuhr R., Smolenov I., Ribeiro A. (Mick)., Panther L., Watson M., Senn J.J., Smith M., Almarsson Ӧ., Pujar H.S., Laska M.E., Thompson J., Zaks T., Ciaramella G. mRNA vaccines against H10N8 and H7N9 influenza viruses of pandemic potential are immunogenic and well tolerated in healthy adults in phase 1 randomized clinical trials. Vaccine. 2019;37(25):3326–3334. https://doi.org/10.1016/J.VACCINE.2019.04.074

81. Gillmore J.D., Gane E., Taubel J., Kao J., Fontana M., Maitland M.L., Seitzer J., O’Connell D., Walsh K.R., Wood K., Phillips J., Xu Y., Amaral A., Boyd A.P., Cehelsky J.E., McKee M.D., Schiermeier A., Harari O., Murphy A., Kyratsous C.A., Zambrowicz B., Soltys R., Gutstein D.E., Leonard J., Sepp-Lorenzino L., Lebwohl D. CRISPR-Cas9 in vivo gene editing for transthyretin amyloidosis. N. Engl. J. Med. 2021;385(6):493–502. https://doi.org/10.1056/nejmoa2107454

82. Soroudi S., Jaafari M.R., Arabi L. Lipid nanoparticle (LNP) mediated mRNA delivery in cardiovascular diseases: Advances in genome editing and CAR T cell therapy. J. Controll. Release. 2024;372:113–140. https://doi.org/10.1016/J.JCONREL.2024.06.023

83. Puthanakit T., Prompetchara E., Gatechompol S., Ketloy C., Thitithanyanont A., Jongkaewwattana A., Buranapraditkun S., Ubolyam S., Kerr S.J., Sophonphan J., Apornpong T., Kittanamongkolchai W., Siwamogsatham S., Sriplienchan S., Patarakul K., Theerawit T., Promsena P., Nantanee R., Manomaisantiphap S., Chokyakorn S., Hong L., Samija M., Montefiori D.C., Gao H., Eaton A., Wijagkanalan W., Alameh M.G., Weissman D., Ruxrungtham K., Tawan M., Sutjarit A., Meepuksom T., Athipunjapong J., Jupimai T., Moonwong J., Nadsasarn R., Khamthi S., Nuncharoen P., Chanpoom Y., Khamkhen P., Narupan N., Thongthip S., Soisoongnern K., Shanyip C., Rachpradit T., Sriphraram K., Somhanwong W., Boondamnern T., Boonnak N., Chomchey N., Tipsuk S., Puttamaswin S., Yewande S., Lertarom P., Uanithirat A., Anuchadbut A., Chanthaburanun S., Tarawat K., Mahanontharit A., Sinthon W., Plakunmonthonw S., Wongmueang S., Dalodom T., Sopa B., Phongam N., Sri-Arunsak A., Chobkarching U., Bouko C., Junseeha S., Phuphalicho B., Pingthaisong P., Khlaiphuengsin A., Pararit P., Eamyoung P., Somjit T., Iampornsin T., Thongchomphunut D., Manopwisedjaroen S., Laopanupong T., Ekronarongchai S., Srisaowakarn C., Jantraphakorn Y., Srisutthisamphan K., Grandin P.V. Phase II prefusion non-stabilised Covid-19 mRNA vaccine randomised study. Sci. Rep. 2024;14(1):2373. https://doi.org/10.1038/s41598-023-49653-6