Downloads

doi: https://doi.org/10.53941/rmd.2024.100003
Li, W., Wang, T., Chen, J., Guo, M., Ling, L., & Higuchi, A. Application of Saccharide Cryoprotectants in the Freezing or Lyophilization Process of Lipid Nanoparticles Encapsulating Gene Drugs for Regenerative Medicine. Regenerative Medicine and Dentistry. 2024. doi: https://doi.org/10.53941/rmd.2024.100003
Forthcoming Issue
Copyright (c) 2024 by the authors.
Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 International License.

Article

Application of Saccharide Cryoprotectants in the Freezing or Lyophilization Process of Lipid Nanoparticles Encapsulating Gene Drugs for Regenerative Medicine

Wanqi Li 1,†, Ting Wang 1,†, Jianyang Chen 1, Minmei Guo 1, Ling Ling 1 and Akon Higuchi 1,2,3,*

1 State Key Laboratory of Ophthalmology, Optometry and Visual Science, Eye Hospital, Wenzhou Medical University,
No. 270, Xueyuan Road, Wenzhou 325027, China

2 Department of Chemical and Materials Engineering, National Central University, No. 300, Jhongda RD., Jhongli District,
Taoyuan 32001, Taiwan

3 R&D Center for Membrane Technology, Chung Yuan Christian University, Chungli District, Taoyuan 32023, Taiwan

* Correspondence: higuchi@ncu.edu.tw or higuchi@wmu.edu.cn

† These authors contributed equally to this work.

Received: 14 November 2024; Revised: 15 December 2024; Accepted: 16 December 2024; Published: 20 December 2024

 

Abstract: Lipid nanoparticles (LNPs) have emerged as highly efficient drug delivery systems in gene therapy and regenerative medicine and have demonstrated great potential in recent years. Notably, LNPs encapsulating mRNA vaccines have achieved remarkable success in combating the COVID-19 epidemic. However, LNPs encapsulating mRNA encounter issues of physical and chemical instability and need to be stored and transported under harsh conditions. Lyophilization technology, which is commonly used to increase the stability of nanomedicines, has been increasingly applied to stabilize mRNA-LNPs. Appropriate cryoprotectants, such as saccharides, glycerin, and dimethyl sulfoxide (DMSO), need to be added to mRNA-LNPs during the freezing or lyophilization process to effectively preserve the physical and chemical properties of mRNA-LNPs, ensuring their stability. Saccharides (i.e., sucrose, trehalose, and maltose) are the most widely used cryoprotectants to protect the integrity of mRNA-LNPs. This is because saccharides are relatively safe molecules compared with other chemical molecules for cells and animals. However, different saccharides have varying levels of protective effects on mRNA-LNP formulations, and the optimal saccharide concentration varies depending on the specific mRNA-LNP. This article reviews the application and mechanisms of saccharide-based cryoprotectants in the freezing or lyophilization process of LNP-delivered gene therapies and regenerative medicines, offering guidance for selecting the most appropriate saccharide-based cryoprotectants for mRNA-LNP drugs during freezing or lyophilization processes.

Keywords:

cryoprotectants stability lipid nanoparticles drug delivery regenerative medicine

References

  1. Ghaemmaghamian, Z.; Zarghami, R.; Walker, G.; et al. Stabilizing vaccines via drying: Quality by design considerations. Adv. Drug Deliv. Rev. 2022, 187, 114313. https://doi.org/10.1016/j.addr.2022.114313.
  2. To, K.K.W.; Cho, W.C.S. An overview of rational design of mRNA-based therapeutics and vaccines. Expert. Opin. Drug Discov. 2021, 16, 1307–1317. https://doi.org/10.1080/17460441.2021.1935859.
  3. Tenchov, R.; Bird, R.; Curtze, A.E.; et al. Lipid Nanoparticles—From Liposomes to mRNA Vaccine Delivery, a Landscape of Research Diversity and Advancement. ACS Nano 2021, 15, 16982–17015. https://doi.org/10.1021/acsnano.1c04996.
  4. Higuchi, A.; Sung, T.-C.; Wang, T.; et al. Material Design for Next-Generation mRNA Vaccines Using Lipid Nanoparticles. Polym. Rev. 2023, 63, 394–436. https://doi.org/10.1080/15583724.2022.2106490.
  5. Packer, M.; Gyawali, D.; Yerabolu, R.; et al. A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems. Nat. Commun. 2021, 12, 6777. https://doi.org/10.1038/s41467-021-26926-0.
  6. Gilleron, J.; Querbes, W.; Zeigerer, A.; et al. Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Nat. Biotechnol. 2013, 31, 638–646. https://doi.org/10.1038/nbt.2612.
  7. Wang, C.; Pan, C.; Yong, H.; et al. Emerging non-viral vectors for gene delivery. J. Nanobiotechnol. 2023, 21, 272. https://doi.org/10.1186/s12951-023-02044-5.
  8. Santana-Armas, M.L.; de Ilarduya, C.T. Strategies for cancer gene-delivery improvement by non-viral vectors. Int. J. Pharm. 2021, 596, 120291. https://doi.org/10.1016/j.ijpharm.2021.120291.
  9. Khalil, I.A.; Sato, Y.; Harashima, H. Recent advances in the targeting of systemically administered non-viral gene delivery systems. Expert. Opin. Drug Deliv. 2019, 16, 1037–1050. https://doi.org/10.1080/17425247.2019.1656196.
  10. Kulkarni, J.A.; Witzigmann, D.; Chen, S.; et al. Lipid Nanoparticle Technology for Clinical Translation of siRNA Therapeutics. Acc. Chem. Res. 2019, 52, 2435–2444. https://doi.org/10.1021/acs.accounts.9b00368.
  11. Tregoning, J.S.; Flight, K.E.; Higham, S.L.; et al. Progress of the COVID-19 vaccine effort: Viruses, vaccines and variants versus efficacy, effectiveness and escape. Nat. Rev. Immunol. 2021, 21, 626–636. https://doi.org/10.1038/s41577-021-00592-1.
  12. Schoenmaker, L.; Witzigmann, D.; Kulkarni, J.A.; et al. mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability. Int. J. Pharm. 2021, 601, 120586. https://doi.org/10.1016/j.ijpharm.2021.120586.
  13. Xu, C.; Lei, C.; Hosseinpour, S.; et al. Nanotechnology for the management of COVID-19 during the pandemic and in the post-pandemic era. Natl. Sci. Rev. 2022, 9, nwac124. https://doi.org/10.1093/nsr/nwac124.
  14. Liu, T.; Tian, Y.; Zheng, A.; et al. Design Strategies for and Stability of mRNA-Lipid Nanoparticle COVID-19 Vaccines. Polymers 2022, 14, 4195. https://doi.org/10.3390/polym14194195.
  15. Shi, R.; Liu, X.; Wang, Y.; et al. Long-term stability and immunogenicity of lipid nanoparticle COVID-19 mRNA vaccine is affected by particle size. Hum. Vaccin. Immunother. 2024, 20, 2342592. https://doi.org/10.1080/21645515.2024.2342592.
  16. Wang, T.; Sung, T.C.; Yu, T.; et al. Next-generation materials for RNA-lipid nanoparticles: Lyophilization and targeted transfection. J. Mater. Chem. B 2023, 11, 5083–5093. https://doi.org/10.1039/d3tb00308f.
  17. Shirane, D.; Tanaka, H.; Sakurai, Y.; et al. Development of an Alcohol Dilution-Lyophilization Method for the Preparation of mRNA-LNPs with Improved Storage Stability. Pharmaceutics 2023, 15, 1819. https://doi.org/10.3390/pharmaceutics15071819.
  18. Ward, K.R.; Matejtschuk, P. The Principles of Freeze-Drying and Application of Analytical Technologies. Methods Mol. Biol. 2021, 2180, 99–127. https://doi.org/10.1007/978-1-0716-0783-1_3.
  19. Izutsu, K.I. Applications of Freezing and Freeze-Drying in Pharmaceutical Formulations. Adv. Exp. Med. Biol. 2018, 1081, 371–383. https://doi.org/10.1007/978-981-13-1244-1_20.
  20. Trenkenschuh, E.; Friess, W. Freeze-drying of nanoparticles: How to overcome colloidal instability by formulation and process optimization. Eur. J. Pharm. Biopharm. 2021, 165, 345–360. https://doi.org/10.1016/j.ejpb.2021.05.024.
  21. Kafetzis, K.N.; Papalamprou, N.; McNulty, E.; et al. The Effect of Cryoprotectants and Storage Conditions on the Transfection Efficiency, Stability, and Safety of Lipid-Based Nanoparticles for mRNA and DNA Delivery. Adv. Healthc. Mater. 2023, 12, e2203022. https://doi.org/10.1002/adhm.202203022.
  22. Isachenko, V.; Rahimi, G.; Mallmann, P.; et al. Technologies of cryoprotectant-free vitrification of human spermatozoa: Asepticity as criterion of effectiveness. Andrology 2017, 5, 1055–1063. https://doi.org/10.1111/andr.12414.
  23. Murray, K.A.; Gibson, M.I. Chemical approaches to cryopreservation. Nat. Rev. Chem. 2022, 6, 579–593. https://doi.org/10.1038/s41570-022-00407-4.
  24. Jiang, P.; Li, Q.; Liu, B.; et al. Effect of cryoprotectant-induced intracellular ice formation and crystallinity on bactria during cryopreservation. Cryobiology 2023, 113, 104786. https://doi.org/10.1016/j.cryobiol.2023.104786.
  25. Li, L.; Tian, Y.; Li, Z.; et al. Effect of non-permeable cryoprotectant sucrose on the development of spotted knifejaw (Oplegnathus punctatus) embryos. Cryobiology 2023, 112, 104555. https://doi.org/10.1016/j.cryobiol.2023.104555.
  26. Poole, C.F. Chapter 15—Application of thin-layer chromatography to the analysis of saccharides. In Instrumental Thin-Layer Chromatography, 2nd ed.; Poole, C.F., Ed.; Elsevier: Amsterdam, the Netherlands, 2023; pp. 413–435.
  27. Li, J.; Wang, H.; Wang, L.; et al. Stabilization effects of saccharides in protein formulations: A review of sucrose, trehalose, cyclodextrins and dextrans. Eur. J. Pharm. Sci. 2024, 192, 106625. https://doi.org/10.1016/j.ejps.2023.106625.
  28. Lamoot, A.; Lammens, J.; De Lombaerde, E.; et al. Successful batch and continuous lyophilization of mRNA LNP formulations depend on cryoprotectants and ionizable lipids. Biomater. Sci. 2023, 11, 4327–4334. https://doi.org/10.1039/d2bm02031a.
  29. Amis, T.M.; Renukuntla, J.; Bolla, P.K.; et al. Selection of Cryoprotectant in Lyophilization of Progesterone-Loaded Stearic Acid Solid Lipid Nanoparticles. Pharmaceutics 2020, 12, 892. https://doi.org/10.3390/pharmaceutics12090892.
  30. Ball, R.L.; Bajaj, P.; Whitehead, K.A. Achieving long-term stability of lipid nanoparticles: Examining the effect of pH, temperature, and lyophilization. Int. J. Nanomed. 2017, 12, 305–315. https://doi.org/10.2147/ijn.S123062.
  31. Zhao, P.; Hou, X.; Yan, J.; et al. Long-term storage of lipid-like nanoparticles for mRNA delivery. Bioact. Mater. 2020, 5, 358–363. https://doi.org/10.1016/j.bioactmat.2020.03.001.
  32. Ohshima, H.; Miyagishima, A.; Kurita, T.; et al. Freeze-dried nifedipine-lipid nanoparticles with long-term nano-dispersion stability after reconstitution. Int. J. Pharm. 2009, 377, 180–184. https://doi.org/10.1016/j.ijpharm.2009.05.004.
  33. Santonocito, D.; Sarpietro, M.G.; Castelli, F.; et al. Development of Solid Lipid Nanoparticles as Dry Powder: Characterization and Formulation Considerations. Molecules 2023, 28, 1545. https://doi.org/10.3390/molecules28041545.
  34. Kamiya, S.; Kurita, T.; Miyagishima, A.; et al. Preparation of griseofulvin nanoparticle suspension by high-pressure homogenization and preservation of the suspension with saccharides and sugar alcohols. Drug Dev. Ind. Pharm. 2009, 35, 1022–1028. https://doi.org/10.1080/03639040802698786.
  35. Shahruzzaman, M.; Hossain, S.; Ahmed, T.; et al. Chapter 7—Biological macromolecules as antimicrobial agents. In Biological Macromolecules; Nayak, A.K., Dhara, A.K., Pal, D., Eds.; Academic Press: Cambridge, MA, USA, 2022; pp. 165–202.
  36. Cummings, R.D. A periodic table of monosaccharides. Glycobiology 2024, 34, cwad088. https://doi.org/10.1093/glycob/cwad088.
  37. Shahgaldian, P.; Gualbert, J.; Aïssa, K.; et al. A study of the freeze-drying conditions of calixarene based solid lipid nanoparticles. Eur. J. Pharm. Biopharm. 2003, 55, 181–184. https://doi.org/10.1016/s0939-6411(02)00196-0.
  38. Curcio, M.; Blanco-Fernández, B.; Costoya, A.; et al. Glucose cryoprotectant affects glutathione-responsive antitumor drug release from polysaccharide nanoparticles. Eur. J. Pharm. Biopharm. 2015, 93, 281–292. https://doi.org/10.1016/j.ejpb.2015.04.010.
  39. Voci, S.; Gagliardi, A.; Salvatici, M.C.; et al. Influence of the Dispersion Medium and Cryoprotectants on the Physico-Chemical Features of Gliadin- and Zein-Based Nanoparticles. Pharmaceutics 2022, 14, 332. https://doi.org/10.3390/pharmaceutics14020332.
  40. Fonte, P.; Araújo, F.; Seabra, V.; et al. Co-encapsulation of lyoprotectants improves the stability of protein-loaded PLGA nanoparticles upon lyophilization. Int. J. Pharm. 2015, 496, 850–862. https://doi.org/10.1016/j.ijpharm.2015.10.032.
  41. Kim, M.; Jeong, M.; Hur, S.; et al. Engineered ionizable lipid nanoparticles for targeted delivery of RNA therapeutics into different types of cells in the liver. Sci. Adv. 2021, 7, abf4398. https://doi.org/10.1126/sciadv.abf4398.
  42. Wan, J.; Wang, Z.; Wang, L.; et al. Circular RNA vaccines with long-term lymph node-targeting delivery stability after lyophilization induce potent and persistent immune responses. mBio 2024, 15, e0177523. https://doi.org/10.1128/mbio.01775-23.
  43. Maretti, E.; Gioia, F.; Rustichelli, C.; et al. Inflammatory-Targeted Lipid Carrier as a New Nanomaterial to Formulate an Inhaled Drug Delivery System. Molecules 2024, 29, 1616. https://doi.org/10.3390/molecules29071616.
  44. Soares, S.; Fonte, P.; Costa, A.; et al. Effect of freeze-drying, cryoprotectants and storage conditions on the stability of secondary structure of insulin-loaded solid lipid nanoparticles. Int. J. Pharm. 2013, 456, 370–381. https://doi.org/10.1016/j.ijpharm.2013.08.076.
  45. Selvaraj, C.; Dinesh, D.C.; Rajaram, K.; et al. Chapter 3—Macromolecular chemistry: An introduction. In In Silico Approaches to Macromolecular Chemistry; Thomas, M.E., Thomas, J., Thomas, S., et al., Eds.; Elsevier: Amsterdam, the Netherlands, 2023; pp. 71–128.
  46. Ruiz-Matus, S.; Goldstein, P. On the universality of viscosity in supersaturated binary aqueous sugar solutions: Cryopreservation by vitrification. Cryobiology 2024, 115, 104886. https://doi.org/10.1016/j.cryobiol.2024.104886.
  47. Kim, B.; Hosn, R.R.; Remba, T.; et al. Optimization of storage conditions for lipid nanoparticle-formulated self-replicating RNA vaccines. J. Control Release 2023, 353, 241–253. https://doi.org/10.1016/j.jconrel.2022.11.022.
  48. Schwarz, C.; Mehnert, W. Freeze-drying of drug-free and drug-loaded solid lipid nanoparticles (SLN). Int. J. Pharm. 1997, 157, 171–179. https://doi.org/10.1016/s0378-5173(97)00222-6.
  49. Kamiya, S.; Nozawa, Y.; Miyagishima, A.; et al. Physical characteristics of freeze-dried griseofulvin-lipids nanoparticles. Chem. Pharm. Bull. 2006, 54, 181–184. https://doi.org/10.1248/cpb.54.181.
  50. Muramatsu, H.; Lam, K.; Bajusz, C.; et al. Lyophilization provides long-term stability for a lipid nanoparticle-formulated, nucleoside-modified mRNA vaccine. Mol. Ther. 2022, 30, 1941–1951. https://doi.org/10.1016/j.ymthe.2022.02.001.
  51. Elbrink, K.; Van Hees, S.; Holm, R.; et al. Optimization of the different phases of the freeze-drying process of solid lipid nanoparticles using experimental designs. Int. J. Pharm. 2023, 635, 122717. https://doi.org/10.1016/j.ijpharm.2023.122717.
  52. Athaydes Seabra Ferreira, H.; Ricardo Aluotto Scalzo Júnior, S.; de Faria, K.K.S.; et al. Cryoprotectant optimization for enhanced stability and transfection efficiency of pDNA-loaded ionizable lipid nanoparticles. Int. J. Pharm. 2024, 665, 124696. https://doi.org/10.1016/j.ijpharm.2024.124696.
  53. Hu, Y.; Liu, X.; Liu, F.; et al. Trehalose in Biomedical Cryopreservation-Properties, Mechanisms, Delivery Methods, Applications, Benefits, and Problems. ACS Biomater. Sci. Eng. 2023, 9, 1190–1204. https://doi.org/10.1021/acsbiomaterials.2c01225.
  54. Weng, L.; Stott, S.L.; Toner, M. Exploring Dynamics and Structure of Biomolecules, Cryoprotectants, and Water Using Molecular Dynamics Simulations: Implications for Biostabilization and Biopreservation. Annu. Rev. Biomed. Eng. 2019, 21, 1–31. https://doi.org/10.1146/annurev-bioeng-060418-052130.
  55. Kamiya, S.; Kurita, T.; Miyagishima, A.; et al. Physical properties of griseofulvin-lipid nanoparticles in suspension and their novel interaction mechanism with saccharide during freeze-drying. Eur. J. Pharm. Biopharm. 2010, 74, 461–466. https://doi.org/10.1016/j.ejpb.2009.12.004.
  56. Kamiya, S.; Takamatsu, H.; Sonobe, T.; et al. The physicochemical interactive mechanism between nanoparticles and raffinose during freeze-drying. Int. J. Pharm. 2014, 465, 97–101. https://doi.org/10.1016/j.ijpharm.2014.02.033.
  57. Wrigley C.; Corke H.; Walker C.E. Carbohydrate Metabolism. In Encyclopedia of Grain Science; Elsevier Academic Press: Oxford, UK, 2004; pp. 168–179.
  58. Zhang, H.; Zhang, F.M.; Yan, S.J. Preparation, in vitro release, and pharmacokinetics in rabbits of lyophilized injection of sorafenib solid lipid nanoparticles. Int. J. Nanomed. 2012, 7, 2901–2910. https://doi.org/10.2147/ijn.S32415.
  59. Sarode, A.; Patel, P.; Vargas-Montoya, N.; et al. Inhalable dry powder product (DPP) of mRNA lipid nanoparticles (LNPs) for pulmonary delivery. Drug Deliv. Transl. Res. 2024, 14, 360–372. https://doi.org/10.1007/s13346-023-01402-y.
  60. Payton, N.M.; Wempe, M.F.; Xu, Y.; et al. Long-term storage of lyophilized liposomal formulations. J. Pharm. Sci. 2014, 103, 3869–3878. https://doi.org/10.1002/jps.24171.
  61. Nail, S.L.; Jiang, S.; Chongprasert, S.; Knopp, S.A. Fundamentals of freeze-drying. Pharm. Biotechnol. 2002, 14, 281–360. https://doi.org/10.1007/978-1-4615-0549-5_6.
  62. Bouchard, A.; Jovanović, N.; Hofland, G.W.; et al. Supercritical fluid drying of carbohydrates: Selection of suitable excipients and process conditions. Eur. J. Pharm. Biopharm. 2008, 68, 781–794. https://doi.org/10.1016/j.ejpb.2007.06.019.
  63. Pegg, D.E. The history and principles of cryopreservation. Semin. Reprod. Med. 2002, 20, 5–13. https://doi.org/10.1055/s-2002-23515.
  64. Pegg, D.E. Principles of cryopreservation. Methods Mol. Biol. 2015, 1257, 3–19. https://doi.org/10.1007/978-1-4939-2193-5_1.
  65. Franzé, S.; Selmin, F.; Samaritani, E.; et al. Lyophilization of Liposomal Formulations: Still Necessary, Still Challenging. Pharmaceutics 2018, 10, 139. https://doi.org/10.3390/pharmaceutics10030139.
  66. Ingvarsson, P.T.; Yang, M.; Nielsen, H.M.; et al. Stabilization of liposomes during drying. Expert. Opin. Drug Deliv. 2011, 8, 375–388. https://doi.org/10.1517/17425247.2011.553219.
  67. Luo, W.C.; Zhang, W.; Kim, R.; et al. Impact of controlled ice nucleation and lyoprotectants on nanoparticle stability during Freeze-drying and upon storage. Int. J. Pharm. 2023, 641, 123084. https://doi.org/10.1016/j.ijpharm.2023.123084.
  68. Pegg, D.E. Principles of cryopreservation. Methods Mol. Biol. 2007, 368, 39–57. https://doi.org/10.1007/978-1-59745-362-2_3.
  69. Boafo, G.F.; Magar, K.T.; Ekpo, M.D.; et al. The Role of Cryoprotective Agents in Liposome Stabilization and Preservation. Int. J. Mol. Sci. 2022, 23, 12487. https://doi.org/10.3390/ijms232012487.
  70. Andersen, H.D.; Wang, C.; Arleth, L.; et al. Reconciliation of opposing views on membrane-sugar interactions. Proc. Natl. Acad. Sci. USA 2011, 108, 1874–1878. https://doi.org/10.1073/pnas.1012516108.
  71. Sydykov, B.; Oldenhof, H.; de Oliveira Barros, L.; et al. Membrane permeabilization of phosphatidylcholine liposomes induced by cryopreservation and vitrification solutions. Biochim. Biophys. Acta Biomembr. 2018, 1860, 467–474. https://doi.org/10.1016/j.bbamem.2017.10.031.
  72. Yu, J.Y.; Chuesiang, P.; Shin, G.H.; et al. Post-Processing Techniques for the Improvement of Liposome Stability. Pharmaceutics 2021, 13, 1023. https://doi.org/10.3390/pharmaceutics13071023.
  73. You, X.; Lee, E.; Xu, C.; et al. Molecular Mechanism of Cell Membrane Protection by Sugars: A Study of Interfacial H-Bond Networks. J. Phys. Chem. Lett. 2021, 12, 9602–9607. https://doi.org/10.1021/acs.jpclett.1c02451.
  74. Golovina, E.A.; Golovin, A.; Hoekstra, F.A.; et al. Water replacement hypothesis in atomic details: Effect of trehalose on the structure of single dehydrated POPC bilayers. Langmuir 2010, 26, 11118–11126. https://doi.org/10.1021/la100891x.
  75. Deng, L.; Wang, Y.; Jiang, H.; et al. Specific protection mechanism of oligosaccharides on liposomes during freeze-drying. Food Res. Int. 2023, 166, 112608. https://doi.org/10.1016/j.foodres.2023.112608.
  76. Aksan, A.; Toner, M. Isothermal desiccation and vitrification kinetics of trehalose-dextran solutions. Langmuir 2004, 20, 5521–5529. https://doi.org/10.1021/la0355186.
  77. Jain, P.; Sen, S.; Risbud, S.H. Effect of glass-forming biopreservatives on head group rotational dynamics in freeze-dried phospholipid bilayers: A 31P NMR study. J. Chem. Phys. 2009, 131, 025102. https://doi.org/10.1063/1.3170927.
  78. Wowk, B. Thermodynamic aspects of vitrification. Cryobiology 2010, 60, 11–22. https://doi.org/10.1016/j.cryobiol.2009.05.007.
  79. Fahy, G.M.; Wowk, B. Principles of Ice-Free Cryopreservation by Vitrification. Methods Mol. Biol. 2021, 2180, 27–97. https://doi.org/10.1007/978-1-0716-0783-1_2.
  80. Koster, K.L.; Webb, M.S.; Bryant, G.; et al. Interactions between soluble sugars and POPC (1-palmitoyl-2-oleoylphosphatidylcholine) during dehydration: Vitrification of sugars alters the phase behavior of the phospholipid. Biochim. Biophys. Acta 1994, 1193, 143–150. https://doi.org/10.1016/0005-2736(94)90343-3.
  81. Li, D.X.; Liu, B.L.; Liu, Y.S.; et al. Predict the glass transition temperature of glycerol-water binary cryoprotectant by molecular dynamic simulation. Cryobiology 2008, 56, 114–119. https://doi.org/10.1016/j.cryobiol.2007.11.003.
  82. Hunt, C.J. Cryopreservation: Vitrification and Controlled Rate Cooling. Methods Mol. Biol. 2017, 1590, 41–77. https://doi.org/10.1007/978-1-4939-6921-0_5.
  83. Elliott, G.D.; Wang, S.; Fuller, B.J. Cryoprotectants: A review of the actions and applications of cryoprotective solutes that modulate cell recovery from ultra-low temperatures. Cryobiology 2017, 76, 74–91. https://doi.org/10.1016/j.cryobiol.2017.04.004.
  84. Teo, S.P. Review of COVID-19 mRNA Vaccines: BNT162b2 and mRNA-1273. J. Pharm. Pract. 2022, 35, 947–951. https://doi.org/10.1177/08971900211009650.
  85. Suzuki, Y.; Ishihara, H. Difference in the lipid nanoparticle technology employed in three approved siRNA (Patisiran) and mRNA (COVID-19 vaccine) drugs. Drug Metab. Pharmacokinet. 2021, 41, 100424. https://doi.org/10.1016/j.dmpk.2021.100424.
  86. Pilkington, E.H.; Suys, E.J.A.; Trevaskis, N.L.; et al. From influenza to COVID-19: Lipid nanoparticle mRNA vaccines at the frontiers of infectious diseases. Acta Biomater. 2021, 131, 16–40. https://doi.org/10.1016/j.actbio.2021.06.023.
  87. Kumar, S.; Gokhale, R.; Burgess, D.J. Sugars as bulking agents to prevent nano-crystal aggregation during spray or freeze-drying. Int. J. Pharm. 2014, 471, 303–311. https://doi.org/10.1016/j.ijpharm.2014.05.060.
  88. Alejo, T.; Toro-Córdova, A.; Fernández, L.; et al. Comprehensive Optimization of a Freeze-Drying Process Achieving Enhanced Long-Term Stability and In Vivo Performance of Lyophilized mRNA-LNPs. Int. J. Mol. Sci. 2024, 25, 10603. https://doi.org/10.3390/ijms251910603.
  89. Khan, A.A.; Abdulbaqi, I.M.; Abou Assi, R.; et al. Lyophilized Hybrid Nanostructured Lipid Carriers to Enhance the Cellular Uptake of Verapamil: Statistical Optimization and In Vitro Evaluation. Nanoscale Res. Lett. 2018, 13, 323. https://doi.org/10.1186/s11671-018-2744-6.
  90. Jakubek, Z.J.; Chen, S.; Zaifman, J.; et al. Lipid Nanoparticle and Liposome Reference Materials: Assessment of Size Homogeneity and Long-Term −70 °C and 4 °C Storage Stability. Langmuir 2023, 39, 2509–2519. https://doi.org/10.1021/acs.langmuir.2c02657.
  91. Wang, T.; Yu, T.; Li, W.; et al. Design and lyophilization of mRNA-encapsulating lipid nanoparticles. Int. J. Pharm. 2024, 662, 124514. https://doi.org/10.1016/j.ijpharm.2024.124514.
  92. Das, S.; Ng, W.K.; Tan, R.B. Sucrose ester stabilized solid lipid nanoparticles and nanostructured lipid carriers. I. Effect of formulation variables on the physicochemical properties, drug release and stability of clotrimazole-loaded nanoparticles. Nanotechnology 2014, 25, 105101. https://doi.org/10.1088/0957-4484/25/10/105101.
  93. Hengherr, S.; Heyer, A.G.; Köhler, H.R.; et al. Trehalose and anhydrobiosis in tardigrades--evidence for divergence in responses to dehydration. Febs J 2008, 275, 281–288. https://doi.org/10.1111/j.1742-4658.2007.06198.x.
  94. Sharma, E.; Shruti, P.S.; Singh, S.; et al. Trehalose and its Diverse Biological Potential. Curr. Protein Pept. Sci. 2023, 24, 503–517. https://doi.org/10.2174/1389203724666230606154719.
  95. Crowe, L.M.; Reid, D.S.; Crowe, J.H. Is trehalose special for preserving dry biomaterials? Biophys. J. 1996, 71, 2087–2093. https://doi.org/10.1016/s0006-3495(96)79407-9.
  96. Bogdanova, E.; Millqvist Fureby, A.; Kocherbitov, V. Hydration enthalpies of amorphous sucrose, trehalose and maltodextrins and their relationship with heat capacities. Phys. Chem. Chem. Phys. 2021, 23, 14433–14448. https://doi.org/10.1039/d1cp00779c.
  97. Doktorovova, S.; Shegokar, R.; Fernandes, L.; et al. Trehalose is not a universal solution for solid lipid nanoparticles freeze-drying. Pharm. Dev. Technol. 2014, 19, 922–929. https://doi.org/10.3109/10837450.2013.840846.
  98. Zuidam, N.J.; Crommelin, D.J. Chemical hydrolysis of phospholipids. J. Pharm. Sci. 1995, 84, 1113–1119. https://doi.org/10.1002/jps.2600840915.
  99. Horn, J.; Friess, W. Detection of Collapse and Crystallization of Saccharide, Protein, and Mannitol Formulations by Optical Fibers in Lyophilization. Front. Chem. 2018, 6, 4. https://doi.org/10.3389/fchem.2018.00004.
  100. Wolkers, W.F.; Oldenhof, H. Principles Underlying Cryopreservation and Freeze-Drying of Cells and Tissues. Methods Mol. Biol. 2021, 2180, 3–25. https://doi.org/10.1007/978-1-0716-0783-1_1.
  101. Hazzah, H.A.; Farid, R.M.; Nasra, M.M.; et al. Lyophilized sponges loaded with curcumin solid lipid nanoparticles for buccal delivery: Development and characterization. Int. J. Pharm. 2015, 492, 248–257. https://doi.org/10.1016/j.ijpharm.2015.06.022.
  102. Thakral, S.; Sonje, J.; Munjal, B.; et al. Mannitol as an Excipient for Lyophilized Injectable Formulations. J. Pharm. Sci. 2023, 112, 19–35. https://doi.org/10.1016/j.xphs.2022.08.029.
  103. Abdelwahed, W.; Degobert, G.; Stainmesse, S.; et al. Freeze-drying of nanoparticles: Formulation, process and storage considerations. Adv. Drug Deliv. Rev. 2006, 58, 1688–1713. https://doi.org/10.1016/j.addr.2006.09.017.
  104. Yong, K.W.; Laouar, L.; Elliott, J.A.W.; et al. Review of non-permeating cryoprotectants as supplements for vitrification of mammalian tissues. Cryobiology 2020, 96, 1–11. https://doi.org/10.1016/j.cryobiol.2020.08.012.
  105. Murray, A.; Kilbride, P.; Gibson, M.I. Trehalose in cryopreservation. Applications, mechanisms and intracellular delivery opportunities. RSC Med. Chem. 2024, 15, 2980–2995. https://doi.org/10.1039/d4md00174e.
  106. Feng, J.; Markwalter, C.E.; Tian, C.; et al. Translational formulation of nanoparticle therapeutics from laboratory discovery to clinical scale. J. Transl. Med. 2019, 17, 200. https://doi.org/10.1186/s12967-019-1945-9.
  107. Mehta, M.; Bui, T.A.; Yang, X.; et al. Lipid-Based Nanoparticles for Drug/Gene Delivery: An Overview of the Production Techniques and Difficulties Encountered in Their Industrial Development. ACS Mater. Au 2023, 3, 600–619. https://doi.org/10.1021/acsmaterialsau.3c00032.
  108. Clarke, S.; Johnston, I.; Braun, A.; et al. Strategies for producing clinical and commercial rna-lnp drug products. Cytotherapy 2024, 26, S147. https://doi.org/10.1016/j.jcyt.2024.03.289.
  109. Roy, A.; Dutta, R.; Kundu, N.; et al. A Comparative Study of the Influence of Sugars Sucrose, Trehalose, and Maltose on the Hydration and Diffusion of DMPC Lipid Bilayer at Complete Hydration: Investigation of Structural and Spectroscopic Aspect of Lipid-Sugar Interaction. Langmuir 2016, 32, 5124–5134. https://doi.org/10.1021/acs.langmuir.6b01115.
  110. Rouco, H.; Diaz-Rodriguez, P.; Guillin, A.; et al. A Traffic Light System to Maximize Carbohydrate Cryoprotectants' Effectivity in Nanostructured Lipid Carriers' Lyophilization. Pharmaceutics 2021, 13, 1330. https://doi.org/10.3390/pharmaceutics13091330.
  111. Fan, Y.; Rigas, D.; Kim, L.J.; et al. Physicochemical and structural insights into lyophilized mRNA-LNP from lyoprotectant and buffer screenings. J. Control Release 2024, 373, 727–737. https://doi.org/10.1016/j.jconrel.2024.07.052.
  112. Wu, Y.; Fletcher, G.L. Efficacy of antifreeze protein types in protecting liposome membrane integrity depends on phospholipid class. Biochim. Biophys. Acta 2001, 1524, 11–16. https://doi.org/10.1016/s0304-4165(00)00134-3.
  113. Ampaw, A. Antifreeze proteins solve cold problems. Nat. Chem. 2022, 14, 1336. https://doi.org/10.1038/s41557-022-01075-z.
  114. Stubbs, C.; Bailey, T.L.; Murray, K.; et al. Polyampholytes as Emerging Macromolecular Cryoprotectants. Biomacromolecules 2020, 21, 7–17. https://doi.org/10.1021/acs.biomac.9b01053.
  115. Chen, L.; Lin, S.; Sun, N. Recent advances in natural peptide-based cryoprotectants in food industry: From source to application. Crit. Rev. Food Sci. Nutr. 2024, 1–17. https://doi.org/10.1080/10408398.2024.2436133.
  116. Bailey, T.L.; Stubbs, C.; Murray, K.; et al. Synthetically Scalable Poly(ampholyte) Which Dramatically Enhances Cellular Cryopreservation. Biomacromolecules 2019, 20, 3104–3114. https://doi.org/10.1021/acs.biomac.9b00681.
  117. Howard, M.D.; Lu, X.; Jay, M.; et al. Optimization of the lyophilization process for long-term stability of solid-lipid nanoparticles. Drug Dev. Ind. Pharm. 2012, 38, 1270–1279. https://doi.org/10.3109/03639045.2011.645835.