Skip to main content

Advertisement

SpringerLink
Log in

Continuous and size-control synthesis of lipopolyplex nanoparticles enabled by controlled micromixing performance for mRNA delivery

  • Full Paper
  • Published:
Journal of Flow Chemistry Aims and scope Submit manuscript

Abstract

Accurate control of core–shell lipopolyplex nanoparticles (LPP NPs) size is crucial for finely adjusting their biomedical performance. However, the synthesis of LPP NPs encounters challenges as two mixing-sensitive processes are involved in the synthesis, rendering precise control over particle size difficult using conventional batch methods. In this study, the formation of the nucleic acid/cationic polymer cores through electrostatic complexation and the subsequent encapsulation by lipid shells via self-assembly were conducted in microreactors, with polyadenylic acid (poly A) and branched polyethylenimine (bPEI) employed as the model system. By assessing the micromixing performance of the microreactors using the Villermaux-Dushman method, the characteristic time scale for electrostatic complexation between poly A and bPEI, as well as the self-assembly of lipids, was determined to be below 1 ms. The Reynolds number, governing micromixing performance, emerged as a crucial factor influencing the sizes of poly A/bPEI cores and LPP NPs. In the kinetic control region, characterized by rapid mixing, the size of poly A/bPEI remained slightly influenced by the N/P molar ratio and volumetric flow rate ratio, irrespective of concentration. The zeta potential, however, was primarily affected by the N/P molar ratio. In the case of LPP NPs, under optimized conditions of anionic lipid molar ratio, the size of LPP NPs was significantly influenced by the composition of lipid shells. This study establishes the foundation for elucidating the structure–activity relationship of LPP NPs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11
Fig. 12
Fig. 13

Similar content being viewed by others

References

  1. Shin MD, Shukla S, Chung YH et al (2020) COVID-19 vaccine development and a potential nanomaterial path forward. Nat Nanotechnol 15:646–655. https://doi.org/10.1038/s41565-020-0737-y

    Article  CAS  PubMed  Google Scholar 

  2. Xiao YF, Tang ZM, Huang XG et al (2022) Emerging mRNA technologies: delivery strategies and biomedical applications. Chem Soc Rev 51:3828–3845. https://doi.org/10.1039/d1cs00617g

    Article  CAS  PubMed  Google Scholar 

  3. Weng YH, Li CH, Yang TR et al (2020) The challenge and prospect of mRNA therapeutics landscape. Biotechnol Adv 40:107534. https://doi.org/10.1016/j.biotechadv.2020.107534

    Article  CAS  PubMed  Google Scholar 

  4. Wang X (2021) Safety and efficacy of the BNT162b2 mRNA COVID-19 vaccine. N Engl J Med 384:1577–1578. https://doi.org/10.1056/NEJMoa2034577

    Article  PubMed  Google Scholar 

  5. Shi YY, Huang JX, Liu Y et al (2022) Structural and biochemical characteristics of mRNA nanoparticles determine anti–SARS-CoV-2 humoral and cellular immune responses. Sci Adv 8:eabo1827. https://doi.org/10.1126/sciadv.abo1827

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Yang R, Deng Y, Huang BY et al (2021) A core-shell structured COVID-19 mRNA vaccine with favorable biodistribution pattern and promising immunity. Signal Transduct Target Ther 6:213. https://doi.org/10.1038/s41392-021-00634-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Ewe A, Schaper A, Barnert S et al (2014) Storage stability of optimal liposome-polyethylenimine complexes (lipopolyplexes) for DNA or siRNA delivery. Acta Biomater 10:2663–2673. https://doi.org/10.1016/j.actbio.2014.02.037

    Article  CAS  PubMed  Google Scholar 

  8. Wan JW, Yang JM, Wang ZM et al (2023) A single immunization with core–shell structured lipopolyplex mRNA vaccine against rabies induces potent humoral immunity in mice and dogs. Emerg Microbes Infect 12:2270081. https://doi.org/10.1080/22221751.2023.2270081

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Geng LJ, Kato N, Kodama Y et al (2023) Influence of lipid composition of messenger RNA-loaded lipid nanoparticles on the protein expression via intratracheal administration in mice. Int J Pharm 637:122896. https://doi.org/10.1016/j.ijpharm.2023.122896

    Article  CAS  PubMed  Google Scholar 

  10. Di JX, Du ZL, Wu KZ et al (2022) Biodistribution and non-linear gene expression of mRNA LNPs affected by delivery route and particle size. Pharm Res 39:105–114. https://doi.org/10.1007/s11095-022-03166-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Cheng MHY, Leung J, Zhang Y et al (2023) Induction of bleb structures in lipid nanoparticle formulations of mRNA leads to improved transfection potency. Adv Mater 35:2303370. https://doi.org/10.1002/adma.202303370

    Article  CAS  Google Scholar 

  12. Wang X, Liu S, Sun YH et al (2023) Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery. Nat Protoc 18:265–291. https://doi.org/10.1038/s41596-022-00755-x

    Article  CAS  PubMed  Google Scholar 

  13. Hassett KJ, Higgins J, Woods A et al (2021) Impact of lipid nanoparticle size on mRNA vaccine immunogenicity. J Control Release 335:237–246. https://doi.org/10.1016/j.jconrel.2021.05.021

    Article  CAS  PubMed  Google Scholar 

  14. Lam K, Schreiner P, Leung A et al (2023) Optimizing lipid nanoparticles for delivery in primates. Adv Mater 35:2211420. https://doi.org/10.1002/adma.202211420

    Article  CAS  Google Scholar 

  15. Linder B, Weirauch U, Ewe A et al (2019) Therapeutic targeting of stat3 using lipopolyplex nanoparticle-formulated siRNA in a syngeneic orthotopic mouse glioma model. Cancers 11:333. https://doi.org/10.3390/cancers11030333

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Persano S, Guevara ML, Li ZQ et al (2017) Lipopolyplex potentiates anti-tumor immunity of mRNA-based vaccination. Biomaterials 125:81–89. https://doi.org/10.1016/j.biomaterials.2017.02.019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Du ZX, Munye MM, Tagalakis AD et al (2014) The role of the helper lipid on the DNA transfection efficiency of lipopolyplex formulations. Sci Rep 4:7107. https://doi.org/10.1038/srep07107

    Article  PubMed  PubMed Central  Google Scholar 

  18. Song HM, Wang G, He B et al (2012) Cationic lipid-coated PEI/DNA polyplexes with improved efficiency and reduced cytotoxicity for gene delivery into mesenchymal stem cells. Int J Nanomedicine 7:4637–4648. https://doi.org/10.2147/IJN.S33923

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhang QY, Ho PY, Tu MJ et al (2018) Lipidation of polyethylenimine-based polyplex increases serum stability of bioengineered RNAi agents and offers more consistent tumoral gene knockdown in vivo. Int J Pharm 547:537–544. https://doi.org/10.1016/j.ijpharm.2018.06.026

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Hu YZ, He ZY, Hao Y et al (2019) Kinetic control in assembly of plasmid DNA/polycation complex nanoparticles. ACS Nano 13:10161–10178. https://doi.org/10.1021/acsnano.9b03334

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Lombardo D, Kiselev MA (2022) Methods of liposomes preparation: formation and control factors of versatile nanocarriers for biomedical and nanomedicine application. Pharmaceutics 14:543. https://doi.org/10.3390/pharmaceutics14030543

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Mitchell MJ, Billingsley MM, Haley RM et al (2020) Engineering precision nanoparticles for drug delivery. Nat Rev Drug Discov 20:101–124. https://doi.org/10.1038/s41573-020-0090-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Chacon WDC, Verruck S, Monteiro AR et al (2023) The mechanism, biopolymers and active compounds for the production of nanoparticles by anti-solvent precipitation: a review. Food Res Int 168:112728. https://doi.org/10.1016/j.foodres.2023.112728

    Article  CAS  Google Scholar 

  24. Liu ZK, Yang M, Zhao QK et al (2023) Scale-up of antisolvent precipitation process with ultrasonic microreactors: cavitation patterns, mixing characteristics and application in nanoparticle manufacturing. Chem Eng J 475:146040. https://doi.org/10.1016/j.cej.2023.146040

    Article  CAS  Google Scholar 

  25. Johnson BK, Prud’homme RK (2003) Mechanism for rapid self-assembly of block copolymer nanoparticles. Phys Rev Lett 91:118302. https://doi.org/10.1103/PhysRevLett.91.118302

    Article  CAS  PubMed  Google Scholar 

  26. Dinter R, Goette K, Gronke F et al (2023) Development of an automated flow chemistry affinity-based purification process for DNA-encoded chemistry. J Flow Chem. https://doi.org/10.1007/s41981-023-00282-0

    Article  Google Scholar 

  27. Kaisin G, Bovy L, Joyard Y et al (2023) A perspective on automated advanced continuous flow manufacturing units for the upgrading of biobased chemicals toward pharmaceuticals. J Flow Chem 13:77–90. https://doi.org/10.1007/s41981-022-00247-9

    Article  Google Scholar 

  28. Heshmatnezhad F, Nazar ARS (2020) On-chip controlled synthesis of polycaprolactone nanoparticles using continuous-flow microfluidic devices. J Flow Chem 10:533–543. https://doi.org/10.1007/s41981-020-00092-8

    Article  CAS  Google Scholar 

  29. Qi TT, Luo GH, Xue HT et al (2023) Continuous heterogeneous synthesis of hexafluoroacetone and its machine learning-assisted optimization. J Flow Chem 13:337–346. https://doi.org/10.1007/s41981-023-00273-1

    Article  Google Scholar 

  30. Ran R, Wang HF, Liu Y et al (2018) Microfluidic self-assembly of a combinatorial library of single- and dual-ligand liposomes for in vitro and in vivo tumor targeting. Eur J Pharm Biopharm 130:1–10. https://doi.org/10.1016/j.ejpb.2018.06.017

    Article  CAS  PubMed  Google Scholar 

  31. Du W, Fu TT, Duan YF et al (2018) Breakup dynamics for droplet formation in shear-thinning fluids in a flow-focusing device. Chem Eng Sci 176:66–76. https://doi.org/10.1016/j.ces.2017.10.019

    Article  CAS  Google Scholar 

  32. Liu ZK, Yang M, Yao W et al (2023) Microfluidic ultrasonic cavitation enables versatile and scalable synthesis of monodisperse nanoparticles for biomedical application. Chem Eng Sci 280:119052. https://doi.org/10.1016/j.ces.2023.119052

    Article  CAS  Google Scholar 

  33. Balbino TA, Serafin JM, Malfatti-Gasperini AA et al (2016) Microfluidic assembly of pDNA/cationic liposome lipoplexes with high pDNA loading for gene delivery. Langmuir 32:1799–1807. https://doi.org/10.1021/acs.langmuir.5b04177

    Article  CAS  PubMed  Google Scholar 

  34. Koh CG, Zhang XL, Liu SJ et al (2010) Delivery of antisense oligodeoxyribonucleotide lipopolyplex nanoparticles assembled by microfluidic hydrodynamic focusing. J Control Release 141:62–69. https://doi.org/10.1016/j.jconrel.2009.08.019

    Article  CAS  PubMed  Google Scholar 

  35. Commenge J, Falk L (2011) Villermaux-Dushman protocol for experimental characterization of micromixers. Chem Eng Process 50:979–990. https://doi.org/10.1016/j.cep.2011.06.006

    Article  CAS  Google Scholar 

  36. Fournier MC, Falk L, Villermaux J (1996) A new parallel competing reaction system for assessing micromixing efficiency-experimental approach. Chem Eng Sci 51:5053–5064. https://doi.org/10.1016/0009-2509(96)00270-9

    Article  CAS  Google Scholar 

  37. Soleymani A, Kolehmainen E, Turunen I (2008) Numerical and experimental investigations of liquid mixing in T-type micromixers. Chem Eng J 135:S219–S228. https://doi.org/10.1016/j.cej.2007.07.048

    Article  CAS  Google Scholar 

  38. Hoffmann M, Schlüter M, Räbiger N (2006) Experimental investigation of liquid–liquid mixing in T-shaped micro-mixers using μ-LIF and μ-PIV. Chem Eng Sci 61:2968–2976. https://doi.org/10.1016/j.ces.2005.11.029

    Article  CAS  Google Scholar 

  39. Bothe D, Stemich C, Warnecke HJ (2006) Fluid mixing in a T-shaped micro-mixer. Chem Eng Sci 61:2950–2958. https://doi.org/10.1016/j.ces.2005.10.060

    Article  CAS  Google Scholar 

  40. Yang HJ, Chu GW, Zhang JW et al (2005) Micromixing efficiency in a rotating packed bed: experiments and simulation. Ind Eng Chem Res 44:7730–7737. https://doi.org/10.1021/ie0503646

    Article  CAS  Google Scholar 

  41. Yang M, Yang LN, Zheng J et al (2021) Mixing performance and continuous production of nanomaterials in an advanced-flow reactor. Chem Eng J 412:128565. https://doi.org/10.1016/j.cej.2021.128565

    Article  CAS  Google Scholar 

  42. Falk L, Commenge JM (2010) Performance comparison of micromixers. Chem Eng Sci 65:405–411. https://doi.org/10.1016/j.ces.2009.05.045

    Article  CAS  Google Scholar 

  43. Li SX, Hu YZ, Li A et al (2022) Payload distribution and capacity of mRNA lipid nanoparticles. Nat Commun 13:5561. https://doi.org/10.1038/s41467-022-33157-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Lokugamage MP, Vanover D, Beyersdorf J et al (2021) Optimization of lipid nanoparticles for the delivery of nebulized therapeutic mRNA to the lungs. Nat Biomed Eng 5:1059–1068. https://doi.org/10.1038/s41551-021-00786-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Ripoll M, Martin E, Enot M et al (2022) Optimal self-assembly of lipid nanoparticles (LNP) in a ring micromixer. Sci Rep 12:9483. https://doi.org/10.1038/s41598-022-13112-5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Kölbl A, Desplantes V, Grundemann L et al (2013) Kinetic investigation of the Dushman reaction at concentrations relevant to mixing studies in stirred tank reactors. Chem Eng Sci 93:47–54. https://doi.org/10.1016/j.ces.2013.01.067

    Article  CAS  Google Scholar 

  47. Wu BC, McClements DJ (2015) Microgels formed by electrostatic complexation of gelatin and OSA starch: potential fat or starch mimetics. Food Hydrocoll 47:87–93. https://doi.org/10.1016/j.foodhyd.2015.01.021

    Article  CAS  Google Scholar 

  48. Pal SK, Dhasmana P, Nigam KDP et al (2019) Tuning of particle size in a helical coil reactor. Ind Eng Chem Res 59:3962–3971. https://doi.org/10.1021/acs.iecr.9b04774

    Article  CAS  Google Scholar 

  49. Luo LM, Yang M, Chen GW (2022) Continuous synthesis of TiO2-supported noble metal nanoparticles and their application in ammonia borane hydrolysis. Chem Eng Sci 251:117479. https://doi.org/10.1016/j.ces.2022.117479

    Article  CAS  Google Scholar 

  50. Clamme JP, Azoulay J, Mély Y (2003) Monitoring of the formation and dissociation of polyethylenimine/DNA complexes by two photon fluorescence correlation spectroscopy. Biophys J 84:1960–1968. https://doi.org/10.1016/s0006-3495(03)75004-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Ziebarth JD, Kennetz DR, Walker NJ et al (2017) Structural comparisons of PEI/DNA and PEI/siRNA complexes revealed with molecular dynamics simulations. J Phys Chem B 121:1941–1952. https://doi.org/10.1021/acs.jpcb.6b10775

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Honoré I, Grosse S, Frison N et al (2005) Transcription of plasmid DNA: influence of plasmid DNA/polyethylenimine complex formation. J Control Release 107:537–546. https://doi.org/10.1016/j.jconrel.2005.06.018

    Article  CAS  PubMed  Google Scholar 

  53. Debus H, Baumhof P, Probst J et al (2010) Delivery of messenger RNA using poly(ethylene imine)–poly(ethylene glycol)-copolymer blends for polyplex formation: biophysical characterization and in vitro transfection properties. J Control Release 148:334–343. https://doi.org/10.1016/j.jconrel.2010.09.007

    Article  CAS  PubMed  Google Scholar 

  54. Kubczak M, Michlewska S, Karimov M et al (2022) Unmodified and tyrosine-modified polyethylenimines as potential carriers for siRNA: biophysical characterization and toxicity. Int J Pharm 614:121468. https://doi.org/10.1016/j.ijpharm.2022.121468

    Article  CAS  PubMed  Google Scholar 

  55. Perche F, Clemencon R, Schulze K et al (2019) Neutral lipopolyplexes for in vivo delivery of conventional and replicative RNA vaccine. Mol Ther Nucleic Acids 17:767–775. https://doi.org/10.1016/j.omtn.2019.07.014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Jovanović AA, Balanč BD, Ota A et al (2018) Comparative effects of cholesterol and β-sitosterol on the liposome membrane characteristics. Eur J Lipid Sci Technol 120:1800039. https://doi.org/10.1002/ejlt.201800039

    Article  CAS  Google Scholar 

  57. Kim J, Jozic A, Lin YX et al (2022) Engineering lipid nanoparticles for enhanced intracellular delivery of mRNA through inhalation. ACS Nano 16:14792–14806. https://doi.org/10.1021/acsnano.2c05647

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Choi S, Kang B, Yang E et al (2023) Precise control of liposome size using characteristic time depends on solvent type and membrane properties. Sci Rep 13:4728. https://doi.org/10.1038/s41598-023-31895-z

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The financial support from the National Natural Science Foundation of China (Nos. 22178336, 22208256, 21991103) and Dalian Institute of Chemical Physics Grant (No. DICP I202216) is gratefully acknowledged.

Author information

Authors and Affiliations

  1. Xi’an Polytechnic University, Xi’an, 710048, China

    Shirong Song & Hongchen Liu

  2. Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, China

    Shirong Song, Zhikai Liu, Letao Guo, Wang Yao, Mei Yang & Guangwen Chen

  3. University of Chinese Academy of Sciences, Beijing, 100049, China

    Zhikai Liu, Letao Guo & Wang Yao

Authors
  1. Shirong Song
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Zhikai Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Letao Guo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Wang Yao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hongchen Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mei Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Guangwen Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Hongchen Liu, Mei Yang or Guangwen Chen.

Ethics declarations

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Additional information

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Highlights

• The micromixing times of two different microreactors were calculated by incorporation model based on Villermaux-Dushman method.

• The effects of different variables on the formation of poly A/bPEI NPs and LPP NPs was systematically studied under controlled mixing.

• The Reynolds number was found to be a crucial factor influencing the sizes of poly A/bPEI cores and LPP NPs.

Supplementary information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 67 KB)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Song, S., Liu, Z., Guo, L. et al. Continuous and size-control synthesis of lipopolyplex nanoparticles enabled by controlled micromixing performance for mRNA delivery. J Flow Chem (2024). https://doi.org/10.1007/s41981-024-00316-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s41981-024-00316-1

Keywords

Search

Navigation