JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Published: February 25th, 2021

DOI:

10.3791/62226

1Biologics Drug Product Development & Manufacturing, Sanofi, Framingham, Massachusetts
* These authors contributed equally

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Drug carriers are used to protect and deliver a therapeutic with typical favorable properties including low cytotoxicity, increased bioavailability, and improved stability1,2,3. Polymeric nanoparticles, micelles, and lipid-based particles have previously been explored for nucleic acid encapsulation and delivery4,5,6,7. Lipids have been used in different types of nanocarrier systems, including liposomes, and lipid nanoparticles, as they are biocompat....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

A schematic of the overall process is provided in Figure 1.

1. Preparation of buffers

NOTE: Sterile filtering of the buffers is highly suggested here to remove any particulates which may impact the nucleic acid and LNP quality.

  1. Phosphate Buffered Saline (PBS)
    1. Prepare 1x PBS using 8 mM Na2HPO4, 2 mM KH2PO4, 137 mM NaCl, and 2.7 mM KCl in nuclease free water and adjust th.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Multiple batches of LNPs with the same lipid formulation and N/P ratio of 6 were developed on separate days to demonstrate reproducibility of the technique. Batch 1 and 2 resulted in overlapping size distributions with similar polydispersity (Figure 2A) No significant difference was observed in the size or encapsulation efficiency between the two different batches (Figure 2B). The encapsulation efficiency was high for each batch .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Reproducibility, speed, and low volume screening are significant advantages of using microfluidic mixing to form LNPs compared to other existing methods (e.g., lipid film hydration and ethanol injection). We have demonstrated the reproducibility of this method with no impact on encapsulation efficiency or particle size observed with different LNP batches. This is an essential criterion for any therapeutic, including LNPs, to become clinically available.

The technique described here employs sta.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Thank you to Atul Saluja, Yatin Gokarn, Maria-Teresa Peracchia, Walter Schwenger, and Philip Zakas for their guidance and contributions towards LNP development.

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

Name Company Catalog Number Comments
1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (C-14 PEG) Avanti Polar Lipids 880151P
10 µl Graduated Filter Tips  (RNase-,DNase-, DNA-free) USA Scientific 1121-3810
1000 µl Graduated Filter Tips (RNase-,DNase-, DNA-free) USA Scientific 1111-2831
20 µl Beveled Filter Tips (RNase-,DNase-, DNA-free) USA Scientific 1120-1810
200 µl Graudated Filter Tips (RNase-,DNase-, DNA-free) USA Scientific 1120-8810
3β-Hydroxy-5-cholestene, 5-Cholesten-3β-ol (Cholesterol) Sigma-Aldrich C8667
BD Slip Tip Sterile Syringes (1 ml syringe) Thermo Fisher Scientific 14-823-434
BD Slip Tip Sterile Syringes (3 ml syringe) Thermo Fisher Scientific 14-823-436
BD Vacutainer General Use Syringe Needles (BD Blunt Fill Needle 18G) Thermo Fisher Scientific 23-021-020
Benchtop Centrifuge Beckman coulter
Black 96 well plates Thermo Fisher Scientific 14-245-177
BrandTech BRAND BIO-CERT RNase-, DNase-, DNA-free microcentrifuge tubes (1.5mL) Thermo Fisher Scientific 14-380-813
Citric Acid Fisher Scientific 02-002-611
Corning 500ml Vacuum Filter/Storage Bottle System, 0.22 um pore Corning 430769
Disposable folded capillary cells Malvern DTS1070
Ethyl Alcohol, Pure 200 proof Sigma-Aldrich 459844
Fisher Brand Semi-Micro Cuvette Thermo Fisher Scientific 14955127
Invitrogen Conical Tubes (15 mL) (DNase-RNase-free) Thermo Fisher Scientific AM12500
MilliporeSigma Amicon Ultra Centrifugal Filter Units Thermo Fisher Scientific UFC901024
NanoAssemblr Benchtop Precision Nanyosystems
Nuclease-free water Thermo Fisher Scientific AM9930
Phosphate Buffered Saline (PBS) Thermo Fisher Scientific AM9624
Quant-iT PicoGreen dsDNA Assay Kit Thermo Fisher Scientific  P7589
Quant-iT RiboGreen RNA Assay Kit Thermo Fisher Scientific R11490
Sodium Chloride Fisher Scientific 02-004-036
Sodium Citrate, Dihydrate, granular Fisher Scientific 02-004-056
SpectraMax i3x Molecular Devices
Zetasizer Nano Malvern

  1. Mitchell, M. J., Billingsley, M. M., Haley, R. M., Wechsler, M. E., Peppas, N. A., Langer, R., et al. Engineering precision nanoparticles for drug delivery. Nature Reviews Drug Discovery. , 1-24 (2020).
  2. Davis, M. E., Chen, Z., Shin, D. M. Nanoparticle therapeutics: an emerging treatment modality for cancer. Nanoscience and technology: A collection of reviews from nature journals. (239), 250 (2010).
  3. Patra, J. K., Das, G., Fraceto, L. F., et al. Nano based drug delivery systems: recent developments and future prospects. J Nanobiotechnol. 16 (71), (2018).
  4. Rai, R., Alwani, S., Badea, I. Polymeric nanoparticles in gene therapy: New avenues of design and optimization for delivery applications. Polymers. 11 (4), 745 (2019).
  5. Bailey, C. M., Nagarajan, R., Camesano, T. A. Designing polymer micelles of controlled size, stability, and functionality for siRNA delivery. ACS Symposium Series. 1271, 35-70 (2017).
  6. Yin, H., et al. Non-viral vectors for gene-based therapy. Nature Reviews Genetics. 15 (8), 541-555 (2014).
  7. Bailey-Hytholt, C. M., Nagarajan, R., Camesano, T. A. Förster resonance energy transfer probing of assembly and disassembly of short interfering RNA/Poly(ethylene glycol)-Poly-L-Lysine polyion complex micelles. Molecular Assemblies: Characterization and Applications. , 47-60 (2020).
  8. Puri, A., Loomis, K., Smith, B. Lipid-based nanoparticles as pharmaceutical drug carriers: from concepts to clinic. Crit Rev Ther Drug Carrier Syst. 26 (6), 523-580 (2009).
  9. Cullis, P. R., Hope, M. J. Lipid nanoparticle systems for enabling gene therapies. Molecular Therapy. 25 (7), 1467-1475 (2017).
  10. Munsell, E. V., Ross, N. L., Sullivan, M. O. Journey to the center of the cell: Current Nanocarrier design strategies targeting biopharmaceuticals to the cytoplasm an nucleus. Current Pharmaceutical Design. 22 (9), 1227-1244 (2016).
  11. Zhao, Y., Huang, L. Lipid nanoparticles for gene delivery. Advances in Genetics. 88, 13-36 (2014).
  12. Chen, S., et al. Influence of particle size on the in vivo potency of lipid nanoparticle formulations of siRNA. Journal of Controlled Release. 235, 236-244 (2016).
  13. Wan, C., Allen, T. M., Cullis, P. R. Lipid nanoparticle delivery systems for siRNA-based therapeutics. Drug Delivery and Translational Research. 4 (1), 74-83 (2014).
  14. Kulkarni, J. A., Cullis, P. R., Van Der Meel, R. Lipid nanoparticles enabling gene therapies: From concepts to clinical utility. Nucleic Acid Therapeutics. 28 (3), 146-157 (2018).
  15. Shin, M. D., et al. COVID-19 vaccine development and a potential nanomaterial path forward. Nature Nanotechnology. 15 (8), 646-655 (2020).
  16. Thanh Le, T., et al. The COVID-19 vaccine development landscape. Nature Reviews. Drug Discovery. 19 (5), 305-306 (2020).
  17. Tam, Y. Y. C., Chen, S., Cullis, P. R. Advances in lipid nanoparticles for siRNA delivery. Pharmaceutics. 5 (3), 498-507 (2013).
  18. Cayabyab, C., Brown, A., Tharmarajah, G., Thomas, A. mRNA lipid nanoparticles. Precision Nanosystems Application Note. , (2019).
  19. Gilleron, J., et al. Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape. Nature Biotechnology. 31 (7), 638-646 (2013).
  20. Suzuki, Y., Ishihara, H. Structure, activity and uptake mechanism of siRNA-lipid nanoparticles with an asymmetric ionizable lipid. International Journal of Pharmaceutics. 510 (1), 350-358 (2016).
  21. Kowalski, P. S., Rudra, A., Miao, L., Anderson, D. G. Delivering the messenger: Advances in technologies for therapeutic mRNA delivery. Molecular Therapy. 27 (4), 710-728 (2019).
  22. Schmid, J. A. The acidic environment in endocytic compartments. Biochemical Journal. 303 (2), 679-680 (1994).
  23. Maugeri, M., et al. Linkage between endosomal escape of LNP-mRNA and loading into EVs for transport to other cells. Nature Communications. 10 (1), (2019).
  24. Kulkarni, J. A., Witzigmann, D., Leung, J., Tam, Y., Cullis, P. R. On the role of helper lipids in lipid nanoparticle formulations of siRNA. Nanoscale. (45), (2019).
  25. Hafez, I. M., Maurer, N., Cullis, P. R. On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids. Gene Therapy. 8 (15), 1188-1196 (2001).
  26. Hafez, I. M., Culis, P. R. Roles of lipid polymorphism in intracellular delivery. Advanced Drug Delivery Reviews. 47 (2-3), 139-148 (2001).
  27. Evers, M. J. W., et al. State-of-the-art design and rapid-mixing production techniques of lipid nanoparticles for nucleic acid delivery. Small Methods. 2 (9), 1700375 (2018).
  28. Mui, B. L., et al. Influence of polyethylene glycol lipid desorption rates on pharmacokinetics and pharmacodynamics of siRNA lipid nanoparticles. Molecular Therapy - Nucleic Acids. 2 (139), (2013).
  29. Zukancic, D., et al. The importance of poly(Ethylene glycol) and lipid structure in targeted gene delivery to lymph nodes by lipid nanoparticles. Pharmaceutics. 12 (11), 1-16 (2020).
  30. NEBioCalculator. New England BioLabs Inc Available from: https://nebiocalculator.neb.com/#!/formulas (2020)
  31. Kastner, E., et al. High-throughput manufacturing of size-tuned liposomes by a new microfluidics method using enhanced statistical tools for characterization. International Journal of Pharmaceutics. 477 (1-2), 361-368 (2014).
  32. Zhigaltsev, I. V., et al. Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing. Langmuir. 28 (7), 3633-3640 (2012).
  33. Belliveau, N. M., et al. Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA. Molecular Therapy - Nucleic Acids. 1 (8), 37 (2012).
  34. Hassett, K. J., et al. Optimization of lipid nanoparticles for intramuscular administration of mRNA vaccines. Molecular Therapy - Nucleic Acids. 15, 1-11 (2019).
  35. Tanaka, H., et al. The delivery of mRNA to colon inflammatory lesions by lipid-nano-particles containing environmentally-sensitive lipid-like materials with oleic acid scaffolds. Heliyon. 4 (12), 00959 (2018).
  36. Singh, J., et al. Nucleic acid lipid nanoparticles. Precision Nanosystems Application Note. , (2018).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved