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Lipid Nanoparticles for Delivery of Nucleic Acids

Ascendia Nucleic Acid header

With the launch of three new nucleic-acid-based Onpattro® neuropathy drugs and Covid-19 vaccines, the pharmaceutical industry is weighing options for evaluating a range of non-viral delivery of nucleic acids as the therapeutics across all modalities. One challenge lies in finding the appropriate excipients/lipids with two aims – maintain longer systemic circulation and deliver drugs to afflicted tissues to avoid adverse effects. Fortunately, Ascendia Pharmaceutical Solutions has advanced proprietary nanotechnologies and state-of-the-art manufacturing capabilities to ensure those aims are true. 

Figure 1 lists the excipients/lipids used in the marketed drug/vaccines.1 As evident, LNPs are comprised of four lipids.2 Interestingly, Onpattro and the Moderna vaccine Spikevax contain a similar PEG-ylated lipids. Comirnaty from Pfizer/BioNtech contains a different class of PEG-ylated lipid. LNPs_Nucleic acids_figure 1

 

Let’s dive a little deeper. Each 1 mL dose of Onpattro (Patisiran) contains 6.2 mg cholesterol USP, 13.0 mg (6Z,9Z,28Z,31Z)-heptatriaconta6,9,28,31-tetraen-19-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA), 3.3 mg 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC), 1.6 mg α-(3'-{[1,2- 2 di(myristyloxy)propanoxy] carbonylamino}propyl)-ω-methoxy, polyoxyethylene (PEG -C-DMG).

 

A 0.5 m dose of Spikevax contains 50 mcg nucleoside modified messenger RNA (mRNA and total lipid content of 1.01 mg (SM-102), polyethylene glycol [PEG] 2000 dimyristoyl glycerol [DMG], cholesterol, and 1,2-distearoyl-sn-glycero-3- phosphocholine [DSPC]). Finally, a 0.3 ml dose of COMIRNATY contains lipids (0.43 mg ((4-hydroxybutyl)azanediyl) bis(hexane-6,1-diyl)bis (2-hexyldecanoate), 0.05 mg (polyethylene glycol 2000)-N,N-ditetradecylacetamide,) 0.09 mg 1,2-distearoyl-snglycero-3-phosphocholine, and 0.19 mg cholesterol.

LNPs for Nucleic Acids

Due to the complex nature of LNPs for nucleic acids requiring higher encapsulation efficiency of mRNA, it is often challenging. Munter et al. (2024) have demonstrated that not all LNPs are encapsulated and there is a substantial percentage of empty LNPs without nucleic acids. Authors conclude that encapsulation efficiency depends upon the lipid components, especially, the cationic /ionizable lipids and nature of helper lipids.3 Thus, these lipids play a crucial role in protecting, delivering and stabilizing of these large macromolecules.4

 

LNPs_Nucleic acids_figure 2As illustrated in Figure 2, the negatively charged nucleic acid is complexed with positively charged ionizable lipids (as a lipoplex) and is entrapped within the LNPs. A recent study suggests that the LNP interior is comprised of electrostatically neutral inverted micelles, in which nucleic acid is surrounded by the ionizable/cationic lipid and other lipid components. Compare this to the surface of the LNP, which is composed of a hydrophilic shell containing helper lipids and PEG‐lipids.5

 

LNPs designed with helper phospholipids, such as di-stearoyl phosphocholine and cholesterol, are distributed asymmetrically to create an outer stable monolayer boundary, whereas, the pegylated lipids are outside for steric stability of core surface. Efforts are still being made by researchers to design better and smarter ionizable lipids to yield greater nucleic acid stability and fusogenicity.6,7

Several PEGylated lipids discovered are aimed at sterically stabilized LNPs with longer circulation time in the blood without being opsonized. Unlike larger mRNA, it is believed that siRNA nucleic acid is encapsulated within spherical multilamellar structure, in which the nucleic acid is sandwiched between bilayer assemblies.8

Cryo-EM and small angle X-ray scattering could shed some light on the packing of nucleic acids within the LNPs; however, since the mass density contrast is not appreciably distinctive enough to resolve RNA from the lipid components, attempts are still continuously made to identify the RNA within the lipid nanoparticles. Brader et al. (2021) used a cationic dye (thionine) as a contrast agent in cryogenic electron microscopy (Cryo-EM) and found that chemical microenvironment of mRNA appears to be located within solvent filled cavities and be also fully stabilized with lipids around, as shown in Figure 2.9

Manufacturing of LNPs

Amphiphilic lipids can spontaneously aggregate into LNPs in aqueous solutions. LNP formation requires direct mixing of lipids in organic solvent and nucleic acids in an aqueous solution by agitation. The result is encapsulation of negatively charged mRNA. Hydrophobic fatty acid chains and polar headgroup help to create lipid assemblies that can further be sized into desired particle sizes.

Top-down and bottom-up approaches are commonly used for generating these particles. Top-down requires high shear and high energy, where the lipid (dried film) is hydrated and homogenized in aqueous buffer to yield desired particle size. The bottom-up approach, such as nanoprecipitation, requires an ethanol injection to form nanoparticles. This method, however, suffers from uncontrolled particle size due to inhomogeneous mixing.10

The microfluidic method is widely used for LNP generation with controlled and uniform particle size distribution under the laminar flow or turbulent flow conditions with higher Reynold’s numbers.11 This technique is fast and easy to scale up or manufacture large batches of LNPs. Precise control of mixing through T-junction or staggard herringbone mixer or ring mixer prevents the premature premixing and results in uniform particle size distribution with low polydispersity under precisely controlled temperature and flow rate.12

McKenzie et al. (2023) used a microfluidic method with ionizable lipid, Dlin-MC3-DMA (MC3), to prepare the mRNA encapsulated LNPs.13 In this nanoprecipitation method, a lipid in organic solvent and mRNA in an acidic buffer yielded stable LNPs with 80-250 nm diameters in size with encapsulation efficiency of > 80%. Jayaraman et al., (2012) prepared stable LNPs from a composition comprised of ionizable lipid/DSCP/Cholesterol/DMG-PEG-2000 (50:10:38.5:1.5), with N/P ratio of 4 (N being the ionizable amine and P being the phosphate associated with mRNA).14 The PEGylated lipids and helper phospholipids are in the 1-2% to 8-12% range, respectively. The typical helper phospholipid used is DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) can aid in yielding highly stable LNPs.

DOPE (dioleoyl sn-glyecro-3-phosphatidyl ethanolamine) is also used to form cone fluidic structure as opposed to cylindrical stable structure of DSPC. The percentage of ionizable lipid could range 40%-50% to create stable LNPs and to promote fusogenicity for greater transfection efficacy. Shepherd et al. (2023) developed microfluid chip method (so-called SCALAR) for producing the LNPs with throughput of >17 L /h at commercial manufacturing scale, as compared to >10 L/h commercially by Precision Nanoassemblr.15 The authors demonstrated the formation of LNPs (comprised of ionic lipid D-Lin-MC3-DMA:DSPC:Cholesterol:DMG-PEG 2000; 50:10:38.5:1.5) in high throughout production with uniform particle size (ca. 70 nm by intensity-weighted average), low polydispersity index (PDI), high encapsulation efficiency and comparable in vivo data in mice between batches.15

Lyophilization technology is another way to help optimize and stabilize the mRNA nucleotide within LNPs.16

LNP Platform Technology

LipidSol® by Ascendia Pharmaceutical Solutions is a LNP platform technology that provides different process methods such as microfluidics, thin film hydrating, extrusion, high pression homogenization, nanoprecipitation, and emulsification/double emulsification to make LNPs for various therapeutic modalities with hydrophilic and lipophilic properties.17 Coupled with lab-scale screening and cGMP sterile manufacturing capabilities, Ascendia Pharmaceutical Solutions is leading the way in design, development and manufacturing of LNPs for novel therapeutics in cancers, infectious, and many rare diseases.

Ascendia Pharmaceutical Solutions’ New Jersey Bioscience Center headquarters boasts sterile manufacturing and filling room space. It is equipped with a platform for the controlled and precise assembly of LNPs and machinery for rapidly scaling nanoparticle formulations for late pre-clinical development. This includes microfluidic systems from NanoAssmblr™ (Cytiva) such as Ignite™, Blaze™, and GMP equipment. They perfectly complement LipidSol-based formulations can be used to prepare the LNPs encapsulated with non-viral nucleic acids as the therapeutics for injectable drugs. Coupled with our lyophilization capabilities, Ascendia is prepared to take the challenges in manufacturing of aseptic drugs to a new height in ISO 5, ISO 6, ISO 7 clean rooms.

Contact us today to learn more about our LNP capabilities.

References

  1. S. Ali and J. Huang, Lipid Nanoparticles, Carriers for nucleic acids delivery, Drug Dev. Delivery, January/February 2024.
  2. NIH: Daily Med (https://dailymed.nlm.nih.gov/dailymed/)
  3. R. Münter, J. B. Larsen and T. L. Andresen, The vast majority of nucleic acid-loaded lipid nanoparticles contain cargo, J. Colloid & Interface Sci., 2024, 674, 139–144.
  4. C. Gueguen, T. Ben Chimol, M. Briand, K. Renaud, M. Seiler, M. Ziesel, P. Erbacher, and M. Hellal, Evaluating how cationic lipid affects mRNA-LNP physical properties and biodistribution, Eur. J. Pharm. Pharmacol., DOI: https://doi.org/10.1016/j.ejpb.2023.08.002
  5. R. N. Kularatne, R. M. Crist and S. T. Stern, The Future of tissue-targeted lipid nanoparticle-mediated nucleic acid delivery, Pharmaceutics, 2022, 15, 897; doi.org/10.3390/ph15070897
  6. X. Hou, T. Zaks, R. Langer and Y. Dong, Lipid Nanoparticles for mRNA delivery, Nature Reviews, 2021, 6, 1078-1094.
  7. F. DeRosa, S. Karve and M. Heartlein, Encapsulation of messenger RNA, US Patent 9,668,980 (June 6, 2017).
  8. M. Gindy, K. DeFelice and Boardman, Mechanisms of macromolecular structure evolution in self-assembled lipid nanoparticles for siRNA delivery, Langmuir, 2014, 30, 4613-4622.
  9. M. L. Brader, S. J. Willims, J. M. Banks, W. H. Hui, Z. H. Zhou, and L. Lin, Encapsulation state of messenger RNA inside lipid nanoparticles, Biophys. J., 2021, 120, 2766-2770.
  10. M. Mehta, T. A. Bui, X. Yang, Y. Aksoy, E. M. Goldys, and W. Deng, 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.
  11. S. Hirota, C. T. De Ilarduya, L. G. Barron and F. C. Szoka, Simple mixing device to reproducibly prepare cationic lipid-DNA complexes (lipoplexes), Biotechniques, 1999, 27, 286-290.
  12. Ripolle, M., Martin, E., Enot, M., Robbe, O., Rapisarda, C., Nicolai, M. C., Deliot, A., Tabeling, P., Authelin, J. R., Nakach, M., & Wils, P. (2022). Optimal self-assembly of lipid nanoparticles (LNP) in a ring micromixer. Scientific Reports, 12(1), 9483.
  13. R. E. McKenzie, J. J. Minnell, M. Ganley, G. F. Painter, and S. L. Draper, mRNA Synthesis and encapsulation in ionizable lipid nanoparticles, Current Protocols, 2023, 3, 1-47.
  14. M. Jayaraman, S. M. Ansell., B. L. Mui, Y.K. Tam, J. Chen, X. Du, D. Butler, L. Eltepu, S. Matsuda, J. K. Narayanannair, K. G. Rajeev, I. M. Hafez, A. Akinc, M. A. Maier, M. A. Tracy. P. R. Cullis, T. D. Madden, M. Manoharan, & M. J. Hope, Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo. Angew. Chemie Inter. Ed. 2012, 51(34), 8529–8533. https://doi.org/10.1002/anie.201203263
  15. S. J. Shepherd, X. Han, A. J. Mukalel, R. El-Mayta, A. S. Thatte, J. Wu, M. S. Padilla, M. G. Alameh, N. Sirkumar, D. Lee, D. Weissman, D. Issadore, and M. J. Mitchell, PNAS, 2023, 120, 1-12.
  16. L. Schoenmaker, D. Witzigmann, J. A. Kulkarni, R. Verbeke, G. Kersten, W. Jiskoot, and D. J. A. Crommelin, mRNA-lipid nanoparticle Covid-19 vaccines: Structure and stability, Int. J. Pharm. 2021, 601, 120586.
  17. J. Huang and S. Ali, LipidSol® - Liposomes: Chemistry, Properties and Applications of lipid nanoparticles, Drug Dev. & Delivery, May 2023.

 


 
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