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  • Elizabeth Klavon

An Introduction and Brief History of mRNA-LNPs

Updated: Mar 29

Lipid nanoparticles (LNPs) have emerged as a pivotal player, revolutionizing the landscape of drug development. These minuscule yet intricate lipid-based carriers have taken center stage in the context of mRNA vaccines, cancer drugs and therapeutics for diseases utilizing saRNA, circRNA, tRNA, miRNA, and ASOs, as well as a whole host of innovation in nucleic acid therapeutics. By encapsulating fragile biological and synthetic molecules within a protective lipid shell, LNPs have not only ushered in the era of rapid vaccine development but are also playing a pivotal role in the global fight against infectious diseases. This Helix Biotech, Inc. article takes a look into mRNA LNPs, unraveling their structure, function, and the transformative impact they've had in medicine and pharmaceutical developments.

The Beginning of Lipid-Based Drug Delivery: Liposomes

Over the past 60 years, the use of mRNA in pharmaceuticals has undergone a remarkable evolution, culminating in the impactful mRNA LNP vaccines we have today. Beginning when mRNA was first discovered in 1961, its potential as a therapeutic was continuously researched throughout the coming decades. This is because the mRNA sequence describes the exact instructions for making a specific protein. Such a sequence can then be engineered to produce an antibody-induced immune response or restore vital protein functions. However, this potential could not be fully realized as foreign mRNA is quickly degraded in humans by ribonucleases. To overcome this, numerous modalities were investigated to act as delivery vehicles, capable of shuttling mRNA into targeted cells. One such modality is liposomes–spherical vesicles composed of a lipid bilayer(s) surrounding an aqueous core. Because liposomes harbor both hydrophobic and hydrophilic environments, they are able to deliver both hydrophobic and hydrophilic drugs, increasing their bioavailability and efficacy. Early pioneers envisioned and then showed how hydrophobic drugs can be embedded within the bilayer of the liposome, while hydrophilic drugs and water-soluble biomolecules, including nucleic acids, can be encapsulated within the aqueous core [1]. In 1995, Doxil, a doxorubicin liposomal formulation, was the first approved liposome-based formulation by the FDA. In subsequent years, the number of FDA-approved liposome drugs and vaccines would steadily rise, delivering much needed antitumor, antibiotic, antifungal, and other therapeutics[2].

A New Dawn has Arrived: Lipid Nanoparticles

Deviating from early, traditional liposomes, LNPs have emerged as a distinct class of drug delivery system. Both liposomes and LNPs protect poorly water soluble drugs via an outer shell of lipids but LNPs typically contain only a single outer layer, enveloping a core of mixed lipids that can entrap hydrophilic species such as nucleic acids (see Fig. 1). Over the next 20 years, the first mRNA LNP therapeutics were formulated and taken through clinical trials, with a large focus on cancer and influenza. This foundational research was crucial to the conception of the COVID-19 mRNA vaccines developed in 2020. Today, mRNA LNPs are being developed to prevent and fight a range of diseases, in gene editing, protein replacement, and vaccines [3].

Fig 1. Basic illustration of lipid nanoparticle and liposome components (left), lipid nanoparticle (middle), and liposome (right) structure. Although some building blocks are shared between them, their structures are quite different.

Earlier generations of liposomes were made with high concentrations of phospholipids, and over time, increasing amounts of the phospholipids were replaced with synthetic, cationic or ionizable lipids [4]. Cationic lipids are known for their constant positive charge, while ionizable lipids typically have a neutral charge at physiological pH. Interestingly, ionizable lipids become cationic in acidic environments due to a protonation event (these types of lipids can therefore be referred to as “ionizable cationic lipids”). The novel ionizable lipids gave LNPs the ability to change their overall charge depending on the external environment, reducing cytotoxic effects and improving drug pharmacokinetics in vivo. This is in contrast to purely cationic nanoparticles that can have nonspecific interactions with negatively-charged cellular components. As demonstrated, a great number of iterations to lipid chemistries and nanoparticle composition and formulation processes were required to produce the LNPs we know today.

The Primary Components of LNPs

LNPs are generally formed by mixing lipids in ethanol with mRNA in an aqueous buffer solution (as seen in Fig 2). First, ethanol facilitates the initial solubilization of lipids–an important step to ensure proper mixing and LNP formation. When mixed with the aqueous phase, the lipids exhibit their hydrophobic nature and self-assemble into supramolecular particles due to the hydrophobic effect. This process mitigates unfavorable polar-nonpolar interactions and helps encapsulate mRNA into the nanoparticle core.

Typically, the lipids in ethanol are a mixture of four different types of lipids that have been identified and developed for the specific function of encapsulating, protecting, and delivering the mRNA to the cell. They consist of:

  1. Ionizable cationic lipid

  2. Helper Phospholipid

  3. Cholesterol

  4. Polyethylene Glycol (PEG) Lipid

It can be said that the defining feature of any LNPs are their ionizable lipids. Some prominent examples include ALC-0315, SM-102, and DLin-MC3-DMA which have all been used in FDA-approved LNP formulations. There are also many new novel ionizable lipids that have been synthesized and researched, many of which improve mRNA encapsulation efficiency, cellular uptake, and drug release. Their overall positive charge in acidic formulation buffer promotes attractive electrostatic interactions with the negatively-charged phosphate backbone of nucleic acids, encouraging nucleic acid incorporation into the developing LNP. Ionizable lipids have a neutral charge at physiological pH of ~7.4 which promotes the endosomal uptake of LNPs. Their eventual re-protonation aids in the release of the encapsulated cargo in cells, as the nanoparticles are internalized via endosomes that naturally see a reduction in pH while trafficking through the cell. Furthermore, the apparent pKa of a LNP is primarily determined by its ionizable lipid component, and it has been shown an apparent pKa between 6 and 7 vastly improves the delivery of RNA [4] compared to LNPs employing purely cationic lipids (ionizable perhaps at only extreme pHs).

Fig 2. Top, left: shows a typical LNP mixing set-up with a lipid ethanolic feed mixing with an aqueous feed containing nucleic acid.. Bottom: with increasing water content, lipids and nucleic acids begin to coalesce based on the hydrophobic effect, eventually forming the lipid nanoparticle structure.

Helper, zwitterionic phospholipids (e.g. DSPC, DOPC, DOPE, and more) help to both stabilize the lipid shell and disrupt the endosomal membrane of the cell, when delivering the mRNA. Cholesterol helps with LNP stability and to control the fluidity of the membrane through differing interactions with unsaturated lipids (decrease fluidity) and saturated lipids (increase fluidity). The addition of cholesterol to the lipid bilayer also helps to prevent the contents from escaping or leaking out. Lastly, PEG lipids, in addition to phospholipids and cholesterol, can help with the stability of the final product for long-term storage and increase LNP circulation time within the bloodstream.

Together, the ratios and types of these four lipid components can be finely tuned to meet specific requirements, such as the target cell/tissue/organ and for the type of drug [5]. Not limited to just four components, researchers have played around with the number of components, different lipid chemistries, and even conjugating different moieties onto the LNP surface for better targeting and pharmacokinetics. Some notable examples include the selective organ targeting (SORT) LNPs that include a fifth SORT lipid component to enable tissue targeting[6], peptide-conjugated LNPs to deliver mRNA through ocular barriers[7], and novel ionizable lipid structure and chemistries to improve RNA release[8].

Concluding Remarks

Liposomes paved the way for LNPs, now the latter is quickly becoming the drug carrier standard for the treatment of many diseases. LNPs, as compared to liposomes, can have increased stability during bloodstream circulation, enhanced cargo delivery, a higher payload capacity, reduced toxicity, enhanced targeting, versatility, and clinical success. However, both LNPs and liposomes have their unique strengths and limitations. Liposomes may still be preferred for certain applications where their properties, such as biocompatibility or cargo release kinetics, align better with the desired outcomes. Ultimately, the choice between LNPs and liposomes depends on the specific goals and challenges of drug delivery or gene therapy in a given context.

As we've explored the functions of these diverse lipids within LNPs, it becomes evident that they are not just passive carriers but active players in mRNA delivery. From the ionizable cationic lipids that facilitate mRNA and mediate endosomal escape to the zwitterionic lipids that provide stability, their collaboration is essential for the successful and precise delivery of genetic information. In the ever-evolving landscape of biotechnology and vaccine development, a deep understanding of these lipid components empowers scientists and researchers to refine the design of mRNA-LNPs, paving the way for innovative solutions to combat diseases and usher in a new era of personalized medicine. With continuous research and innovation, the promise of mRNA LNPs holds great potential in addressing some of the most pressing challenges in medicine and therapeutics, offering hope for a healthier and more resilient future.


  1. Tenchov, R., Bird, R., Curtze, A. E., Zhou, Q. Lipid Nanoparticles- From Liposomes to mRNA Vaccine Delivery, a Landscape of Research Diversity and Advancement. ACS Nano 15, 11, 16982-17015. (2021).

  2. Herrera-Barrera, M., Ryals, R. C., Gautam, M., Jozic, A., Landry, M., Korzun, T., Gupta, M., Acosta, C., Stoddard, J., Reynaga, R., Tschetter, W., Jacomino, N., Taratula, O., Sun, C., Lauer, A. K., Neuringer, M., & Sahay, G. (2023). Peptide-guided lipid nanoparticles deliver mRNA to the neural retina of rodents and nonhuman primates. Science Advances, 9(2).

  3. Hou, X., Zaks, T., Langer, R. et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater 6, 1078–1094 (2021).

  4. Patel, P., Ibrahim, N. M., & Cheng, K. (2021). The Importance of Apparent pKa in the Development of Nanoparticles Encapsulating siRNA and mRNA. Trends in Pharmacological Sciences, 42(6), 448–460.

  5. Albertsen, C. A., Kulkarni J. A., Witzigmann, D., Lind, M., Petersson, K., Simonsen, J. B. (2022). The role of lipid components in lipid nanoparticles for vaccines and gene therapy, Advanced Drug Delivery Reviews, Volume 188,114416, ISS N 0169-409X. (2022).

  6. Wang, X., Liu, S., Sun, Y., Yu, X., Lee, S. M., Cheng, Q., Wei, T., Gong, J., Robinson, J., Zhang, D., Lian, X., Basak, P., & Siegwart, D. J. (2023). Preparation of selective organ-targeting (SORT) lipid nanoparticles (LNPs) using multiple technical methods for tissue-specific mRNA delivery. Nature Protocols, 18(1), 265–291.

  7. Herrera-Barrera, M., Ryals, R. C., Gautam, M., Jozic, A., Landry, M., Korzun, T., Gupta, M., Acosta, C., Stoddard, J., Reynaga, R., Tschetter, W., Jacomino, N., Taratula, O., Sun, C., Lauer, A. K., Neuringer, M., & Sahay, G. (2023). Peptide-guided lipid nanoparticles deliver mRNA to the neural retina of rodents and nonhuman primates. Science Advances, 9(2).

  8. Herrera-Barrera, M., Ryals, R. C., Gautam, M., Jozic, A., Landry, M., Korzun, T., Gupta, M., Acosta, C., Stoddard, J., Reynaga, R., Tschetter, W., Jacomino, N., Taratula, O., Sun, C., Lauer, A. K., Neuringer, M., & Sahay, G. (2023). Peptide-guided lipid nanoparticles deliver mRNA to the neural retina of rodents and nonhuman primates. Science Advances, 9(2).


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