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Mechanisms of LNP-Mediated mRNA Delivery

Updated: Jan 8


The delivery of genetic material holds profound implications for therapeutic treatment of a variety of diseases and ailments. Among the many drug delivery systems, lipid nanoparticles (LNPs) have emerged as the premier option for mRNA delivery. In 2018, the first LNP-based RNA therapeutic, Onpattro®, was approved by the US FDA for the treatment of hereditary transthyretin amyloidosis[1]. Not long after, in 2021 and 2022, the worldwide administration of mRNA-LNP COVID-19 vaccines by Pfizer-BioNTech and Moderna further demonstrated the applicability and potential of RNA-based LNPs. With mRNA-LNP vaccines paving the way, there have been recent FDA clearances for LNP-based gene editing clinical trials for rare diseases and cancer treatment. These include clinical trials of RNA-LNP drugs to treat hepatocellular carcinoma by Omega Therapeutics, cystic fibrosis by Vertex Pharmaceuticals, and transthyretin amyloidosis with cardiomyopathy by Intellia Therapeutics. 

Understanding the intricate mechanisms by which LNPs package, protect, transport, and deliver mRNA is crucial for advancing the next generation of nucleic acid therapeutics. In this article, we at Helix Biotech, Inc. will explore the prevailing theories behind LNP-mediated mRNA delivery, shedding light on the complex processes involved in harnessing the potential of these complex particles for targeted gene expression.

Fig 1. This figure illustrates a lipid nanoparticle and syringe, representing a crucial mode of LNP administration. LNPs, loaded with the desired therapeutic payload, are drawn into the syringe and can then be administered in a targeted and controlled manner for various medical applications.

Packaging of mRNA into LNPs 

Naked mRNA is destabilized and quickly degraded by host ribonucleases in the bloodstream. Thus, the first step in LNP-mediated mRNA delivery is the encapsulation of mRNA within the LNP core. The prevailing theory suggests that LNPs form stable complexes with mRNA through electrostatic interactions. This interaction is facilitated by the use of ionizable lipids–one of the four main LNP components with the others being cholesterol, a helper lipid, and a poly(ethylene glycol)-(PEG)ylated lipid–whose charge is dependent on the environmental pH[2]. At acidic pH, typical of mRNA-LNP formulation buffers, the ionizable lipid is positively-charged, allowing for favorable interactions with the negatively-charged phosphate groups of mRNA. This electrostatic binding allows the mRNA to be entrapped within the core of the LNP, shielding it from degradation and clearance by the host’s immune system. 

Protection of mRNA 

LNPs provide a protective shield for mRNA during its journey through the extracellular environment. This protective mechanism involves the lipid layers and structures encapsulating the mRNA, preventing enzymatic degradation by ribonucleases. One of the biggest factors affecting mRNA-LNP stability is passive mRNA degradation, occurring via hydrolysis of the phosphodiester backbone or oxidation by water, acids, and bases[3]. It was initially believed that the internal LNP core possessed little to no water; however, it was shown that the core can comprise up to 24% water[3]. LNPs indeed reduce the presence of water in the surrounding mRNA environment via mRNA-lipid complexation. Despite this, further research is required to precisely gauge the extent of this effect and to explore optimal strategies for maximizing it. This is crucial for enhancing the protective measures shielding mRNA from degradation.  

Additionally, LNPs prevent immune recognition of mRNA, allowing them to evade the host's immune system and reach the target cells with minimal interference. It has been proposed that the lipid composition and surface modifications of LNPs contribute to their ability to avoid immune recognition. PEGylation, the attachment of poly(ethylene glycol) chains to the surface of LNPs, is a common strategy. The PEG chains create a hydrophilic surface, reducing particle aggregation, minimizing phagocytosis by macrophages, and improving the circulation time of LNPs in the bloodstream. Despite making up only a small percentage (<1-3%) of any given LNP formulation, there are growing concerns about the immunogenicity of PEG lipids[4], necessitating research and development to improve on this component and enable broader application of mRNA+LNP pharmaceuticals.

Transport of mRNA-LNPs in the Body 

Once mRNA is encapsulated and protected within the lipid shell, LNPs are loaded into medical syringes for administration in hospitals, pharmacies, and healthcare centers (refer to Fig. 1). Noteworthy routes of mRNA-LNP delivery include intravenous, intramuscular, intranasal, and intradermal administrations, reflecting the versatility of LNPs in targeted therapeutic applications. A few notable LNP drugs and their route of administration include: the Pfizer-BioNtech and Moderna COVID-19 vaccines are delivered intramuscularly; and Onpattro is administered via intravenous injection. Many researchers are also looking at local and topical administration routes for respiratory or ocular therapies and dermatological applications. Depending on the application, the route of administration can also have an effect on mRNA transfection, expression, and subsequent immune response[5].

Once administered, LNPs must navigate through the complex biological milieu to reach the target cells. What makes LNPs such an attractive mRNA carrier is that their physicochemical properties can be easily tuned by chemical reactions or by simply substituting their individual components with those possessing unique characteristics[6]. With this in mind, active targeting strategies involve surface modifications of LNPs with ligands that can recognize specific receptors on target cells. This targeted approach aims to enhance the selectivity and efficiency of LNP-mediated mRNA delivery to specific tissues or cells. On the other hand, passive targeting methods employ changes in the LNP size, surface charge, and pKa, which can be achieved by substituting the lipid components with those containing the desired properties[6]. 

Other researchers comment on the role of the enhanced permeability and retention (EPR) effect, which details how long-circulating LNPs passively accumulate in tumor tissues with leaky vasculature. This effect is particularly relevant for cancer therapeutics, where LNPs can exploit the abnormal vasculature of tumors for targeted delivery[7]. 

Delivery of mRNA in the Target Cells


One of the final stages in LNP-mediated mRNA delivery is the release of mRNA into the cytoplasm of target cells. This release is necessary for the mRNA to reach the cellular machinery that translates its genetic code into proteins. It is believed that LNPs enter cells through endocytosis, where the lipid nanoparticles are engulfed by the cell membrane and enclosed in endosomes. The pH-dependent ionizable lipids play a crucial role at this stage by facilitating the disruption of the endosomal membrane, a process often referred to as “endosomal escape”. The microenvironment of maturing endosomes becomes more acidic than the surrounding cytoplasm, leading to protonation of the ionizable lipids and a change in the nanoparticle structure that promotes disruption of the endosomal membrane. Importantly, the ionizable lipids in released mRNA+lipid complexes are re-neutralized from the cationic form - this ultimately eliminates the binding of mRNA to cationic lipids. 

Fig. 2 Lipid nanoparticles, loaded with nucleic acid molecules, can travel through the bloodstream, transporting their drug payload to their targeted destination.

Upon endosomal escape, mRNA released into the cytoplasm can engage with cellular machinery for translation. Naturally occurring ribosomes bind starting sites on the mRNA, and engineered proteins are produced naturally as designed for the target therapeutic or immunological application. The efficiency of this step may be a determinant of the overall success of LNP-mediated mRNA delivery; although, only a small amount of RNA are ever translated by cellular ribosomes[8], these therapeutics continue to work and show great potential.   

Challenges and Future Directions 

While significant progress has been made in elucidating the mechanisms of LNP-mediated mRNA delivery, several challenges persist. As mentioned above, more research must be done on minimizing contact between water and mRNA in the LNP core, a potential source of mRNA degradation. Additionally, innovative surface modifications and ligand targeting strategies need to be further explored to refine the precision of LNP-mediated mRNA delivery. Less than 2% of mRNA appear to ever achieve endosomal escape[8], highlighting another key area to improve potency and reduce expensive mRNA costs by requiring lower mRNA content in these therapeutics. Future directions in this field include the development of next-generation LNPs with improved biocompatibility, reduced toxicity, and enhanced biodegradability as well. We at Helix Biotech, Inc. are dedicated to tackling and helping solve these challenges for our clients and the rest of the field. 


In conclusion, LNP-mediated mRNA delivery represents a groundbreaking approach in the field of nucleic acid therapeutics. The prevailing theories emphasize the complex interplay of electrostatic interactions, ionizable lipids, and surface modifications that govern the packaging, protection, transport, and delivery of mRNA by LNPs. As our understanding of these mechanisms deepens, the potential for developing highly effective and targeted therapies using LNP-mediated mRNA delivery continues to expand. The ongoing research in this area holds the promise of revolutionizing the treatment of various diseases, paving the way for a new era in genetic and personalized medicine.


  1. Shepherd, S. J., Issadore, D., & Mitchell, M. J. (2021). Microfluidic formulation of nanoparticles for biomedical applications. Biomaterials, 274, 120826.

  2. Wilson, B., & Geetha, K. M. (2022). Lipid nanoparticles in the development of mRNA vaccines for COVID-19. Journal of Drug Delivery Science and Technology, 74, 103553.

  3. Kon, E., Elia, U., & Peer, D. (2022). Principles for designing an optimal mRNA lipid nanoparticle vaccine. Current Opinion in Biotechnology, 73, 329–336.

  4. Ju, Y., Carreño, J. M., Simon, V., Dawson, K., Krammer, F., & Kent, S. J. (2023). Impact of anti-PEG antibodies induced by SARS-CoV-2 mRNA vaccines. Nature Reviews Immunology, 23(3), 135–136.

  5. Anderluzzi, G., Lou, G., Woods, S., Schmidt, S. T., Gallorini, S., Brazzoli, M., … Perrie, Y. (2022). The role of nanoparticle format and route of administration on self-amplifying mRNA vaccine potency. Journal of Controlled Release, 342, 388–399.

  6. Cao, S., Zhang, W., Pan, H., Huang, Z., Guo, M., Zhang, L., Xu, X., & Saw, P. E. (2023). Bioactive lipid-nanoparticles with inherent self-therapeutic and anti-angiogenic properties for cancer therapy. Acta Biomaterialia, 157, 500–510.

  7. Ju, Y., Carreño, J. M., Simon, V., Dawson, K., Krammer, F., & Kent, S. J. (2023). Impact of anti-PEG antibodies induced by SARS-CoV-2 mRNA vaccines. Nature Reviews Immunology, 23(3), 135–136.

  8. Gilleron, J., Querbes, W., Zeigerer, A., Borodovsky, A., Marsico, G., Schubert, U., Manygoats, K., Seifert, S., Andree, C., Stöter, M., Epstein-Barash, H., Zhang, L., Koteliansky, V., Fitzgerald, K., Fava, E., Bickle, M., Kalaidzidis, Y., Akinc, A., Maier, M., & Zerial, M. (2013). Image-based analysis of lipid nanoparticle–mediated siRNA delivery, intracellular trafficking and endosomal escape. Nature Biotechnology, 31(7), 638–646.

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