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Application Note

RNA Introduction for Lipid Nanoparticle 

Lipid nanoparticles (LNPs) have emerged as one of the most versatile and clinically successful drug delivery platforms, revolutionizing therapeutic applications ranging from mRNA vaccines to gene editing systems. Their unique structure and composition enable the encapsulation and targeted delivery of a remarkably diverse range of therapeutic payloads, making them a cornerstone technology in modern nanomedicine.

Payload Types.png

Fig 1. Types of payloads delivered by LNPs with their applications in drug delivery.

1. Overview of LNP Structure and Functionality

LNPs are sophisticated delivery vehicles composed of four key lipid components: ionizable cationic lipids, helper phospholipids, cholesterol, and PEGylated lipids. The ionizable lipids are particularly crucial, as they can switch from neutral at physiological pH to positively charged at low pH, facilitating both cargo encapsulation during formulation and endosomal release within target cells. This pH-responsive behavior enables LNPs to protect their cargo during circulation while efficiently releasing it intracellularly.​

 

The assembly mechanism of LNPs involves rapid mixing of an aqueous phase (containing the therapeutic cargo) with an organic phase (containing the lipid mixture.) This triggers self-assembly of stable nanoparticles typically ranging from 80-200 nm in diameter. The resulting particles have a complex internal structure where ionizable lipids entrap negatively charged nucleic acids in the core, surrounded by a lipid shell containing helper lipids, cholesterol, and PEGylated components.

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Fig 2. LNP synthesis with microfluidic mixing.

2. Nucleic Acid Payloads

2.1 mRNA

Messenger RNA (mRNA) represents the most common and clinically advanced payload for LNPs, exemplified by the COVID-19 vaccines that have demonstrated global efficacy and safety. LNPs protect mRNA from degradation by serum nucleases and facilitate its intracellular delivery, where it undergoes translation to produce the encoded protein.

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Fig 3. Recent advances in mRNA-LNP therapeutics. Image adapted from Journal of nanobiotechnology. 2022 Jun 14;20(1):276.

Recent single-particle analysis revealed that benchmark mRNA LNP formulations typically contain 2-3 mRNA molecules per loaded particle, with 40-80% of particles being empty depending on assembly conditions. Remarkably, payload capacity correlates with mRNA mass rather than copy number, meaning smaller mRNAs can achieve higher copy numbers per particle.

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Some clinical applications of mRNA-LNPs include:

  • Vaccines: COVID-19 vaccines (Pfizer-BioNTech, Moderna), with expanding applications to other infectious diseases and cancer immunotherapy

  • Protein replacement therapy: Delivering mRNA encoding therapeutic proteins for genetic diseases

  • Cancer therapy: mRNA encoding tumor antigens, cytokines (IL-21, IL-7), or immune modulators

2.2 siRNA

Small interfering RNA (siRNA) is one alternative to mRNA. siRNA-LNPs have achieved FDA approval with Onpattro (patisiran) for treating hereditary transthyretin-mediated amyloidosis. These smaller nucleic acids (~21 base pairs) show different packaging characteristics compared to mRNA, with studies indicating no empty LNPs in siRNA formulations.

siRNA-LNP Schematic.png

Fig 4. Schematic representation of mechanism of LNP-mediated siRNA delivery into cells resulting in gene silencing. Image adapted from Pharmaceutics. 2022 Nov 19;14(11):2520.

Recent single-particle analysis revealed that benchmark mRNA LNP formulations typically contain 2-3 mRNA molecules per loaded particle, with 40-80% of particles being empty depending on assembly conditions. Remarkably, payload capacity correlates with mRNA mass rather than copy number, meaning smaller mRNAs can achieve higher copy numbers per particle.

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Some clinical applications of siRNA-LNPs include:

  • Genetic diseases: Targeting specific disease-causing genes in the liver

  • Cancer therapy: Silencing oncogenes such as Bcl-2, MYC, or growth factor receptors

  • Metabolic disorders: Targeting cholesterol metabolism genes like PCSK9

2.3 Gene Editing Systems

LNPs enable reliable delivery of CRISPR-Cas9 components, whether as mRNA precursors or preformed ribonucleoprotein (RNP) complexes.

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  • mRNA Delivery: This method involves co-encapsulation of Cas9 mRNA and guide RNA. This may be done in either single or separate particles, and can achieve median editing rates of 38.5% in liver hepatocytes.

CRISPR-LNP Schematic.png

Fig 5. Schematic illustration of LNP-CRISPR. Image adapted from Nature communications. 2021 Dec 8;12(1):7101.

  • RNP Delivery: This method involves direct delivery of preformed Cas9-gRNA complexes. Some advantages include reduced off-target effects, improved editing efficiency, and shorter intracellular half-life. Recent advances using thermostable genome editors enable robust RNP encapsulation and tissue-selective editing in both liver and lung tissues.

RNP-LNP Schematic.png

Fig 5. Schematic illustration of LNP-CRISPR. Image adapted from Nature communications. 2021 Dec 8;12(1):7101.

2.4 circRNA

Circular RNAs (CircRNA) represent a paradigm shift in RNA therapeutics due to their unique covalently closed-loop structure that lacks the 5' and 3' ends found in linear RNA. This circular topology confers remarkable stability against exonuclease degradation, as these enzymes typically require free RNA ends to initiate cleavage. Endogenously produced circRNAs demonstrate 2-5 times greater stability compared to their linear counterparts, with potential therapeutic applications benefiting from this enhanced durability.

 

CircRNA vaccines have demonstrated robust immunogenicity in preclinical models, with mannose-modified LNP-circRNA vaccines showing comparable immune responses to conventional platforms in both rabies virus and SARS-CoV-2 models. The enhanced stability profile enables room temperature storage after lyophilization, addressing critical cold-chain limitations of current mRNA vaccines.

circRNA.png

Fig 7. Transfection with Circular RNA. Image adapted from MBio, 15(1), pp.e01775-23.

2.5 SaRNA

Self-amplifying RNAs (SaRNA) are considerably larger molecules (9-12 kb) compared to conventional mRNA, containing the basic mRNA elements (cap, 5' UTR, 3' UTR, poly(A) tail) plus a substantial open reading frame encoding four non-structural proteins (nsP1-4) and a subgenomic promoter. Upon cellular delivery, saRNA undergoes translation to produce the four components of an RNA-dependent RNA polymerase (RDRP) complex. This RDRP complex, tethered to plasma membrane invaginations, synthesizes complementary negative-strand RNA, which subsequently serves as a template for producing both genomic and subgenomic positive-strand RNA in excess. The self-amplifying nature enables sustained protein expression for 20-26 days compared to 2-3 days for conventional mRNA.

 

Multiple saRNA vaccine candidates have advanced to clinical trials, including GEMCOVAC-OM (Gennova Biopharmaceuticals) and ARCT-154, both targeting SARS-CoV-2. The KOSTAIVE (ARCT-154) saRNA COVID-19 vaccine has shown promising safety and efficacy profiles in Phase I/II trials. Recent clinical data demonstrate that optimized saRNA vaccines can achieve protective immunity at ultra-low doses, with some formulations effective at doses 100-1000 times lower than conventional mRNA vaccines.

SaRNA.png

Fig 8. Transfection with SaRNA. Image adapted from Scientific Reports, 11(1), p.21308.

References:

  1. https://www.xenocs.com/exploring-saxs-applications-in-mrna-lnps-drug-delivery-systems/

  2. https://www.biorxiv.org/content/10.1101/2025.06.11.659145v1

  3. https://pmc.ncbi.nlm.nih.gov/articles/PMC9363157/

  4. https://pubs.acs.org/doi/10.1021/acsmaterialsau.3c00032

  5. https://www.precigenome.com/lipid-nanoparticles-lnp/lipid-nanoparticle-synthesis-system-application

  6. https://www.precigenome.com/lipid-nanoparticles-lnp/lipid-nanoparticle-lnp-mrna-concept-introduction

  7. https://pmc.ncbi.nlm.nih.gov/articles/PMC10636777/

  8. https://pubmed.ncbi.nlm.nih.gov/38621638/

  9. https://www.sciencedirect.com/science/article/abs/pii/S1748013224004420

  10. https://www.biochempeg.com/article/283.html

  11. https://pmc.ncbi.nlm.nih.gov/articles/PMC9691360/

  12. https://www.nature.com/articles/s41467-022-33157-4

  13. https://pubs.acs.org/doi/10.1021/acs.nanolett.4c05186

  14. https://www.precigenome.com/lipid-nanoparticles-lnp/introduction-lnp-formulation-design-preparation

  15. https://pmc.ncbi.nlm.nih.gov/articles/PMC2676808/

  16. https://www.nature.com/articles/s41598-024-52685-1

  17. https://www.nature.com/articles/s41598-021-99180-5

  18. https://www.sciencedirect.com/science/article/abs/pii/S0378517325001334

  19. https://www.biorxiv.org/content/10.1101/2024.03.17.584721v1.full-text

  20. https://mitchell-lab.seas.upenn.edu/wp-content/uploads/2025/01/NanoLetters_Han.pdf

  21. https://www.nature.com/articles/s41587-024-02437-3

  22. https://pmc.ncbi.nlm.nih.gov/articles/PMC10407046/

  23. https://ehoonline.biomedcentral.com/articles/10.1186/s40164-025-00602-1

  24. https://pmc.ncbi.nlm.nih.gov/articles/PMC10823087/

  25. https://www.frontiersin.org/journals/chemistry/articles/10.3389/fchem.2020.589959/full

  26. https://mitchell-lab.seas.upenn.edu/wp-content/uploads/2023/04/1-s2.0-S2211383522003239-main-3.pdf

  27. https://pmc.ncbi.nlm.nih.gov/articles/PMC9176214/

  28. https://www.nature.com/articles/s41467-024-54877-9

  29. https://pmc.ncbi.nlm.nih.gov/articles/PMC11040677/

  30. https://pubs.acs.org/doi/10.1021/acs.molpharmaceut.1c00916

  31. https://pmc.ncbi.nlm.nih.gov/articles/PMC11510967/

  32. https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2024.1466337/full

  33. https://pmc.ncbi.nlm.nih.gov/articles/PMC8069344/

  34. https://pmc.ncbi.nlm.nih.gov/articles/PMC11921523/

  35. https://www.abcam.com/en-us/stories/articles/circrna-therapeutics-the-next-leap-after-mrna

  36. https://journals.asm.org/doi/10.1128/mbio.01775-23

  37. https://www.nature.com/articles/s41598-021-00830-5

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Related Resources

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Lipid nanoparticle applications and technical notes

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Publications

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Lipid nanoparticle publications 

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Premade siRNA-LNP

Product

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Premade siRNA-LNP 

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