In the delivery journey of mRNA-LNP therapeutics, particle size stands as a critical parameter that determines success or failure. Unlike mRNA, which carries genetic instructions, or ionizable lipids that respond intelligently to the microenvironment, even a minor change in particle diameter profoundly impacts every performance aspect of LNPs, including physical stability, encapsulation efficiency, in vivo distribution and cellular uptake. So what is the most favorable particle size for LNPs? What lies behind this scientifically validated figure?
The Consensus: The Gold Standard Range of 60 to 100 Nanometers
A review of approved mRNA vaccines and extensive academic research shows that the particle size of LNPs predominantly falls within the narrow range of 60 to 100 nm. For instance, the two leading COVID-19 mRNA vaccines feature an average particle size between 80 and 100 nm, with the formulations developed by Pfizer/BioNTech and Moderna both adhering to this specification.
LNP particles formed by lipids complexed with mRNA via microfluidic mixing typically measure 60 to 100 nm. To cite examples, the average particle sizes of the SARS-CoV-2 vaccine candidates nCoVsaRNA and ARCoV are 75 nm and 89 nm respectively.
This range is not arbitrarily selected, but the optimal solution balanced by multiple physical, chemical and biological constraints. The U.S. FDA classifies particle size and particle size distribution as Critical Quality Attributes (CQAs) for liposomal drugs. The industry widely recognizes 60–100 nm as the ideal particle size range, paired with a polydispersity index (PDI) below 0.2. Systematic reviews also indicate that LNP manufacturing platforms can stably produce particles ranging from 60 to 180 nm, and a PDI no higher than 0.30 is the basic requirement to meet regulatory standards. Undoubtedly, 60–100 nm has become the universally accepted gold standard for particle size in mRNA-LNP formulation development.
Drawbacks of Undersized Particles: Renal Clearance and Reduced Encapsulation Efficiency
When LNP particles are smaller than 60 nm, a series of issues emerge. For one thing, smaller particles have a limited internal volume for mRNA loading. To deliver a sufficient drug dose with the same lipid input, more particles need to be produced, which increases formulation volume and production costs.
For another, such particles risk crossing the renal filtration barrier. The kidneys normally filter out small molecules below 10 nm, while particles smaller than 50 nm are prone to rapid renal clearance. This shortens their circulation half-life in the bloodstream and impairs tissue targeting capability.
More importantly, excessively small particles are usually accompanied by decreased encapsulation efficiency. The lipid bilayer of tiny particles cannot provide adequate internal space to encapsulate and protect mRNA molecules. As a result, more RNA is exposed to the external environment and vulnerable to enzymatic degradation.
In contrast, particles larger than 200 nm face another set of challenges: they are readily recognized and rapidly eliminated by macrophages in the liver and spleen. As professional phagocytes, macrophages are highly sensitive to particulate foreign substances. Oversized LNPs are preferentially internalized by these cells, leading to massive accumulation in the reticuloendothelial system of the liver and spleen instead of reaching target tissues. Particles that are too small lack sufficient protection and get metabolized quickly, while oversized ones are cleared prematurely by phagocytes. The 60–100 nm range strikes a perfect balance between these two extremes.
Underlying Physical Mechanisms: Hepatic Sinus Fenestrae and Cellular Uptake
The particle size range below 100 nm also aligns precisely with the anatomical structures of the liver and target cells.
After intravenous administration, most LNPs need to pass through the endothelial lining of hepatic sinusoids to reach hepatocytes. The hepatic sinusoid walls are covered with numerous fenestrae measuring 100 to 200 nm in diameter. Particles larger than 200 nm can hardly traverse these pores, whereas those under 100 nm easily penetrate the sinusoids and enter the perisinusoidal space. This explains why LNP-siRNA therapeutics such as Onpattro and numerous liver-targeted mRNA therapies adopt a particle size below 100 nm.
At the cellular level, particle size directly governs the efficiency of LNP internalization. Most therapeutic LNPs enter target cells via endocytosis, a process highly dependent on particle dimensions. The optimal size window for efficient endocytosis is approximately 80 to 120 nm. Notably, influenza virus (around 80 nm) and reovirus (around 120 nm) naturally fall within this range. Through long-term evolution, these viruses have acquired the ability to invade cells efficiently via receptor-mediated endocytosis. Similarly, LNPs sized between 80 and 100 nm can leverage this naturally optimized cellular entry mechanism to achieve high cellular uptake efficiency.
Strategies for Precise Particle Size Control: From Formulation to Manufacturing Processes
The particle size of LNPs is not fixed upon production and can be finely tuned via multiple approaches.
Microfluidic mixing is currently the dominant manufacturing technology for LNPs. By adjusting the flow rate ratio and total flow rate between the organic phase (lipids dissolved in ethanol) and the aqueous phase (mRNA in acidic buffer), particle size can be accurately controlled within milliseconds. Higher flow rates enable faster mixing and yield smaller particles, while lower flow rates result in larger particles. Combined with design of experiments (DoE) for parameter optimization, this technique enables the reliable production of LNPs with custom particle sizes ranging from 60 to 180 nm.
Lipid formulation is another core factor for size regulation. The molar ratio of PEG-lipids plays a vital role, with the typical dosage maintained at 1–2 mol%. An excessively high PEG-lipid ratio creates excessive aqueous interfacial areas and lowers encapsulation efficiency, while an insufficient ratio leads to particle aggregation and increased particle size. Fine modulation of particle size can also be achieved by adjusting the molecular weight of PEG lipids and the nitrogen-to-phosphorus (N/P) ratio between ionizable lipids and mRNA.
In recent years, innovative size modulation methods have emerged. Studies conducted in the early 2020s demonstrated that adding salts such as sodium chloride to the buffer serves as a simple and effective approach. Salts can increase particle size to over 200 nm in a concentration-dependent manner following the Hofmeister series. Although LNPs sized 200–300 nm are not ideal for intravenous delivery, they facilitate uptake by dendritic cells and exhibit unique advantages in applications such as cancer vaccines.
Particle Size: A Key Determinant of In Vivo Biodistribution
LNPs of different sizes naturally accumulate in distinct tissues after administration, a property known as passive targeting.
Particles smaller than 50 nm tend to accumulate gradually in the bone marrow;
Particles ranging from 50 to 100 nm achieve favorable liver targeting;
Particles between 100 and 200 nm possess strong immunogenicity and are ideal for activating immune cells in vaccines;
Particles larger than 200 nm are mechanically trapped by pulmonary capillaries and thus enriched in lung tissues.
For systemic administration, particle size acts as a decisive factor governing the in vivo tissue distribution of LNPs.
Conclusion
Particle size is far more than a mere numerical parameter; it serves as a critical physical bridge connecting molecular design to the in vivo fate of LNPs. Constrained by multiple biological barriers including the glomerular filtration threshold, hepatic sinus fenestra dimensions, the optimal window for endocytosis and phagocytic activity of macrophages, the 60–100 nm range stands out as the most balanced choice.
A thorough understanding of how particle size affects delivery performance not only facilitates the optimization of existing formulations, but also provides scientific guidance for the development of next-generation LNPs with organ-tropic and cell-specific targeting capabilities. When LNPs travel throughout the body with an optimal particle size, half the battle for superior efficacy of mRNA therapeutics is already won.
