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  • Michael Nguyen

Exploring Lipid Nanoparticle Formulation Strategies: How to Make the Best LNPs

Updated: Mar 29



The field of mRNA-based therapeutics has witnessed remarkable advancements in recent years, particularly with the development of lipid nanoparticles (LNP). Central to the success of these therapies is effective drug delivery into target cells. LNPs have emerged as promising delivery vehicles due to their ability to protect a wide range of biomolecules from degradation and facilitate their entry into cells. However, there exists a vast array of different LNP compositions and formulation strategies, which makes it difficult to navigate this exciting field. In this article, we at Helix Biotech, Inc. discuss the broad design considerations involved in LNP formulation, focusing on strategies to optimize drug loading and ensure the successful delivery and translation of mRNA.


Initial Formulation Strategies


Traditional methods of LNP formulations relied on thin lipid film hydration. Here, the desired lipid components are pre-mixed in organic solvent, which is then evaporated under vacuum leaving a thin lipid film. The film, ideally homogenous after the evaporation step, is then hydrated with an aqueous buffer containing the nucleic acid to form a LNP suspension. The resulting LNPs however possess poor critical quality attributes, such as large particle diameters (>100 nm), a broad particle size distribution (i.e. high polydispersity index, PDI), and low nucleic acid encapsulation efficiency. Furthermore, the morphological structure of these particles are overwhelmingly multilamellar, that is multiple lipid bilayers that prevent efficient nucleic acid loading and release.


Seeking to improve both the poor critical quality attributes and mRNA transfection efficiency, researchers pursued different approaches and methods for LNP production. Methods like conventional pipette mixing, in-line and batch solvent injection, and microfluidic nanoparticle production rose to prominence due to their ease of use and potential scalability. The popular solvent injection methods, e.g. ethanol loading, involve the rapid injection of an organic solution containing lipids into an aqueous solution containing the nucleic acid. The speed of LNP component mixing here is ideally faster than the rate of nanoparticle formation which helps ensure LNP monodispersity and homogeneity–critical factors not only for product potency and quality, but also when one considers scaling up in manufacturing. Microfluidic mixing is another popular method used to manufacture LNPs. Simply, they involve the use of channels and wells on the micro-scale to survey small volumes of sample. Its strength lies in the multitude of geometries that can be employed for mixing and their relatively small laboratory footprint and resource requirements. The rise of these innovative techniques has enabled researchers more flexibility in terms of modifying LNP formulations, manufacturing parameters, and optimization.


The Role of Lipid Composition on LNP Performance


An initial step in development of any LNP-based drug product is consideration of the overall composition. The lipid composition of LNPs is a critical factor in determining mRNA encapsulation and translation efficiency. Several components typically go into a single formulation. The most well-known mRNA-LNP vaccines (Pfizer’s BNT162b2 and Moderna’s mRNA-1273) contain four major components at various ratios, i.e. ionizable lipid, polyethylene glycol (PEG) lipid, zwitterionic helper lipid, and cholesterol.

Figure 1. Kinetic analysis of the effect of ionizable lipids on the expression of green fluorescent protein (GFP) in different cell lines. Each culture was treated with 500 ng/mL of the indicated LNP formulations with (left) or without (right) FBS. The LNP composition is ionizable cationic lipid/DMG-PEG2000/DSPC/Cholesterol (50/1.5/10/38.5). Adapted with permission from Helix Biotech and Cayman Chemical’s application note.


Ionizable lipids play a pivotal role in forming the core structure of LNPs, facilitating electrostatic interactions with the negatively charged mRNA. These interactions are essential for the encapsulation of mRNA within the LNP core. An ionizable lipid can be broken down into three subunits called the headgroup, linker region and tails, which can be chemically-modified to produce LNPs with desirable traits. With such potential tunability, whole libraries of novel ionizable lipids have been synthesized and screened for enhanced nucleic acid encapsulation efficiency, loading capacity, LNP size and polydispersity, lipid headgroup pKa, cellular targeting, LNP stability and cytotoxicity [1]. In Fig. 1, we show the ionizable lipid has a tremendous effect on in vitro GFP expression in several different cell lines. The signal is often attenuated when using FBS alone without serum proteins, now known to be related to the formation of protein coronas around the nanoparticles


Additionally, PEG lipids, zwitterionic helper lipids, and cholesterol contribute to the overall stability of LNPs, enhancing their circulation time in the bloodstream and aiding in evading the immune system. Adjusting the ratio or identity of these components can have an effect on LNP properties as well.


The physical characteristics of LNPs, specifically their size and surface charge, significantly impact their interactions with biological systems. LNPs with a diameter in the range of 50-100 nm are considered optimal for systemic delivery. This size range allows for effective cellular uptake while avoiding rapid clearance by the immune system. Researchers were able to finely adjust the LNP particle size to achieve these diameters by altering the amount of PEG lipid within the LNP formulation, which was also achieved without compromising encapsulation and transfection efficiencies [2]. The other helper lipids (typically distearoylphosphatidylcholine, DSPC) and cholesterol are also critical in the stable encapsulation of nucleic acids, actively participating in the formation of RNA-lipid complexes [3]. At mol percentages of less than 40% for DSPC and cholesterol combined, the encapsulation efficiency of siRNA was found to decrease; such effects were most pronounced at 5 mol percent where the encapsulation was essentially zero [3]. Replacing DSPC with dioleoylphosphatidylcholine (DOPC) or stearoyloleoylphosphatidylcholine (SOPC) can potentially result in a ~50-fold increase in transfection rates due to a slight increase in unsaturation, fluidity or lipid shape[9].


The Crucial Role of LNP Buffers


Buffers play a crucial role in the stability and formulation of mRNA and mRNA-LNPs. The buffer can also affect the success of LNP formulations as efficient encapsulation of mRNA and storage is critical to the proper delivery and transfection/translation of the therapeutic.


Naked mRNA are sensitive to changes in pH and thus require an aqueous buffer with sufficient salts for stabilization and to prevent degradation. Sodium acetate and sodium citrate are the most commonly used salts in buffers during the LNP formulation process. The concentration varies but is typically in the 1-100 mM range. The pH of the buffer also influences LNP charge and the formulation process. An acidic pH of roughly 4-5 is typically used in the mixing step between lipids in ethanol and mRNA in aqueous buffer. At this pH, the ionizable lipid component is primarily positively charged which allows for favorable electrostatic interactions with the mRNA.


Once the LNP is fully formed, the newly formed solution has a high percentage of solvent (typically ethanol) and must be replaced with a new buffer via dialysis, spin concentrators, or tangential flow filtration. Typically, the newly exchanged buffer contains components and ions that better stabilize the mRNA-LNP and raises the pH to ~7.4, a physiologically relevant isotonic pH which also neutralizes ionizable lipids on the exterior of nanoparticles. Phosphate buffered saline (PBS) is generally the buffer of choice, but saline alone, DPBS, Tris, and HEPES are also used depending on which minimizes effects on LNP morphology, and promotes mRNA transfection efficiency and cryoprotection[7].


Another critical step in the LNP pipeline is long-term LNP storage in buffer to maximize stability. Such stability is a key consideration in LNP formulation to ensure the integrity of the nanoparticles during storage, transportation, and in physiological conditions. Physical stability involves preventing aggregation and maintaining the structural integrity of the LNP, ensuring that LNPs remain intact until reaching the target cells. These stability considerations are crucial for preventing premature release or degradation of mRNA and maintaining the therapeutic efficacy of the LNP formulation.


The storage buffer pH typically does not affect LNP stability and thus they can be stored at the physiological pH of 7.4 for direct use after thawing [4]. To avoid lipid and nucleic acid degradation via oxidation and hydrolysis, LNPs are often stored in sub-zero temperatures, often at -80℃. However, the freezing and subsequent thawing of the mRNA-LNP aliquots can significantly affect their physicochemical properties[7]. As a result, the storage buffer often includes cryoprotectants such as sucrose or trehalose to help maintain LNP integrity.


Adjusting Formulation Process Parameters to Yield Optimal LNPs

Depending on the LNP manufacturing method, there are several process parameters that can be manipulated to optimize the nanoparticle size, PDI, and mRNA encapsulation efficiency. For mixing techniques, the LNP size and PDI can be controlled by adjusting the total flow rate (TFR) and the flow rate ratio (FRR, i.e. the ratio between the flow rate of the aqueous mRNA feed to the flow rate of the ethanol-lipid feed). The effect of TFR on LNP size and PDI can be observed in Fig. 2 where increasing TFR leads to a reduction in size of empty-cargo ionizable-lipid based LNPs. This parameter depends on the type of nanoparticle and its cargo; as a result, systematic study is required to determine the optimal flow rates for each unique formulation.

Figure 2. Influence of total flow rate on LNP size and polydispersity (PDI). Performed using the Helix Biotech’s Nova™ benchtop impinged jet mixer (IJM) System. The LNP composition is ALC-0315/ALC-0159/DSPC/Cholesterol (46.3/1.6/9.4/42.7).


The FRR typically varies from 3:1 to 9:1. At higher FRRs and lower stock lipid concentrations, the LNP size is small due to a rapid dilution of the ethanol and rising polarity which limits nanoparticle growth [6]. The FRR is also closely tied to the N/P ratio, the ratio of nitrogen amines (N) in the ionizable lipid to the number of phosphate groups (P) in the nucleic acid. The N/P ratio serves to optimize the electrostatic interactions between the two groups and is important for the composition and formation of stable mRNA-LNPs.


Concluding Remarks

It is often desired for mRNA-LNP drug products to possess an mRNA encapsulation efficiency that is greater than 90%. However, there is growing evidence that translation efficiency decreases with increasing EE. This may be due to the tighter mRNA packing within the LNP core, inhibiting proper endosomal escape and translation by cellular machinery [8]. At the end of the day, the most optimal formulation and drug product characteristic will be dependent on the active pharmaceutical ingredient, medical application, and target.   


In conclusion, the formulation of LNPs for mRNA delivery is a complex and multidimensional process that requires careful consideration of various design parameters. Optimizing drug loading and ensuring the successful delivery and translation of mRNA involve a delicate balance of factors, from the lipid composition and physical characteristics of LNPs to the formulation process parameters that make them. Addressing these considerations is essential for the development of safe, effective, and scalable mRNA-based therapeutics that can revolutionize the treatment of various diseases, from infectious diseases to genetic disorders. As research in this field continues to advance, the potential for LNP-based therapies to become a cornerstone of modern medicine becomes increasingly evident.




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  2. Belliveau, N. M., Huft, J., Lin, P. J., Chen, S., Leung, A. K., Leaver, T. J., … Cullis, P. R. (2012). Microfluidic Synthesis of Highly Potent Limit-size Lipid Nanoparticles for In Vivo Delivery of siRNA. Molecular Therapy - Nucleic Acids, 1, e37.

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  4. Kulkarni, J. A., Witzigmann, D., Leung, J., Tam, Y. Y. C., & Cullis, P. R. (2019). On the role of helper lipids in lipid nanoparticle formulations of siRNA. Nanoscale, 11(45), 21733–21739.

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  8. Carrasco, M. J., Alishetty, S., Alameh, M.-G., Said, H., Wright, L., Paige, M., … Buschmann, M. D. (2021). Ionization and structural properties of mRNA lipid nanoparticles influence expression in intramuscular and intravascular administration. Communications Biology, 4(1), 956.

  9. Kulkarni, J. A., Myhre, J. L., Chen, S., Tam, Y. Y. C., Danescu, A., Richman, J. M., & Cullis, P. R. (2017). Design of lipid nanoparticles for in vitro and in vivo delivery of plasmid DNA. Nanomedicine: Nanotechnology, Biology and Medicine, 13(4), 1377–1387.


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