Application Note
Introduction to Lipid Nanoparticle: LNP Formulation Design and Preparation
Lipid nanoparticles (LNPs) have emerged as revolutionary drug delivery systems, gaining widespread recognition through their role in COVID-19 mRNA vaccines. These sophisticated delivery vehicles protect delicate therapeutic molecules and enable their precise delivery to target cells. Additionally, they offer several advantages over conventional drug delivery methods, such as increased drug stability, high target specificity, and controlled release.
This technical introduction explores the formulation and preparation of LNPs, highlighting common types, their typical composition, supported cargoes, and basic preparation considerations.
1. Types of Lipid Based Nanoparticles
Lipid-based nanoparticles constitute a diverse family of delivery systems that share a fundamental spherical structure composed of lipids, typically ranging from 10 to 1000 nanometers in diameter. Within this broader category, several specialized subclasses exist (Fig. 1), including liposomes, lipid nanoparticles, solid lipid nanoparticles, nanostructured lipid carriers, lipomers, and nano-emulsion.
Fig 1. Types of lipid based nanoparticles. Image from Pharmaceutics 16.1 (2024): 131.
1.1 Liposomes
Liposomes are spherical vesicles with one or more phospholipid bilayers surrounding an aqueous core. Their structure closely mimics biological membranes, making them highly biocompatible. Liposomes have been widely used for drug delivery, including anticancer agents and vaccines, and can be engineered for targeted delivery or controlled release of both hydrophilic and hydrophobic drugs.
1.2 Lipid Nanoparticles (LNPs)
The term "LNP" can be broadly used to describe all lipid-based nanoparticles, but most often refers specifically to the sophisticated delivery systems used in mRNA vaccines and therapeutics. While similar to liposomes, they instead feature a solid lipid core which leads to greater stability. LNPs also feature a unique combination of ionizable lipids that respond to pH changes, enabling efficient encapsulation and release of many cargoes, most notably nucleic acids.
1.3 Solid Lipid Nanoparticles (SLNs)
SLNs possess a surfactant shell over a solid lipid core matrix. The solid lipid core can effectively solubilize lipophilic (fat-soluble) molecules with controlled release and high stability, though SLNs may still be used with hydrophilic cargoes [1].
1.4 Nanostructured Lipid Carriers (NLCs)
NLCs feature a surfactant shell much like SLNs, though their core matrix includes both solid and liquid lipids. This leads to a less ordered structure which can increase drug loading capacity while minimizing drug leakage [2].
1.5 Lipomers
Lipomers are hybrid nanoparticles that integrate features of liposomes and polymeric nanoparticles. They typically have a polymeric core surrounded by a lipid shell, combining the structural stability of polymers with the biocompatibility of lipids. Lipomers can be used for otherwise poorly soluble drugs, and can be engineered for controlled or stimuli-responsive release.
1.6 Nano-emulsions (O/W and W/O)
Nano-emulsions are kinetically stable dispersions of two immiscible phases (water and oil) which are further stabilized with surfactants or proteins [3]. Both water-in-oil and oil-in-water emulsions are possible, in which case the role of dispersed and continuous phase is switched. This allows both hydrophilic or hydrophobic drugs to be solubilized in emulsions to suit different application requirements.
2. Structural Components of LNPs
Modern LNPs comprise four essential components: cationic/ionizable lipid, helper lipid, cholesterol and PEGylated lipid. Each serves specific functions in the delivery system (Fig. 2).
Fig 2. Essential components of LNPs
2.1 Ionizable Lipids
These lipids are the cornerstone of effective nucleic acid delivery systems. With a pH-triggered charge shift, they will become positively charged under acidic conditions while remaining neutral at physiological pH. This enables efficient encapsulation of nucleic acids during LNP synthesis at acidic conditions without the toxicity of permanently charged lipids at physiological pH [4]. Several examples are commonly used (Fig. 3), including but not limited to SM102, D-Lin-MC3-DMA, and ALC0315.
Fig 3. Major types of ionizable lipids. Images adapted from Nature communications 12.1 (2021): 7233.
2.2 Phospholipids
Phospholipids, also known as helper lipids, provide critical structural support for LNPs, contributing to overall particle morphology and integrity [4]. Multiple studies have highlighted the significant impact of phospholipid selection on transfection outcomes across different cell types (Fig. 4).
Fig 4. Structures of commonly used phosphatidylethanolamine (PE), phosphatidylcholine (PC) and cholesterol helper lipids. Images adapted from Pharmaceutical research 40.1 (2023): 27-46.
2.3 Cholesterol
Cholesterol works alongside phospholipids to stabilize LNP structure. This is due to cholesterol's mechanism of increasing membrane fluidity, which favors the formation of a liquid-ordered phase [4]. It also facilitates payload encapsulation and delivery, aiding the LNP to interface with a target cell. Notably, while cholesterol is also a type of helper lipid, that term is generally reserved for phospholipids to avoid confusion.
2.4 PEGylated Lipids
PEGylated lipids provide several critical functions. They are key to preventing aggregation between LNPs and reducing immune response to the LNPs, as they will form a hydrophilic shield around the LNP surface. This in turn will increase LNP circulation time. PEGylated lipids may also be used for conjugating additional ligands to increase target specificity [4].
Additional components are used in other types of lipid based nanoparticles:
2.5 Surfactants
Surfactants in the lipid shell of SLNs and NLCs improve stability by reducing the interfacial tension between the lipophilic core and surrounding aqueous phase.
2.6 Solid Lipids
Solid lipids are solid at ambient temperatures and used to produce both SLNs and NLCs. They are typically saturated and also referred to as fats.
2.7 Liquid Lipids
Liquid lipids are fluid at ambient temperatures and used to produce NLCs. They are typically unsaturated and also referred to as oils.
3. Types of LNP Cargo
LNPs can encapsulate various therapeutic molecules, including nucleic acids, proteins/peptides, small molecules.
3.1 Nucleic Acids
Nucleic acids, including mRNA, siRNA, and DNA, are the most prominent cargo used with LNPs (Fig. 5). The ionizable cationic lipid present in LNPs is ideal for encapsulating these negatively charged payloads, while the overall lipid shell provides much needed protection and guidance. Of these types of nucleic acid, mRNA is the most prominent due to its role in several COVID-19 vaccines and ease of incorporation with LNPs [5]. siRNA may be used instead when the goal is RNA interference rather than protein expression [6], while DNA is preferrable for longer term treatments.
Fig 5. Common nucleic acid payloads of LNPs and points of optimization
3.2 RNP Complexes
Ribonucleoprotein (RNP) complexes feature RNA conjugated with a protein. A notable example is the Cas9 complex used in CRISPR genome editing, where Cas9 is conjugated with a guide RNA targeting a site of interest. LNPs are also ideal for encapsulating these complexes and providing targeted delivery. However, additional care must be taken to ensure adequate encapsulation since RNP complexes lack the negative charge density of free RNA. Furthermore, the large size of these complexes (150-400 kDa) inherently limits payload capacity compared to other types of cargo.
PreciGenome's NanoGenerator platform has been used successfully to produce LNPs with an RNP payload. In a paper by Chen and colleagues at the University of California, Berkeley, thermostable iGeoCas9 was incorporated with LNPs to accomplish genome-editing levels of 16-37% in liver and lungs from a single dose (Fig. 6) [7]. The LNPs were produced using the NanoGenerator Flex-M when larger volumes were required.
Fig 6. Synthesis of LNP with RNP for gene editing.
Image from Nature Biotechnology (2024): 1-13.
3.3 Proteins/Peptides
Several types of protein ranging from enzymes to antibodies may be delivered using LNPs. Due to their diversity, the size of these cargoes can vary greatly (5-150 kDa). LNP mediated protein delivery can offer much needed protection against proteases along with the typical advantages of targeted delivery. When encapsulating proteins though, special attention must be paid to the risk of denaturation during LNP formulation.
PreciGenome's NanoGenerator has also been used to encapsulate protein. Using the NanoGenerator Flex-S, Wang and colleagues at the University of Michigan, Ann Arbor developed a proteolysis-targeting vaccine (PROTAV) that promoted both quantity and quality of CD8+ T cells against various peptides (Fig. 7) [8].
Fig 7. Using a method to efficiently and stably encapsulate proteins in the lumen of LNPs, protein therapeutics may be delivered to thousands of heretofore unavailable intracellular targets.
Image from Communications Materials 6.1 (2025): 34.
3.4 Small Molecules
The last major category of therapeutic molecules is broadly referred to as "small molecules". These may be either hydrophilic or hydrophobic drugs, and typically are no more than 1 kDa in size. LNPs provide a major advantage in extending the circulation time of small molecules through controlled release, while hydrophobic drugs in particular benefit from enhanced solubility.
As with other cargo types, small molecules may also be encapsulated with PreciGenome's NanoGenerator. Lamparelli and colleagues at the University of Salerno demonstrated superior size and encapsulation efficiency with the NanoGenerator Flex-M vs. traditional methods when loading LNPs with triiodothyronine (T3) (Fig. 8) [9].
Fig 8. Mean sizes, standard deviation, zeta potential, and drug loading (Triiodothyronine (T3) or FITC) of Phosphatidylcholine (PC) nano-vesicles obtained by microfluidic mixing. Values are reported as means ± standard deviation (SD) (n = 3).
Image from International Journal of Pharmaceutics 624 (2022): 122007.
4. LNP Formulation Design
Before producing LNPs, several interrelated factors must be considered during the formulation process. These will affect downstream properties such as encapsulation efficiency, particle size, and safety of the final product. Normally, lipid libraries and payload libraries are set up for formulation screening (Fig. 9).
Fig 9. Library preparation functions in the NanoGenerator Flex-S Plus automated high throughput LNP synthesis system. (A) Lipid library preparation. (B) Payload library preparation.
4.1 Lipid Formulation
The foundational architecture of lipid nanoparticles consists of a precise combination of four essential components, each contributing uniquely to the overall transfection capability. Understanding the role and optimal proportion of each component provides critical insights into designing high-efficiency LNP formulations. Ionizable cationic lipids represent the core functional component of modern LNP formulations (Fig. 10), facilitating both nucleic acid encapsulation and endosomal escape; Helper lipids and phospholipids play crucial roles in determining LNP membrane stability, fusion properties, and transfection efficiency; Cholesterol serves as a critical structural component in LNP formulations, enhancing membrane rigidity, reducing permeability, and improving overall particle stability; PEGylated lipids constitute a critical surface component that influences circulation time, colloidal stability, and immune recognition of LNPs.
Fig 10. Selected ionizable lipids under clinical development for RNA therapeutics. Ionizable lipids used in on-going clinical trials have not been publicly disclosed, so one of the possible structures. Image from Nature communications 12.1 (2021): 7233.
4.2 Lipid Molar Ratio
The molar ratio of the four primary lipid components will determine various attributes such as structural integrity, encapsulation efficiency, and particle size [10]. As previously described, these components are ionizable lipid, helper lipid, cholesterol, and PEGylated lipid. While each of these components has a generally accepted range for their molar ratio, the exact value will require optimization for the specific application at hand.
4.3 Lipid-Nucleic Acid Weight Ratio
When the payload of interest is a nucleic acid, the relative mass of lipid to nucleic acid is key for efficient encapsulation. Ideally, the ratio would permit complete or near-complete encapsulation with minimal empty LNPs and maximum payload per LNP [10]. Therefore, excess nucleic acid will reduce encapsulation efficiency, while excess lipid will lead to more empty LNPs.
4.4 N/P Ratio
The N/P ratio refers to the molar ratio of amine groups (N) to phosphate groups (P) in the system. Amine groups are present on ionizable lipid, while phosphate groups are present on nucleic acids. N/P ratio therefore reflects the charge balance of the system, with a higher ratio indicating more positive charge and a lower ratio a more negative charge [10]. While the optimal N/P ratio is payload dependent, it is generally around 3 for siRNA and 6 for mRNA.
4.5 Lipid pKa
The pKa of the ionizable lipid is the pH where it transitions between its charged and neutral state. As previously described, this phenomenon allows it to be positively charged under acidic conditions and neutral at physiological pH [10]. The ideal pKa will therefore typically range from 6.0 to 6.5, maximizing encapsulation while minimizing toxicity.
4.6 Aqueous Buffer Properties
Finally, while separate from the lipid phase, the aqueous phase is still important to consider during the overall formulation process. The choice of buffer used to suspend the payload will affect the assembly, stability, and delivery performance of LNPs. Some important properties include the composition (e.g. citrate, phosphate, acetate), pH, and ionic strength [11]. While several buffer compositions have been successfully used, the pH is nearly always mildly acidic during LNP synthesis to take advantage of the lipid pKa.
4.7 High-throughput Screening
High-throughput screening has emerged as a transformative approach in the development and optimization of lipid nanoparticles for RNA delivery, addressing the critical bottleneck of formulation optimization that has historically limited the pace of innovation in this field. The advanced platforms and methodologies have dramatically accelerated the discovery process, enabling researchers to efficiently navigate the vast parameter space of LNP formulation and identify optimal compositions for specific therapeutic applications (Fig. 11). From blood-brain barrier penetration to lung-targeted delivery, high-throughput screening has facilitated remarkable advances in tissue-specific LNP development. The continued evolution of high-throughput screening technologies, particularly through integration with artificial intelligence, advanced in vitro models, and structural analysis techniques, promises to further enhance the efficiency and effectiveness of LNP development. As these technologies mature, they will likely enable the creation of increasingly sophisticated delivery systems tailored to the unique requirements of different RNA therapeutics and target tissues. In an era where RNA-based medicines are poised to revolutionize the treatment of numerous diseases, high-throughput screening will remain an indispensable tool for translating the promise of these therapeutics into clinical reality.
Fig 11. NanoGenerator Flex-S Plus, an automated high throughput LNP synthesis system. (A) Instrument overview. (B) LNP screening result (size & PDI of 32 different samples) from NanoGenerator Flex-S Plus.
5. Basic LNP Preparation Workflow
Once the formulation is decided, LNPs may be produced and tested. This involves several critical steps that must be carefully controlled to ensure consistent particle size, payload encapsulation, and stability (Fig. 12).
Fig 12. LNP preparation workflow
5.1 Reagent Preparation
During reagent preparation, the lipid mix is dissolved in organic solvent (most often anhydrous ethanol) while the payload is dissolved in aqueous buffer (most often at weakly acidic pH.) At this stage, it is crucial that all reagents are RNase-free to protect RNA payloads from degradation. Additionally, the component ratio for each phase must be carefully considered as this will affect several critical quality attributes (CQAs) of the resulting LNPs.
5.2 LNP Synthesis
Once both reagent phases are prepared, they may be mixed under controlled conditions to produce LNPs. While many mixing methods are available, microfluidic mixing is increasingly common due to its several advantages for product uniformity and scalability (Fig. 13). Furthermore, it allows easy tuning of both the total flow rate (TFR) and flow rate ratio (FRR), which in turn may be used to optimize the resulting LNPs.
Fig 13. LNP synthesis with microfluidic chaotic mixing
5.3 Purification
After synthesis, the LNP product must be purified to reduce potential side effects and improve dosing consistency. This step will remove unwanted components such as unencapsulated payload, empty LNPs, and organic solvent. Several purification methods are available, but will generally fall under filtration, dialysis, or chromatography.
5.4 Characterization
Various CQAs must be analyzed after purification to verify LNP quality remains intact. The most common include size, polydispersity, surface charge, and encapsulation efficiency. Methods and assays used for characterization vary by the CQA of interest. Regardless, it remains important to incorporate several to gain a multi-faceted view of LNP quality, especially before administration to biological models.
5.5 Surface Modification
In some cases, it can be desirable to incorporate additional functional groups onto the LNP surface to improve targeted delivery or reduce unwanted immune responses. Examples include ligands such as antibodies or peptides, which may be embedded in the membrane, typically at the PEGylated lipids (Fig. 14).
Fig 14. Antibody conjugation on LNPs for targeted drug delivery
5.6 Assessment
Fig 15. Scaled up LNP synthesis systems for LNP assessment at different stages. Examples include LNP characterization, in vitro study, animal model, and human tests.
5.7 Storage
If the finished LNPs are not used immediately, they will need special storage conditions to prevent degradation. Generally, while purified LNPs may be stored at 4°C for up to 1 week, they will require lyophilization or storage at -80°C for the long term. This will also typically require cryoprotectants due to the very low temperatures.
Traditional approaches for LNP storage include specialized containers or glass vials, which can pose challenges at the commercial scale. One innovative solution was investigated by Takeda and colleagues at Sartorius Inc., where single-use Flexboy bags showed promising results at preserving LNPs produced with the PreciGenome Mix-4 mixing chip (Fig. 16) [12].
Fig 16. Stability study of mRNA in Flexboy bags. (A) mRNA concentration, was stable for six months. (B) Agarose gel electrophoresis indicates that mRNA integrity was maintained in both SU bags and cryovials at -80 °C for six months. (C, D) HeLa cells were transfected with cryopreserved mRNA using liposome. (C) mRNA effectively transfected cells after cryostorage for four months in both SU bags and cryovials, and EGFP signal retained for 72 h. (D) mRNA samples stored for six months showed reduced potency.
Image from Physics of Fluids 37.3 (2025).
References:
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Nanostructured Lipid Carriers for Improved Delivery of Therapeutics via the Oral Route - Wiley
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The Role of Lipid Components in Lipid Nanoparticles for Vaccines and Gene Therapy - PMC
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Optimization of Lipid Nanoformulations for Effective mRNA Delivery - PMC
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siRNA Functionalized Lipid Nanoparticles (LNPs) in Management of Diseases - PMC
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Proteolysis-targeting vaccines (PROTAVs) for robust combination immunotherapy of melanoma - bioRxiv
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Comprehensive analysis of lipid nanoparticle formulation and preparation for RNA delivery - PMC
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Leveraging biological buffers for efficient messenger RNA delivery via lipid nanoparticles - PMC
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Single-use bags as a viable solution for long-term stability of lipid nanoparticles - AIP
