The document summarizes the key components of lipid nanoparticles (LNPs), which are important for mRNA vaccine and nucleic acid therapy delivery. LNPs generally consist of four components - ionizable cationic lipids, phospholipids, cholesterol, and PEG lipids. Each component plays an important role in LNP stability, transfection efficacy, and safety. Ionizable cationic lipids determine LNP surface charge and toxicity. Phospholipids and cholesterol contribute to LNP formation and endosomal escape. PEG lipids influence particle size and stability but can also reduce efficacy and cause immune responses. The properties and roles of each component are described.

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The Role of Four Lipid Components Of LNPs
Lipid nanoparticles (LNPs) play an important role in COVID-19 mRNA vaccines. In
addition, many preclinical and clinical studies have shown that LNPs have great potential
in the field of nucleic acid therapy. The structural and biological properties of LNPs are not
solely attributable to a single lipid component, but rather a combination of various types of
lipids. Therefore, we will focus on describing the role of different lipid components in LNP
preparation.
Lipid Components of LNPs
Generally, LNP consists of four components: ionizable cationic lipids,
phospholipids, cholesterol, and PEG lipids (Figure 1). Each component plays a key
role in terms of LNP stability, transfection efficacy and safety.
Figure 1. Components of LNPs

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Ionizable Cationic Lipids
Ionizable cationic lipids are key components in the LNP formulation, and their acid
dissociation constants (pKa) determine the ionization and surface charge of LNP,
further affecting its stability and toxicity. Conventional permanently charged cationic
lipids previously used for nucleic acid delivery (e.g., DOTAP) readily interact with
negatively charged serum proteins and aggregate in the bloodstream, which can lead to
rapid clearance of LNP by mononuclear phagocytes, resulting in a short half-life in the
bloodstream. In addition, the relatively high hemolytic activity of cationic lipids increases
the risk of toxic side effects, such as hemoglobin release due to red cell membrane
damage. To avoid these problems, ionizable cationic lipids with pKa values typically
ranging from 6.0 to 7.0 have been developed. This ionizable lipid-based LNP (iLNP)
ensures efficient encapsulation of nucleic acids under acidic conditions and
reduces toxicity during recycling under physiological conditions. Entering
endosomes/lysosomes (pH below surface pKa), LNP can be positively charged again to
facilitate endosome escape and release mRNA into the cytoplasm. It was found that LNP
with pKa values of 6.2-6.5 and 6.6-6.9 favored hepatic delivery of siRNA in vivo and
intramuscular administration of mRNA vaccines, respectively.
Structurally, synthetic lipids usually contain three parts: (i) cationic or ionizable head
groups, (ii) linker groups, and (iii) hydrophobic tails. The chemical diversity of each part
results in a number of structurally distinct ionizable lipids that can be produced by
combinatorial chemistry. Depending on the number of amino heads, ionizable cationic
lipids can be classified as either monoamino or polyamino lipids. DLin-MC3-DMA (MC3),
SM-102, and ALC-0315 are the best known monoamino acid lipids. They are also the
three FDA-approved ionizable cationic lipids for RNA delivery. Researchers usually focus
on tuning the lipid tail structure to confer enhanced potency or specific functions by
changing the number of tails, designing linear or branching structures, and introducing
unsaturated or biodegradable bonds, e.g., the unsaturated tails in MC3 and ester bonds in
L319 play important roles in facilitating endosomal escape of siRNAs and accelerating
intracellular degradation of lipids. By providing larger head groups, polyamino-ionizable
cationic lipids have greater variability than monoamino-ionizable cationic lipids, and a

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number of multitude of lipids have been designed and studied, e.g., 306O i10, cKK-E12,
C12-200, 5A2-SC8, TT3, FTT5.
Figure 2. The ionizable lipids used Comirnaty® (ALC-0315) and Spikevax® (SM-102) and
in Onpattro® (MC3). Source: reference [1]
PEG-lipids
Even though PEG-lipids constitute the smallest molar percentage of the lipid components
in LNPs (typically ∼1.5 mol%), they have several effects on the properties of lipid
nanoparticles.
 1. The amount of PEG-lipids can influence particle size and zeta potential;
 2. PEG-lipids can better contribute to particle stability by decreasing particle aggregation;
 3. Certain PEG modifications extend the blood circulation time of nanoparticles by
lowering clearance mediated by the kidneys and the mononuclear phagocyte system
(MPS).
 4. PEG-lipids can be used to conjugate particular ligands to the particle for targeted
delivery.
The short chained diacyl PEG-lipid PEG-carbamate-1,2-dimyristoyl-sn-glycerol
(PEG-c-DMG) is introduced in the Onpattro® formulation, which is rapidly dissociated
from the lipid membrane. When dissociated from their PEG, LNPs can interact with blood
proteins, including ApoE proteins, for delivery to the hepatocytes. In this context, two short
lipids-PEG (C14) were recently used in the Comirnaty® and Spikevax® COVID-19
vaccines: ALC-0159 (DTA-PEG2000, ditetradecylacetamide) and DMG-PEG2000,
respectively.

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Figure 3. The PEG-lipids used in Onpattro® (PEG-c-DMG) , Spikevax® (PEG-DMG) and
Comirnaty® (ALC-0159). Source: reference [1]
However, PEGylation also presents a "PEG dilemma" that hinders interaction with
target cells and subsequent endosomal escape, resulting in reduced transfection
efficiency.
Accelerated blood clearance ("ABC phenomenon") is an unexpected immunogenic
response observed in PEG conjugates that results in rapid clearance of PEGylated
nanocarriers. The ABC phenomenon has been widely observed after repeated dosing and
reduces the effectiveness of PEG conjugates and nanocarriers.
Another unexpected immune response is the hypersensitivity reaction known
as complement activation-related pseudoallergy (CARPA), which significantly
reduces the safety of PEGylated nanocarriers and has been associated with reduced
efficacy of PEGylated treatments in clinical trials. The CARPA phenomenon has been
categorized as a non-IgE-mediated pseudoallergic reaction caused by activation of the
complement system.
Various parameters of the PEG–lipid chemical structure including PEG size and
architecture, lipid structure including hydrocarbon chains, headgroup, lipid–PEG linkage,
and PEG terminal groups, are significant determinants of the PEGylated LNPs safety and
bioactivity.

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Figure 3. Scheme of the PEG–lipid structure. Source: reference [3]
 ▶ PEG length is a key structural factor influencing the immunologic safety profile. The
effect is biphasic, with both long and short chain PEG conjugates being more likely to
induce the ABC phenomenon.
 ▶ Like PEG length, PEG density (i.e., the percentage of PEG in the LNP) also exhibits a
biphasic effect. However, both lower and higher densities of PEG exhibit a reduced ABC
phenomenon.
 ▶ Differences in PEG structure may have an effect, with branched PEG lipid conjugates
conferring a higher degree of invisibility to LNP compared to linear PEG.
 ▶ The functional terminal groups attached to the chain of the PEG particles are another
factor that affects their immunogenicity and clearance.
 ▶ Parameters such as size and surface charge also affect immunogenicity. For example,
PEGylated carriers containing negatively charged phospholipids are more capable of
stimulating the immune system through complement activation than uncharged vesicles.
 ▶ As with the PEG fraction, the structure and length of the hydrophobic chains of lipids can
determine not only the degree of immunogenicity but also its efficacy.
 ▶ PEGylated LNP produced using a specific lipid anchoring group (e.g., cholesterol as an
anchoring group) has more sustained permeability and higher systemic bioavailability in
circulation.
 ▶ Lipid linkage is an important parameter in the design and performance of lipids, where
ester bonds replace carbamate bonds for the formation of unstable vesicles.

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A thorough understanding of the correlation between the structural parameters of PEG
lipids and the immune-related adverse effects of PEGylated LNPs, as well as their
biophysical and physiological background, is an essential requirement. With this
knowledge, optimal drug formulations that reduce and eliminate adverse immune
reactions and improve the safety and efficiency of PEGylated drugs can be prepared.
Helper Lipids - Cholesterol
The inclusion of cholesterol in nucleic acid-containing LNP formulations is based primarily
on two major findings obtained with liposomal formulations of small molecule therapeutics:
1) cholesterol is an exchangeable molecule that can accumulate within liposomes during
circulation, 2) cholesterol dramatically reduces the amount of surface-bound proteins and
improves the circulating half-life. Therefore, equimolar amounts of cholesterol are
included in LNP formulations relative to endogenous membranes; this prevents net efflux
or influx and maintains membrane integrity.
Lipids such as cholesterol are also essential for encapsulating nucleic acids. Since
cholesterol increases membrane rigidity, it reduces drug leakage from the liposomal core.
However, for large cargo such as nucleic acids, this effect may not be important. As the
design of ionizable lipids has improved, providing more potency and less toxicity, the total
proportion of helper lipid has decreased. However, a recent study noted that a certain
threshold amount of helper lipids is still needed to promote stable encapsulation.
Biopharma PEG can supply high-quality cholesterol (plant-derived) from lab to large
scale to meet your research and drug development needs.

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Figure 4. The molecular shape of standard helper lipids, including DSPC, DOPE, and
cholesterol, used in LNPs. Source: reference [1]
Helper Lipids – Phospholipids
Phospholipids are helper lipids that contribute to the formation of lipid nanoparticles and
the escape of endosomes. Commonly used phospholipids in preclinical studies and
clinical applications are DSPC and DOPE. Phospholipids can spontaneously organize
into lipid bilayers and the higher phase transition temperature enhances the membrane
stability of LNPs. Similar to cell membranes, phospholipids are located at the periphery of
the LNP. These lipids are usually semisynthetic. For example, phosphatidylcholine is
usually derived from natural sources such as egg yolk and soybeans and can be
chemically modified (e.g., by adding fatty acid tails).
DSPC is a structural lipid used in LNPs for siRNA therapy (Onpattro) and SARS-CoV-2
mRNA vaccines. DSPC consists of a phosphatidylcholine head group and two saturated
18-carbon tails, with the two tails forming a tightly packed lipid bilayer. In LNP, DSPC is
mainly located on the surface of the nanoparticles and in more marginal positions in the
core of the nanoparticles. DOPE is another phospholipid commonly used in preclinical
studies of LNP. The unsaturated tails of DOPE not only form a more fluid lipid bilayer but
also are capable of forming a hexagonal-phase (HII) organized form, which facilitates the
fusion of lipid membranes with endosomal membranes, leading to cytoplasmic nucleic
acid release.

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Conclusion
Under the premise of consistent preparation conditions, the nucleic acid composition and
the proportion of lipids have a decisive influence on the structure of nanoparticles.
Understanding the function of various types of lipids is a prerequisite for the optimization
of LNP formulations, and the roles of four common components of LNPs are briefly
described in the paper.
Biopharma PEG is a dynamic science company dedicated to PEG derivatives. We are
capable of supplying small to large quantities of a rich selection of PEG derivatives with
GMP & non-GMP standards for your mRNA vaccine R&D. We can produce and provide
classic PEG lipids, such as mPEG-DMG, ALC-0159 and mPEG-DSPE for your LNPs
R&D.
References:
[1] Hald Albertsen C, Kulkarni JA, Witzigmann D, Lind M, Petersson K, Simonsen JB. The
role of lipid components in lipid nanoparticles for vaccines and gene therapy. Adv Drug
Deliv Rev. 2022 Sep;188:114416. doi: 10.1016/j.addr.2022.114416. Epub 2022 Jul 3.
PMID: 35787388; PMCID: PMC9250827.
[2] Hou, X., Zaks, T., Langer, R. et al. Lipid nanoparticles for mRNA delivery. Nat Rev
Mater 6, 1078–1094 (2021). https://doi.org/10.1038/s41578-021-00358-0
[3] PEGylated Lipid Nanoparticle Formulations: Immunological Safety and Efficiency
Perspective, Rumiana Tenchov, Janet M. Sasso, and Qiongqiong Angela Zhou,
Bioconjugate Chemistry 2023 34 (6), 941-960, DOI: 10.1021/acs.bioconjchem.3c00174
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