COMMENT

https://doi.org/10.1038/s41467-021-27493-0 OPEN

An ionizable lipid toolbox for RNA

delivery
Xuexiang Han1,8, Hanwen Zhang1,8, Kamila Butowska1,2, Kelsey L. Swingle 1,

Mohamad-Gabriel Alameh 3, Drew Weissman3 &

Michael J. Mitchell 1,4,5,6,7 ✉
1234567890():,;

Recent years have witnessed incredible growth in RNA therapeutics, which has
benefited significantly from decades of research on lipid nanoparticles, specifi-
cally its key component—the ionizable lipid. This comment discusses the major
ionizable lipid types, and provides perspectives for future development.

Need for ionizable lipids

Broadly speaking, ribonucleic acid (RNA) therapeutics include antisense oligonucleotides
(ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), messenger RNAs (mRNAs),
and single-guide RNAs (sgRNAs)-mediated CRISPR-Cas9 system, which can manipulate
essentially any gene of interest through distinct modes of action1. However, RNA therapeutics
are susceptible to nucleases and cannot permeate cells due to their large size and negative charge.
Delivery of RNAs to target cells by clinically translatable lipid nanoparticles (LNPs) provides vast
opportunities to tackle a series of life-threatening diseases including COVID-192. LNPs typically
consist of four components—ionizable lipid, phospholipid, cholesterol, and PEGylated lipid,
among which, the ionizable lipid plays a major role in protecting RNAs and facilitating their
cytosolic transport. Ionizable lipids are positively charged at acidic pH to condense RNAs into
LNPs, but are neutral at physiological pH to minimize toxicity. They can be protonated in the
acidic endosome after cellular uptake, and interact with anionic endosomal phospholipids to
form cone-shaped ion pairs that are not compatible with a bilayer (Fig. 1). These cationic-
anionic lipid pairs drive the transition from the bilayer structure to the inverted hexagonal HII
phase, which facilitates membrane fusion/disruption, endosomal escape and cargo release into
the cytosol3. Since 2008, ionizable lipids with diverse chemical identities have been created.
Systematic categorization of these lipids based on their structures can greatly benefit the field and
facilitate the development of next-generation ionizable lipids. Currently, there are five major
ionizable lipid types that are widely used for RNA delivery (Fig. 1).

Unsaturated ionizable lipids

Tail saturation greatly influences the fluidity and delivery efficiency of ionizable lipids. Increasing
the tail unsaturation from 0 to 2 cis double bonds correlates with an increased tendency for
bilayer lipids to form a nonbilayer phase4, leading to enhanced membrane disruption and

1 Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA. 2 Laboratory of Biophysics, Intercollegiate Faculty of Biotechnology,

University of Gdańsk and Medical University of Gdańsk, Abrahama 58, 80-307 Gdańsk, Poland. 3 Department of Medicine, University of Pennsylvania,
Philadelphia, PA 19104, USA. 4 Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. 5 Institute
for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. 6 Cardiovascular Institute, Perelman School of
Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. 7 Institute for Regenerative Medicine, Perelman School of Medicine, University of
Pennsylvania, Philadelphia, PA 19104, USA. 8These authors contributed equally: Xuexiang Han, Hanwen Zhang. ✉email: mjmitch@seas.upenn.edu

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Fig. 1 Mechanism for endosome disruption by ionizable lipids and five major structural classes of ionizable lipids. The cone-shaped ion pairs formed by
anionic endosomal phospholipids and protonated ionizable lipids can disrupt the bilayer structure to promote endosomal escape. Based on their structural
properties, RNA-delivering ionizable lipids can be categorized into unsaturated (containing unsaturated bond), multi-tail (containing more than two tails),
polymeric (containing polymer or dendrimer), biodegradable (containing biodegradable bond) and branched-tail (containing branched tail) ones.

payload release. For this reason, the linoleyl tail was chosen as the deliver mRNA vaccines robustly but also activate the stimulator
structural basis during the optimization process of the ionizable of interferon genes (STING) pathway9. However, the inclusion of
lipid DLin-MC3-DMA (MC3)3,5. This ionizable lipid confers unsaturated bonds in the ionizable lipid does not always corre-
robust hepatic gene silencing and is used in the first FDA- spond with potent in vivo RNA delivery7,8,10, indicating that both
approved siRNA drug Onpattro® for the treatment of hereditary rational design and screening are necessary.
transthyretin amyloidosis (hATTR)6. Importantly, both the
structure and formulation of MC3 laid the groundwork for fur-
ther LNP development. Multi-tail ionizable lipids
Unsaturated ionizable lipids have also been demonstrated to Multi-tail ionizable lipids are structurally distinguished from
enhance mRNA delivery. Anderson and colleagues showed a two-tail ones by having three or more tails. Such ionizable lipids
linoleic acid-derived ionizable lipid (OF-02) achieved the highest are expected to produce a more cone-shaped structure with
hepatic mRNA delivery and protein expression among its class7. enhanced endosome-disrupting ability due to the increased cross-
Later, they demonstrated that an alkyne ionizable lipid (A6) could section of the tail region. Multi-tail ionizable lipids can be easily
greatly increase membrane fusion and facilitate albumin- synthesized by combinatorial chemistry and subjected to high-
mediated mRNA delivery when incorporated into benchmark throughput screening. Since the first combinatorial library created
LNPs (e.g., MC3)8. Interestingly, in a heterocyclic ionizable lipid in 2008, several multi-tailed leads (e.g., 98N12-5, C12-200, and
library prepared by a three-component reaction, they identified cKK-E12) have been identified to potently knockdown hepatic
an alkylene ketone-derived A18-Iso5-2DC18 that could not only genes11–13.

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Although initially developed for siRNA delivery, multi-tail Notably, the position and steric effect of the ester groups can
ionizable lipids can be repurposed for efficient mRNA delivery by greatly affect ionizable lipid clearance and potency30,31.
optimizing the LNP composition via design of experiment Alternative to rational design, diversely structured biodegradable
methodologies with significantly reduced workload14,15. For ionizable lipids can be combinatorially synthesized using alkyl
example, the optimized formulation of C12-200 increased mRNA amines and acrylate tails11,32. The analysis of structure–activity
expression 7-fold compared to the standard formulation14. Later, relationships indicated that acrylate-based ionizable lipids with
this formulation was widely adopted by multi-tail ionizable lipids tertiary amines, at least three O13 tails, and a suitable pKa (5.5–7.0)
for various mRNA delivery purposes such as chimeric antigen confer robust in vivo gene silencing32. The lead ionizable lipid
receptor T-cell engineering and prenatal protein replacement (304O13) had similar potency to the non-degradable C12-200
therapy16,17. Recently, a class of multi-tail ionizable phospholi- benchmark, but posed much lower toxicity at high doses. It is
pids was created; the top-performing 9A1P9, comprising one noteworthy that ester-containing ionizable lipids typically demon-
zwitterionic head and three tails, assisted to promote membrane strate lower potency compared to non-degradable analogs due to
destabilization and cargo release, which greatly enhanced LNP- inefficient delivery caused by rapid hydrolysis3,11. This suggests an
mediated tissue-selective mRNA delivery and gene editing18. activity-degradability tradeoff that needs to be balanced to max-
Nevertheless, lead multi-tail ionizable lipids often have stable imize total benefits. One viable approach to overcome this dilemma
backbones and low degradability, so their toxicity and immuno- is adopting the more stable secondary ester instead of the primary
genicity should always be taken into consideration19. one31. Interestingly, OF-Deg-Lin, a degradable analog of OF-02,
selectively transfected splenic lymphocytes and induced >85% of
total protein expression in the spleen (an organ with a relative low-
Ionizable polymer-lipids abundance of hydrolases)33, suggesting that degradable ionizable
Substitution of free amines on cationic polymers with alkyl tails lipids could be promising vectors for spleen transfection.
affords ionizable polymer-lipids, which can enhance particle To further improve biodegradability and accelerate RNA
formation through hydrophobic aggregation. From 500 structu- release, disulfide bonds that are sensitive to the reductive intra-
rally diverse synthetic polymer-lipid hybrids, Anderson et al. cellular environment can be incorporated into acrylate tails34.
identified that C15 epoxide-modified low-molecular-weight The resulting bioreducible ionizable lipids have been shown to
polyethyleneimine (7C1) conferred the most efficient non-liver efficiently deliver ASOs and CRISPR/Cas9 systems for gene
siRNA delivery20. 7C1 preferentially transfected endothelial cells silencing and editing with good tolerability in mice35–37. For
—rather than hepatocytes and pulmonary epithelial cells—in example, 306-O12B outperformed MC3 in CRISPR-Cas9–based
multiple organs, with the highest endothelial gene silencing in the liver genome editing of Angptl3, and negligible toxicity or off-
lung. In non-human primates, ~80% endothelial gene knockdown target mutagenesis was observed38. Despite the promise of dis-
in the lungs was achieved by 7C1 without significant toxicity21, ulfide bond-containing ionizable lipids, the difficulty of synthesis
indicating its potential to treat dysfunctional endothelium-related and risk of premature release could limit their applications.
diseases. Recently, its re-optimized formulation was further
demonstrated to achieve potent gene silencing in bone marrow
Branched-tail ionizable lipids
endothelial cells, which could further regulate hematopoietic cell
Along with tail length and saturation, tail branching can greatly
activity22.
influence ionizable lipid performance. Ionizable lipids with metha-
Additionally, lead ionizable polymer-lipids derived from
crylate tails (a 1C branch near the head) generally showed reduced
poly(amido amine) and poly(propylenimine) dendrimers have
efficacy compared to acrylate-based ones39. Interestingly, ionizable
also shown strong avidity to transfect liver endothelium and lung
lipids containing isodecyl acrylate (Oi10, a 1C branch at the end)
vasculature23,24, implying preferential transfection of endothe-
dramatically boosted hepatic mRNA expression (>10-fold) compared
lium for polycation-based ionizable lipids. However, such cell
to their isomers with linear tails, despite their similar
population tropism can be varied depending on the LNP
biodistribution40. This increased potency can be explained by
formulation23,25. For example, when co-formulated with other
enhanced endosomal escape due to the stronger protonation of
accessory excipients, G0-C14 confers high accumulation and
spaced ionizable lipids at endosomal pH and the increased cross-
effective transfection of various RNA therapeutics in tumors26–29,
section of the lipid tails, allowing for adoption of a more cone-shaped
demonstrating the promise of LNPs for cancer therapy. Never-
structure.41 The lead ionizable lipid (306Oi10) efficiently co-delivered
theless, ionizable polymer-lipids, even after purification, usually
multiple RNA constructs to the liver and transfected >80% of
comprise a mixture of different substitution compounds20,23,
hepatocytes, Kupffer and endothelial cells, demonstrating the
which increase their complexity. Moreover, the toxic polycation
potential for integrating different therapeutic modalities (e.g., gene
core and non-degradable backbone pose extra hurdles for clinical
silencing, expression, and editing) to treat liver dysfunction42.
translation.
Recently, Dong and coworkers developed ionizable lipids with
branched ester chains that had higher transfection efficiency in the
liver than their analogs with linear ester chains, presumably due to
Biodegradable ionizable lipids
the slower degradation rate of the secondary ester10,31. Their top-
In order to reduce accumulation and potential side effects,
performing ionizable lipid (FTT5) demonstrated efficient delivery
ionizable lipids should be readily degraded into non-toxic meta-
of large mRNA constructs for protein supplementation and base
bolites after successful intracellular cargo delivery, which is
editing therapies. Notably, increased tail branching is one of the
especially crucial for RNA therapeutics requiring repeated dosing.
major features investigated by LNP companies31.
A common strategy to introduce biodegradability into ionizable
lipids is through the inclusion of ester bonds that are stable at
physiological pH, but are enzymatically hydrolyzed within tissues Clinical development of ionizable lipids
and cells. For example, due to the slow degradability of the dili- Before the approval of Onpattro® in 2018, scientists have screened
noleyl tail in MC3, a biodegradable substitution (L319) was numerous ionizable lipids and LNP formulations for more than a
created by replacing one of the double bonds in each tail with a decade. The success of MC3 has re-ignited the enthusiasm for
primary ester30. L319 not only maintained in vivo potency, but RNA delivery and greatly accelerated the clinical development of
also displayed rapid elimination and improved tolerability. other LNP-based RNA therapeutics, particularly mRNA vaccines

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Fig. 2 Selected ionizable lipids under clinical development for COVID-19 mRNA vaccines and other RNA therapeutics. Ionizable lipids used in on-going
clinical trials have not been publicly disclosed, so one of the possible structures (Acuitas A946, Arcturus Lipid 2,2 (8,8) 4C CH347, Genevant CL148 and
LP0149,50) is shown, respectively. i.v. intravenous, i.m. intramuscular.

(Fig. 2). Specifically, the COVID-19 pandemic has caused mil- targeting transthyretin (TTR) and Cas9 mRNA led to a 87%
lions of deaths. However, in less than one year, two LNP-based reduction of serum TTR in patients with hATTR, and only few or
mRNA vaccines (mRNA-1273 and BNT162b2) underwent mild adverse events were observed. This study heralds an era for
unprecedented speed of development and received the historic LNP-mediated in vivo gene editing.
approval for emergency use after demonstrating protection effi- When reviewing the approved MC3, SM-102, and ALC-0315
cacies above 94%43,44. Interestingly, Moderna’s SM-102 and structures as well as those patented by Acuitas et al.46–48,50, the
BioNTech’s Acuitas ALC-0315 have some shared features, ester-based biodegradable structure appears in every ionizable
including a tertiary amine, branched tails and ester linkers. lipid; the next most commonly shared features are the multi/
Moreover, both of them bear extended aliphatic branches, making branched-tail and unsaturated structures. This suggests that
them appear like multi-tail structures. Currently, additional biodegradability is a key feature for the clinical translation of
COVID-19 mRNA vaccines delivered by LNPs are undergoing ionizable lipids. Additionally, increased branching or tail number
clinical development. Although these ionizable lipid structures is a common characteristic for newly developed ionizable lipids.
have not been publicly disclosed, the probable ones are shown However, these multi/branched-tail ionizable lipids typically
based on available patents and literature (Fig. 2). possess only one tertiary amine, which is a sharp contrast to those
Recently, positive results from the first human trial of LNP- discussed in Fig. 1. Interestingly, while the unsaturated structure
enabled CRISPR-Cas9 in vivo gene editing were disclosed45. A is important to ionizable lipids with long tails, it appears less
single dose of LP000001-formulated LNPs carrying sgRNA important for those with multiple short tails.

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