A Systematic Study of Unsaturation in Lipid Nanoparticles Leads to Improved mRNA Transfection In Vivo

Article abstract of DOI:10.1002/anie.202013927

Lipid nanoparticles (LNPs) represent the leading concept for mRNA delivery. Unsaturated lipids play important roles in nature with potential for mRNA therapeutics, but are difficult to access through chemical synthesis. To systematically study the role of unsaturation, modular reactions were utilized to access a library of 91 amino lipids, enabled by the synthesis of unsaturated thiols. An ionizable lipid series (4A3) emerged from in vitro and in vivo screening, where the 4A3 core with a citronellol-based (Cit) periphery emerged as best. We studied the interaction between LNPs and a model endosomal membrane where 4A3-Cit demonstrated superior lipid fusion over saturated lipids, suggesting its unsaturated tail promotes endosomal escape. Furthermore, 4A3-Cit significantly improved mRNA delivery efficacy in vivo through Selective ORgan Targeting (SORT), resulting in 18-fold increased protein expression over parent LNPs. These findings provide insight into how lipid unsaturation promotes mRNA delivery and demonstrate how lipid mixing can enhance efficacy.

Full text of DOI:10.1002/anie.202013927

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
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Accepted Article
Title: A Systematic Study of Unsaturation in Lipid Nanoparticles Leads
to Improved mRNA Transfection In Vivo
Authors: Sang Lee, Qiang Cheng, Xueliang Yu, Shuai Liu, Lindsay
Johnson, and Daniel John Siegwart
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202013927
Link to VoR: https://doi.org/10.1002/anie.202013927

10.1002/anie.202013927
Angewandte Chemie International Edition
COMMUNICATION
A Systematic Study of Unsaturation in Lipid Nanoparticles Leads
to Improved mRNA Transfection In Vivo
Sang M. Lee, Qiang Cheng, Xueliang Yu, Shuai Liu, Lindsay T. Johnson, Daniel J. Siegwart*
S.M. Lee, Dr. Q. Cheng. Dr. X. Yu, Dr. S. Liu, L.T. Johnson, Professor D.J. Siegwart
Department of Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd., Dallas, Texas, 75390-9038 (USA)
Email: daniel.siegwart@utsouthwestern.edu
Supporting information for this article is given via a link at the end of the document.
The universal importance of unsaturated lipids in natural membranes support
a LNP design emulating such a chemical composition. However, there has
been no robust study on the optimization of unsaturation for LNPs nor its
purpose.
Abstract: Lipid nanoparticles (LNPs) represent the leading concept for
mRNA delivery. Unsaturated lipids play important roles in nature with
potential for mRNA therapeutics but are difficult to access through chemical
synthesis. To systematically study the role of unsaturation, modular reactions
were utilized to access a library of 91 amino lipids, enabled by the synthesis
of unsaturated thiols. An ionizable lipid series (4A3) emerged from in vitro
and in vivo screening, where the 4A3 core with a citronellol-based (Cit)
periphery emerged as best. We studied the interaction between LNPs and a
model endosomal membrane where 4A3-Cit demonstrated superior lipid
fusion over saturated lipids, suggesting its unsaturated tail promotes
endosomal escape. Furthermore, 4A3-Cit significantly improved mRNA
delivery efficacy in vivo via Selective ORgan Targeting (SORT), resulting in
18-fold increased protein expression over parent LNPs. These findings
provide insight into how lipid unsaturation promotes mRNA delivery and
demonstrate how lipid mixing can enhance efficacy.
To study the role of unsaturated lipids in LNPs, we focused on an
ionizable amino lipid platform created using modular dendrimer growth
reactions, where we could systematically introduce unsaturation. Our lipid
design consisting of an ionizable amine core, ester-based degradable linker,
and alkyl thiol tail periphery[10] was revisited with attention on unsaturation
for the periphery. To incorporate unsaturation into the ionizable lipid, we
synthesized alkenyl thiols and inserted them as the hydrophobic tail domain,
mimicking natural fatty acids. These newly synthesized lipids were
formulated into LNPs and compared to their saturated parents. In doing so,
we aimed to understand why unsaturation may be important for LNPs and
explore the potential applications of unsaturated LNPs.
Modular reactions were utilized to create a chemically diverse library of
unsaturated amino lipids. Our synthetic route towards the ionizable lipid
involves a nucleophilic amine addition to an ester-based linker, followed by
a Michael addition with the thiols. Previous studies solely involved alkyl
thiols because unsaturated thiols are not commercially available. Thus, we
hypothesized bridging this gap by synthesizing unsaturated thiols from
alkenyl alcohols and terpenes found in nature (Scheme 1A). However, initial
attempts for the unsaturated thiols proved difficult, resulting in undesired
products and low yields.
After employing multiple strategies, including the Mitsunobu
reaction/reduction and Bunte salt, two methods emerged as optimal based on
the alcohol, reaction scale, and yield (Figure S1). For most allylic alcohols,
conversion of the alcohol to a bromide and subsequent reaction with NaSH
provided thiols at 48% to 91% yield. For non-allylic alcohols and farnesol,
tosyl protection of the alcohol and subsequent treatment with NaSH afforded
the desired thiols at 19% to 67% yields. Thiol nomenclature was based on
the carbon chain length (6/8), position of unsaturation (2-5), configuration
(cis/trans), and/or their natural product derivative (Citronellol, Nerol, and
Farnesol). In tandem, seven ionizable amines were selected as candidates
based on the established optimal pKa of 6.2-6.5 for ionizable lipids.[11] The
amines were prepared by reacting with an ester-based linker synthesized as
previously described.[10] With the amine cores and thiols in hand, appropriate
equivalents of thiols per amine were combined with the modified amines and
dimethyl phenyl pyridine as a catalyst to create the desired ionizable,
unsaturated lipids (Scheme 1B).
Beyond functioning as a link between the genetic code of DNA and
functional proteins, messenger mRNA (mRNA) has emerged as a versatile
tool to produce proteins for therapeutic applications towards cancer, vaccines,
and other areas.[1] However, the major challenge of RNA therapeutics
remains efficacious delivery. mRNAs are incapable of passing through
cellular membranes on their own due to their physiochemical attributes and
propensity for degradation. Methods are needed to encapsulate and deliver
mRNA inside cells.[2] To address this challenge, lipid nanoparticles (LNPs)
represent the leading concept for mRNA delivery. LNPs are composed of
multiple lipids, including ionizable amino lipids, which acquire charge
during endosomal maturation and allow endosomal escape of RNA into the
cytoplasm to enable delivery of the genetic material.[3]
LNPs were initially established as carriers for siRNAs,[4] and have
increasingly been explored for delivery of mRNA.[1a, 5] Studies have shown
that optimization of LNP carriers for mRNA versus siRNA often require
chemical changes to the lipids involved,[4c, 6] as well as the molar relationship
of lipids that compose of the formulations.[7] Despite this progress, much
remains unknown about structure-activity relationships (SAR) uniting
chemistry and efficacy for mRNA delivery. Recently, the field of mRNA
therapeutics has started to include unsaturated lipids in LNPs in the form of
natural unsaturated fatty acids and its motifs.[8] In nature, unsaturated lipids
play a pivotal role in maintaining the stability and fluidity of membranes.[9]
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10.1002/anie.202013927
Angewandte Chemie International Edition
COMMUNICATION
A
Ts-Cl
Pyridine
DMAP
NaSH * X H2O
PBr3
NaSH * X H2O
R
OH
R
OTs
R
SH
R
OH
4T
R
Br
R
SH
DCM
DMF
0°C
DCM
0°C
DMF
0°C
0°C-RT
R = Unsaturated Hydrocarbon Chain
SH
Me
SH
Me
SH
Me
SH
5
2T
-Br: 47%
-SH: 48%
3T
-OTs: 90%
-SH: 20%
-OTs: 99%
-SH: 24%
-OTs: 92%
-SH: 42%
SH
Me
Me
Me
2C
-Br: 65%
-SH: 53%
3C
-OTs: 82%
-SH: 19%
SH
4C
-OTs: 83%
-SH: 30%
SH
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
SH
Me
SH
Cit-SH
Ne-SH
(Nerol)
-Br: 86%
-SH: 66%
Far-SH
SH
SH
8/2
-Br: 54%
-SH: 91%
(Citronellol)
-OTs: 94%
-SH: 53%
(Farnesol)
-OTs: 61%
-SH: 67%
O
B
O
Me
Me
O
P
G1
G1
G1 SR
G1 SR
(G1)
BHT
N2, 50°C
R
SH
O
Me
NH2
N
N
DMPP
DMPP
50°C
Me
N
Me
N
H
N
NH2
N
NH2
NH2
NH2
HO
Me
NH2
Me
2A2: 54% 3A4: 48%
2A9: 59% 4A1: 36%
2A9V: 61% 4A3: 44%
6A3: 69%
N
2A2
N
2A9
2A9V
3A4
Me
Me
Me
SH
SH
N
NH2
Me
N
SC6
H2N
H2N
NH2
N
H2N
NH2
4A1
4A3
6A3
2A2: 16% 3A4: 5%
2A9: 23% 4A1: 10%
2A9V: 15% 4A3: 6%
6A3: 9%
2A2: 39% 3A4: 30%
2A9: 36% 4A1: 22%
2A9V: 36% 4A3: 57%
6A3: 36%
2A2: 39% 3A4: 30%
2A9: 50% 4A1: 60%
2A9V: 43% 4A3: 46%
6A3: 46%
Me
SH
Me
Me
SH
2T
2C
5
3T
4T
2A2: 7% 3A4: 28%
2A9: 2% 4A1: 7%
2A9V: 3% 4A3: 62%
6A3: 11%
2A2: 31% 3A4: 13%
2A9: 41% 4A1: 56%
2A9V: 37% 4A3: 48%
6A3: 37%
2A2: 22% 3A4: 32%
2A9: 22% 4A1: 5%
2A9V: 14% 4A3: 12%
6A3: 21%
SH
Me
Me
SH
SH
3C
4C
2A2: 34% 3A4: 18%
2A9: 48% 4A1: 56%
2A9V: 61% 4A3: 15%
6A3: 43%
2A2: 57% 3A4: 27%
2A9: 59% 4A1: 35%
2A9V: 66% 4A3: 39%
6A3: 60%
2A2: 42% 3A4: 24%
2A9: 40% 4A1: 61%
2A9V: 42% 4A3: 39%
6A3: 39%
SH
Me
SH
Me
Me
SH
SH
SC8
SC8/2
Me
Me
2A2: 28% 3A4: 5%
2A9: 50% 4A1: 7%
2A9V: 40% 4A3: 4%
6A3: 35%
Me
Me
2A2: 4% 3A4: 5%
2A9: 21% 4A1: 7%
2A9V: 19% 4A3: 4%
6A3: 9%
Me
Me
2A2: 34% 3A4: 8%
2A9: 18% 4A1: 6%
2A9V: 14% 4A3: 8%
6A3: 4%
Me
Me
2
Cit-SH
(Citronellol)
Ne-SH
(Nerol)
Far-SH
(Farnesol)
SH
SH
Scheme 1. Synthesis of unsaturated thiols allowed access to unsaturated lipids. A) Scheme and reaction scope for synthesis of alkenyl thiols using non-allylic
and allylic alcohols. B) Scheme and reaction scope for synthesis of ionizable amino lipids using 7 different amine cores with isolated yields reported.
The new lipids were formulated into LNPs using a mix of the
synthesized lipid, DOPE, cholesterol, and DMG-PEG2k (15:15:30:3,
mol:mol), encapsulating Firefly Luciferase (Luc) mRNA. This ratio was
selected based on parameters previously optimized for mRNA delivery.[7c]
We evaluated the LNPs in IGROV-I cells (25 ng/well) for cell viability and
Luc expression (Figure 1). Assessment of the heat map allowed for
determination of SARs with respect to hydrophobic domain and amine core
chemical structure. While introducing unsaturation in the tail produced some
lipids with improved transfection over parent compounds, this chemical
alteration was not a change that automatically and universally improved
performance. Rather, the placement and cis/trans configuration of the
unsaturation were important in determining whether in vitro mRNA delivery
would be improved. For example, 8/2 showed comparable Luc expression to
its saturated counterpart (SC8). Among all cores examined in the initial
screen, 4A3 was the most efficacious. Therefore, we selected the entirety of
the 4A3 series to further evaluate its capabilities and study SAR with respect
to the hydrophobic domain.
Figure 1. 4A3-derived lipids performed best across the lipid series for Luc mRNA delivery to IGROV-I cells. A) Bar graph of the in vitro Luc expression
assay shows 4A3-4T as the most potent. B) Heat map of the in vitro Luciferase assay data revealed differences based on position and configuration of the
unsaturation.
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10.1002/anie.202013927
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COMMUNICATION
Next, we evaluated the 4A3 series to assess how the unsaturation
variations affected mRNA delivery in vivo, which is the more relevant setting
for mRNA therapeutic testing. LNPs were formulated using the same
components for in vitro testing in the same molar ratio as described above.
C57BL/6 mice were administered with each of the 4A3-based LNP series
carrying Luc mRNA via tail-vein injections (Figure 2). Clear differences
from the in vitro data appeared. While the six-carbon chain series showed
few significant differences amongst each other, the eight-carbon tail series
exhibited striking distinctions.
duo suggests that the “stiffness” of the components and its ability to promote
membrane permeability may be an important factor in improving mRNA
delivery when tailoring to its unsaturation. With 4A3-8/2, it exhibited lower
Luc expression compared to its alkyl partner, suggesting again that the
introduction of unsaturation alone is not sufficient to increase efficacy.
Efficacy did not significantly correlate with size, PDI, or encapsulation
efficiency (Figure S5), suggesting that chemical structure was the major
driver of efficacy. Moreover, the introduction of unsaturation did not change
the biodistribution of 4A3-SC8 and saturated 4A3-Cit based LNPs (Figure
S8). As endosomal escape of LNPs remains a major challenge that correlates
with amino lipid chemistry, we postulated that these LNPs may differ based
on their ability to escape the endosome.[3]
Despite being structurally similar, 4A3-Cit performed better than 4A3-Ne,
differing only by a prenyl motif per tail. This disparity is likely due to the
increased rigidity based on this structural difference. The comparison of the
Figure 2. Ex vivo imaging shows highest mRNA expression in eight carbon series (0.25 Luc mg/kg). A) Whole body images 6 hours after i.v. administration
of LNPs. B) Ex vivo organs imaged 6 hours after administration of LNPs. C) Quantified total radiance of the liver. D) Representation of the 4-component
standard LNP in vivo formulation method.
To gain mechanistic insight into this foray, the following assay was
conducted to study how our LNPs interacted with a model endosomal
membrane. We employed a fluorescence resonance energy transfer (FRET)-
based assay to determine the LNP’s ability to disrupt and fuse with the
endosomal membrane (Figure 3). DOPE-conjugated FRET probes (NBD-
PE and N-Rh-PE) were formulated into endosome-mimicking nanoparticles.
NBD is normally quenched by the rhodamine, but the NBD signal would rise
if disruption of the membrane occurred.. In accordance with the in vivo data,
4A3-Cit proved to be the best. As citronellol is a known membrane disrupter,
we believe 4A3-Cit’s unique structure promotes better endosomal escape
than the other LNPs.[12]
internalized at 4 hours and 24 hours (Figure S10 A and B). There were
differences at 4 hours, with the two unsaturated lipids (4A3-Far and 4A3-Cit)
LNPs internalizing slightly faster than the saturated (4A3-SC8) LNPs. We
further tracked the progression of Cy5 mRNA from the cell membrane to
endosomes to lysosomes and quantified co-localization of Cy5 mRNA with
lysosomes. Interestingly, there was significantly higher colocalization
between 4A3-Far and lysosomes compared to 4A3-SC8 and 4A3-Cit. These
results suggest that 4A3-Far LNPs lead to mRNA accumulation in lysosomes
and unproductive delivery. Moreover, the reduced colocalization with
lysosomes for 4A3-SC8 and 4A3-Cit LNPs helps to explain why they are
efficacious. The overall results connect chemical structure to factors
including cellular uptake, endosomal escape, and intracellular trafficking.
Building on the discovery of unsaturated 4A3-Cit and understanding of
increased lipid fusion, we sought to explore how 4A3-Cit could be used to
enhance mRNA delivery in vivo to the liver. We recently reported a
technology called Selective Organ Targeting Nanoparticles (SORT) for
selective mRNA delivery to different tissues.[13] In one application, mixing
two ionizable lipids into a 5-component LNP increased delivery efficacy to
[13a, 14]
the liver.
We hypothesized that 4A3-SC8 and 4A3-Cit could be
combined into a SORT LNP to engineer improved lipid formulations for the
liver. We previously showed that mRNA delivery efficacy and tissue tropism
correlated with the chemical identity and percent incorporation of the added
SORT molecule while keeping the molar amount of other molecules constant.
We hypothesized increasing the occupied percentage of our unsaturated lipid
would promote endosomal escape and improve mRNA delivery. Thus, the
following primary formulations were devised: 4A3-SC8 with additional
parent lipid, 4A3-Cit with additional parent lipid, and a mixture of the two
lipids where one would serve as the parent lipid and the other served as
additional supplemental SORT lipid (Figure S2).ꢀꢀ
Figure 3. A lipid fusion assay shows 4A3-Cit with the highest lipid fusion
percent. A) Visual assay representation showing the emission changes based
on fusion. B) Percent of lipid fusion of LNPs with the model endosomal
membrane.
To further examine cellular uptake and intracellular trafficking, we
performed in vitro experiments using Cy5-labeled mRNA LNPs. The results
showed that 4A3-SC8, 4A3-Far, and 4A3-Cit LNPs are all effectively
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10.1002/anie.202013927
Angewandte Chemie International Edition
COMMUNICATION
First, we aimed to optimize the SORT lipid required to improve mRNA
transfection by evaluating formulations across a range of SORT lipid
amounts. Additional 4A3-SC8 yielded only mild improvements (Figure S3),
but formulations involving additional Cit achieved significant improvements
(Figure 4). Once we identified the ideal percentages for the SORT lipids (+5
and established benchmarks including 5A2-SC8 LNPs and DLin-MC3-
DMA LNPs (Figure S9). Interestingly, 8+Cit (30%) performed worse than
its Cit (+30%) counterpart, and the reverse was observed with its 20% variant.
The data revealed that improvement is accessible by increasing occupancy
of the 4A3-Cit, but there is a threshold for the additive effect of Cit. The
% SC8 and +20/30% of Cit), we tested the cross-over mixtures. Cit+SC8 (5%) comparison of the average luminescence suggests that there needs to be a
performed only slightly better than its parent formulation (Figure S3), but
SC8+Cit (20%) surpassed all others with an 18-fold increase of the average
luminescence. SC8+Cit (20%) was also superior to its saturated parent lipid
careful balance of ionizable lipids for LNPs to achieve successful increased
transfection.
Figure 4. 4A3-SC8 + Cit (20%) improved Luc mRNA expression in liver 18-fold over the saturated base LNP formulation (4A3-Cit). A) Optimization of Cit
SORT lipid. B) Evaluation of cross-over mix using the identified lipids percentages. C) Quantified average luminescence of the liver after 6 hours. D) Molar
ratio and percentage of the 5-component SORT LNP formulations. D) Visual representation of formulation. E) Table of details for the base LNP and SORT
LNP formulations (Total lipids/mRNA ratio = 40; wt/wt).
In summary, we synthesized unsaturated thiols that enabled access to a
library of 91 ionizable lipids. In vitro mRNA delivery screening results
revealed differences and SAR that correlated with the location/configuration
of the unsaturated bond(s) rather than the simple presence of unsaturation.
Selecting the 4A3 amine core to study in vivo, 4A3-Cit demonstrated the
highest efficacy. Mechanistic studies using model endosomal membranes
indicated that 4A3-Cit demonstrated the highest lipid fusion ability,
suggesting its unique tail structure may enhance endosomal escape. 4A3-Cit
further established its exceptional utility for mRNA delivery through
application in SORT LNPs. SC8+Cit (20%) SORT LNPs improved mRNA
delivery 18-fold over parent formulations. The findings from this work aid a
deeper understanding of how unsaturation may promote mRNA delivery by
increasing endosomal fusion, identify 4A3-Cit as a potent new lipid, and
further expand the utility of SORT LNPs for efficacious mRNA delivery.
Acknowledgements. D.J.S. acknowledges financial support from the
National Institutes of Health (NIH) National Institute of Biomedical Imaging
and Bioengineering (NIBIB) (R01 EB025192-01A1), the Cystic Fibrosis
Foundation (CFF) (SIEGWA18XX0), the Cancer Prevention and Research
Institute of Texas (CPRIT) (RP190251), the American Cancer Society (ACS)
(RSG-17-012-01), and the Welch Foundation (I-1855). We acknowledge the
UTSW Small Animal Imaging Shared Resource (NIH NCI P30CA142543).
S.M.L. acknowledges support from NIH (T32GM127216).
Keywords: lipid nanoparticles • mRNA • unsaturated lipids •
unsaturated thiols • nanomaterials
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5

10.1002/anie.202013927
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
Unsaturated amino lipids were synthesized to study the role of unsaturation in lipid nanoparticle (LNP)-mediated mRNA delivery. A lipid series
with a Citronellol tail (4A3-Cit) was identified from a library screen. Employing 4A3-Cit in new Selective ORgan Targeting (SORT) formulation
resulted in an 18-fold increase in mRNA expression over saturated LNPs. The findings provide insights to how unsaturation and SORT mixing
can promote mRNA delivery.
Institute and/or researcher Twitter usernames: @DJSiegwart
This article is protected by copyright. All rights reserved.