Simplifying the Chemical Structure of Cationic Lipids for siRNA-Lipid Nanoparticles

Article abstract of DOI:10.1021/acsmedchemlett.8b00652

We report a potent cationic lipid, SST-02 ((3-hydroxylpropyl)dilinoleylamine), which possesses a simple chemical structure and is synthesized just in one step. Cationic lipids are key components of siRNA-lipid nanoparticles (LNP), which may serve as potential therapeutic agents for various diseases. For a decade, chemists have given enhanced potency and new functions to cationic lipids along with structural complexity. In this study, we conducted a medicinal chemistry campaign pursuing chemical simplicity and found that even dilinoleylmethylamine (SST-01) and methylpalmitoleylamine could be used for the in vitro and in vivo siRNA delivery. Further optimization revealed that a hydroxyl group boosted potency, and SST-02 showed an ID₅₀ of 0.02 mg/kg in the factor VII (FVII) model. Rats administered with 3 mg/kg of SST-02 LNP did not show changes in body weight, blood chemistry, or hematological parameters, while the AST level decreased at a dose of 5 mg/kg. The use of SST-02 avoids a lengthy synthetic route and may thus decrease the future cost of nucleic acid therapeutics.

Full text of DOI:10.1021/acsmedchemlett.8b00652

Letter
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Simplifying the Chemical Structure of Cationic Lipids for siRNA-Lipid
Nanoparticles
Takeshi Kuboyama,* Kaori Yagi, Tomoyuki Naoi, Tomohiro Era, Nobuhiro Yagi, Yoshisuke Nakasato,
Hayato Yabuuchi, Saori Takahashi, Fumikazu Shinohara, Hiroto Iwai, Ayumi Koubara-Yamada,
Kazumasa Hasegawa, and Atsushi Miwa
Research Functions Unit, R&D Division, Kyowa Hakko Kirin, Co., Ltd., 3-6-6, Asahi-machi, Machida-shi, Tokyo 194-8533, Japan
S
* Supporting Information
ABSTRACT: We report a potent cationic lipid, SST-02 ((3-hydroxylpropyl)dilinoleylamine), which possesses a simple
chemical structure and is synthesized just in one step. Cationic lipids are key components of siRNA-lipid nanoparticles (LNP),
which may serve as potential therapeutic agents for various diseases. For a decade, chemists have given enhanced potency and
new functions to cationic lipids along with structural complexity. In this study, we conducted a medicinal chemistry campaign
pursuing chemical simplicity and found that even dilinoleylmethylamine (SST-01) and methylpalmitoleylamine could be used
for the in vitro and in vivo siRNA delivery. Further optimization revealed that a hydroxyl group boosted potency, and SST-02
showed an ID50 of 0.02 mg/kg in the factor VII (FVII) model. Rats administered with 3 mg/kg of SST-02 LNP did not show
changes in body weight, blood chemistry, or hematological parameters, while the AST level decreased at a dose of 5 mg/kg. The
use of SST-02 avoids a lengthy synthetic route and may thus decrease the future cost of nucleic acid therapeutics.
KEYWORDS: Cationic lipid, siRNA, lipid nanoparticle, LNP, drug-delivery system
hort interfering RNAs (siRNAs) are 20−30 mer double-
1,2
Sstranded RNAs
that form a complex with endogenous
Ago2 protein in animal cells and effectively degrade the
mRNAs of disease-relevant genes in a sequence-specific
manner.3 siRNAs can facilitate inhibition of biological
processes that are neither targeted by small molecules nor
antibodies; therefore, they are potentially useful for medicinal
usage. However, due to their large molecular weight, high
hydrophilicity, and enzymatic instability, in vivo delivery of
siRNA into cells has been a major research topic since their
discovery.
Figure 1. Structures of DLinDMA, SST-01, and SST-02.
Lipid-mediated delivery vehicles such as siRNA-lipid
nanoparticles (LNPs) are one of the most successful drug
delivery systems for siRNA. LNPs are about 100 nm in size
and are composed of siRNA, neutral lipid(s), PEG-lipid, and
cationic lipid. Cationic lipids are protonated in acidic
endosomes, increase the electric charge of nanoparticle
surfaces, and induce siRNA migration into the cytosol. From
a chemical perspective, cationic lipids used in LNPs generally
have one “titrable amino group”, two “aliphatic chains” (mostly
unsaturated unbranched C18 hydrocarbons), and a “core”
structure that connects them (Figure 1).
cationic lipids (Figure S1). This “medicinal chemistry
research” started with conversion of the dimethylamino moiety
of DLinDMA to cyclic amines.5 Delivery potency to the liver is
boosted by DLin-KC2-DMA6 and the U.S. Food and Drug
Administration (FDA)-approved DLin-MC3-DMA,7 wherein
two aliphatic chains are connected via a carbon atom. YSK05 is
an example of an acetal derivative that can be synthesized in
one step.8 A serine-derived cationic lipid is suitable for delivery
to macrophages and dendritic cells.9 Addition of a small
Since pioneering work on DLinDMA by researchers at
Protiva4 revealed that small alternation of cationic lipid
chemical structures alters the whole nature of LNPs, many
efforts have been made to optimize the chemical structure of
Received: December 27, 2018
Accepted: April 5, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.8b00652
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ACS Medicinal Chemistry Letters
Letter
portion of quaternary cationic lipids to LNPs is effective for
targeting leukemia cells.10 In some cases, asymmetric aliphatic
chains work better than symmetric chains,11 especially when
one is short.12,13 Cyclopropane is used as a bioisostere of a
double bond in an unsaturated aliphatic chain.13 Ester bonds
introduced in the midst of the aliphatic chain enhance
elimination from hepatocytes and thus avoid hepatotoxic-
ity.14,15 In this research history, medicinal chemists have
provided cationic lipids with enhanced potency and new
functions; however, structural complexity, lengthy-synthetic
routes, and increased costs have become seemingly indis-
pensable.
Table 1. In Vitro and in Vivo Activities of DLinDMA,
DODMA, and DPalDMA LNPs
a
aliphatic
chain
in vitro IC50 (95%
relative serum
cholesterol level
b
c
cationic lipid
C.I.) [nM]
1
linoleyl
0.653 (0.617−0.690)
52%
(DLinDMA)
2 (DODMA)
3 (DPalDMA) palmitoleyl
oleyl
18.4 (17.7−19.0)
4.23 (4.00−4.47)
no data
81%
a
Each of LNPs were prepared using lipids 1−3 using a one-step
b
method. ApoB-siRNA was incorporated. 95% C.I.; 95% confidence
intervals. Serum cholesterol levels relative to the saline-treated group.
c
Dosed at 3 mg/kg.
However, pursuit of the minimum components required for
activity is also an important aspect of medicinal chemistry. In
this research,16 we started from DLinDMA, modified aliphatic
chain and core, and successfully found the structurally simplest
cationic lipid for siRNA-LNPs. We also boosted the delivery
potency by adding one hydroxy functional group.
We first focused on the aliphatic chain. The chain of
DLinDMA is a linoleyl ((Z, Z)-9, 12-octadecadien-1-yl) group
with 18 carbons and 2 unsaturated bonds per chain. According
to the literature,4 linoleyl, compared to the one-less-
unsaturated oleyl ((Z)-9-octadecen-1-yl) group, gives a lower
melting temperature to the cationic lipid and decreases the
phase-transition temperature of LNPs, resulting in potentiation
of in vitro and in vivo transfection efficiency.
When designing new aliphatic chains, we considered
avoiding the de novo chemical synthesis of lipid analogues
because it may increase the costs and potential risks of drug
metabolism. Among commercially available lipid derivatives,
which are mainly derived from natural sources, we took
particular note of the palmitoleyl ((Z)-9-hexadecen-1-yl)
group. This aliphatic chain has the same unsaturation as
oleyl but has two less carbon atoms (16 for palmitoleyl, 18 for
oleyl). Therefore, we assumed that palmitoleyl has an effect
similar to linoleyl. In fact, the melting temperatures of
corresponding fatty acids is reported to be in the order of
oleic acid (16.2 °C) > palmitoleic acid (0.5 °C) > linoleic acid
(−6.5 °C).17
cholesterol level,18 with 70 to 80% suppression as a peak.
DLinDMA-LNP and DPalDMA-LNP were intravenously
administered to Balb/c mice at 3 mg/kg, and the blood
cholesterol levels were measured at 48 h after administration
(Table 1). These in vivo results were parallel to those obtained
in vitro; DPalDMA-LNP decreased blood cholesterol but to a
lower extent compared to DLinDMA-LNP.
Aiming to investigate further aspects of palmitoleyl, we
compared the stability of DLinDMA and DPalDMA under UV
irradiation. Thin-layer chromatography analysis showed that
DLinDMA was decomposed almost completely on day 7,
whereas DPalDMA showed superior stability (Figure S3). The
reason for this is not completely clear; however, non-
conjugated double bonds in DLinDMA may lead to photo-
chemical instability, whereas DPalDMA has isolated double
bonds. These experiments showed that the palmitoleyl group
could provide increased delivery potency to cationic lipids
compared to the oleyl group, and showed more stability to
light compared to the linoleyl group.
Next, we proceeded to the bioisosteric conversion and
simplification of the core. In DLinDMA, a 1,2-dihydroxypro-
pane (PRO, Figure 2) unit can be regarded as the core,
The synthesis of palmitoleyl congener of DLinDMA (3, or
DPalDMA) was achieved following the method reported for
DLinDMA,4 utilizing palmitoleyl mesylate instead of linoleyl
mesyate. (For the synthetic scheme, see Figure S2)
For the screening cascade, we considered that an in vitro
assay was beneficial at least as the first screening before an in
vivo assay. An LNP formulation incorporating ApoB-siRNA18
for in vitro transfection experiments was prepared using a
SNALP-like one-step method,4 i.e., by mixing an ethanolic
solution of lipids (cationic lipids, DSPC, cholesterol, and PEG-
DMPE) and an aqueous solution of siRNA, followed by
dilution and ultrafiltration. Serially diluted LNP solutions were
added to HepG2 cells and after incubation for 24 h, and
ApoB mRNA levels were determined using real-time
quantitative polymerase chain reaction.
Figure 2. Design of “core” structures.
connecting its titrable amine (dimethylamino moiety) and
aliphatic chains (two linoleyl moiety). First, we designed a 3,4-
dihydroxypyrrolidine (PYR, Figure 2) core by connecting the
N-methyl carbon and C1 of 1, 2-dihydroxypropane. This ring
closure generated three possible stereoisomers and among
them, trans-R,R isomer (R,R-4) gave the best in vitro result
(Table 2). It is natural that stereoisomerism of cationic lipids
affects the delivery potency of LNPs in a chiral environment of
mixtures with other stereopure lipids and oligonucleotides.19
In the second step, the ring opening of PYR yielded a
diethanolamine (DEA, Figure 2) core. An advantage of DEA is
that it is achiral and, thus, simple. Interestingly, when the three
As shown in Table 1, consistent with the literature,4
DLinDMA yielded better results compared to DODMA in
our experiment even though we used a different PEG-lipid,
siRNA, and component ratio. In addition, DPalDMA, a newly
synthesized palmitoleyl congener, showed intermediate results
(Table 1). These in vitro results show a good correlation with
the melting temperatures of corresponding fatty acids.
ApoB-siRNA, when delivered to the liver, induces reduction
of ApoB mRNA in hepatocytes and decreases the blood
B
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We fixed the di-“aliphatic chain”-amine structure (DA core),
and synthesized a series of dilinoleylamine derivatives with
various substituents instead of the methyl group. FVII-siRNA
incorporating LNPs were prepared in a one-step process using
these cationic lipids and their in vivo delivery potency was
measured. We found that addition of one hydroxyl group (16)
resulted in drastic improvement (Table 3). One hypothesis for
this phenomenon is that the hydroxyl group adds a hydrogen
bonding interaction between the cationic lipid and siRNA. In
addition, a more probable hypothesis is that a hydrophilic
balance of hydroxyl and propylene groups plays an important
role in the nanoparticle surface and/or cationic lipid-siRNA
interaction because use of more hydrophilic 13 (two hydroxyl
groups) resulted in a nearly complete loss of activity.
Formulation design is known to affect the in vivo delivery
potency of LNPs. To enhance the potency of 16-incorporating
LNPs, we adopted a previously reported two-step mixing
process, or Wrapsome (WS) process with minor adjustment in
the amount of cationic lipid.9,22,23 We prepared 16-
incorporating LNP using this two-step process and compared
the in vivo delivery potency with that of 16-incorporating LNP
prepared using the one-step SNALP-like process described
above. In the case of 16, the former (relative serum factor VII
protein level was 9%/21% for 0.1 and 0.03 mg/kg dose)
showed much better results than the latter (35%/79%).
Although the details are not clear, one cause may be the
different average particle sizes arising from the different
formulation processes. In this case, the two-step process
generated slightly smaller (88 nm) particle than the one-step
process (124 nm). Another cause, which likely is more
dominant, may be the difference in the ratio of each lipid and
the ratio of total lipid to siRNA. In this case, the LNP
generated by the two-step process contained more cationic
lipids and other lipids than the LNP generated by the one-step
process.
Table 2. In Vitro and in Vivo Activities of LNPs Using
Cationic Lipids with Various Cores and Aliphatic Chains
a
aliphatic
chain
in vitro IC50 (95%
relative serum
c
b
no.
core
C.I.) [nM]
cholesterol level
R,R-4 PYR
linoleyl
linoleyl
linoleyl
oleyl
palmitoleyl
linoleyl
oleyl
palmitoleyl
linoleyl
oleyl
1.01 (0.963−1.07)
28.8 (27.9−29.6)
5.67 (5.41−5.94)
16.4 (15.5−17.3)
0.561 (0.538−0.586)
25.4 (24.2−26.7)
0.922 (0.881−0.965)
0.667 (0.632−0.703)
5.96 (5.24−6.69)
7.30 (6.63−8.01)
0.839 (0.752−0.927)
44%
cis-4
S,S-4
PYR
PYR
no data
no data
no data
no data
86%
64%
42%
37%
82%
R,R-5 PYR
R,R-6 PYR
7
8
9
10
11
12
DEA
DEA
DEA
DA
DA
DA
palmitoleyl
42%
a
Each of the LNPs were prepared using a one-step method. ApoB-
b
c
siRNA was incorporated. 95% C.I.; 95% confidence intervals. Serum
cholesterol levels relative to saline-treated group. Dosed at 3 mg/kg.
chains were attached to the DEA core, oleyl gave good result
both in vitro and in vivo (Table 2). Although the reason for
this inconsistency with the other “core” is unclear, some
physical property of oleyl is better than that of the others,
when combined with the DEA core.
In the last step, we deleted the ether functional group of
DEA. This simplification resulted in di-“aliphatic chain”-amine
compounds (DA, Figure 2). These can be regarded as the
simplest compounds that satisfy the structural requisites of the
cationic lipids in LNPs. However, to the best of our
knowledge, there is no report of 10−12 being applied to
oligonucleotide (ex. siRNA) delivery. In addition, linoleyl (10)
and palmitoleyl (12) DA compounds gave better results
(Table 2) compared to DLinDMA (1, Table 1) upon in vivo
usage.
The ApoB-siRNA/blood cholesterol system has a poor
dynamic range due to a plateau observed at high dose, whereas
the FVII-siRNA/blood FVII protein system is reported to have
a good dynamic range and is, therefore, used in many studies
on LNPs.6,7,13−15,20,21 We prepared FVII-siRNA incorporating
LNPs using cationic lipid 10 in an one-step mixing process
similar to the method described above. We then administered
this formulation to mice and measured the FVII protein
coagulation activity in blood after 48 h. At the dose of 0.3 mg/
kg, 35% suppression was observed (Table 3), which was
inferior to the reported potency (IC50 ≈ 0.03 mg/kg) of DLin-
MC3-DMA.7
Furthermore, we synthesized a series of cationic lipids in
which the length between the nitrogen atom and oxygen atom
in 16 was altered and the aliphatic chain were changed. LNPs
incorporating these lipids and FVII-siRNA were prepared using
the two-step WS process. As shown in Table 4, propylene
linker (16) was found to be the best. Elongation of the linker
(18, 19) affected the potency, likely because of the high
hydrophobicity. Regarding aliphatic chains, the palmitoleyl or
oleyl groups reduced the delivery potency.
We next focused on the enhancement of delivery potency,
under the condition of adding limited structural complexity.
Table 4. Effects of N−O Linker Length and Aliphatic
a
Chains
a
Table 3. Effects of Various Substituents on the DA Core
relative serum factor
b
no.
chain
-R group
VII protein level
b
no.
R
relative serum factor VII protein level
16
17
18
19
20
21
linoleyl
linoleyl
linoleyl
linoleyl
oleyl
-CH2CH2CH2OH
-CH2CH2OH
-CH2CH2CH2CH2OH
-CH2CH2CH2CH2CH2OH
-CH2CH2CH2OH
-CH2CH2CH2OH
21%
25%
34%
65%
82%
66%
10
13
14
15
16
-CH3
65%
91%
96%
95%
12%
-CH2CH(OH)CH2OH
-CH2CH2CO2Et
-CH2CH2CO2NH2
-CH2CH2CH2OH
palmitolyel
a
a
Each of the LNPs were prepared using a one-step method. FVII-
Each of the LNPs were prepared using the two-step WS method.
b
b
siRNA was incorporated. Serum factor VII protein level relative to
the saline-treated group. Dosed at 0.3 mg/kg.
FVII-siRNA was incorporated. Serum factor VII protein level relative
to the saline-treated group. Dosed at 0.03 mg/kg.
C
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An independent experiment was conducted to calculate the
IC50 value of 16-LNP, which was 0.02 mg/kg (Table S1). To
assess tolerability, 16-LNP incorporating non-targeting control
siRNA instead of FVII-siRNA was prepared in the same
manner. For comparison, an LNP utilizing DLinDMA as a
cationic lipid was also prepared with the same formulation
design and process. Rats administered with 3 mg/kg of
DLinDMA-LNP showed significant body weight loss, while no
significant body weight loss was observed in the 16-LNP
treated groups even at 5 mg/kg dose (Figures S4). A dose of 3
mg/kg of DLinDMA-LNP resulted in a decrease in platelet
and an increase in ALT and total bilirubin. In contrast, A dose
of 3 mg/kg of 16-LNP does not show these changes, and a
decrease of AST was observed at 5 mg/kg dose (Table S2).
Because rats are known to show lower tolerance to siRNA-
LNPs than mice and humans,24 these results suggest the
favorable safety profile of 16-LNP in humans.
In summary, we conducted a medicinal chemistry study
pursuing structurally simpler cationic lipid(s) for siRNA-LNPs.
Through the design and synthesis of one new “aliphatic chain”
and three novel “core” structures, 10 (SST-01) and 12 were
found to demonstrate good in vitro and in vivo effects.
Although these showed inferior delivery potency, at least in
delivery to the liver, compared with the cationic lipids
described in latest literatures, we were satisfied with the
simplest chemical structure of 10 and 12 among known
cationic lipids.
Among this series of cationic lipids, a mRNA LNP utilizing
17 was recently reported by Sabnis et al.,25 while its potency
was weaker than that of tritailed cationic lipids tailored for long
single-stranded mRNA. Although more-detailed studies
including pharmacology, toxicology, and manufacturing need
to be conducted, these structurally simple cationic lipids may
be helpful for decreasing the future costs of LNP-based nucleic
acid therapeutics.26
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.8b00652.
Figures showing cationic lipids, a synthetic scheme,
photostability analysis, and body weight change; tables
showing dose−response curves and blood chemistry and
hematological parameters; details on materials and
1
methods; and H-NMR charts (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: takeshi.kuboyama@kyowa-kirin.co.jp.
■
ORCID
Takeshi Kuboyama: 0000-0002-4138-144X
Author Contributions
Moreover, the addition of one more functional group (−OH
group) into 10 yielded the surprisingly potent cationic lipid 16
(SST-02). This still has a simple chemical structure but
showed a robust (IC50 ≈ 0.02 mg/kg) delivery potency to the
liver, which is comparable to that of the FDA-approved DLin-
MC3-DMA (IC50 ≈ 0.03 mg/kg).7 Rats administered with 3
mg/kg of 16 (SST-02) LNP did not show changes in body
weight, blood chemistry, or hematological parameters, while
the AST level decreased at a dose of 5 mg/kg. Our results
suggest the favorable safety and efficacy profile for application
of 16 (SST-02) LNP.
Simple compounds can potentially be synthesized easily. In
addition to the diversity-oriented medicinal chemistry route,
we developed an efficiency-oriented synthetic route in which
16 (SST-02) can be easily synthesized from commercially
available ethanolamine and linoleyl mesylate in just one step
(Figure 3 and the Supporting Information).
The design and synthesis of cationic lipids were done by T.K.,
K.Y., T.Y., and Y.N. The design and preparation of LNP
formulations were done by T.N. and N.Y. The in vitro and in
vivo assay were done by F.S., H.Y., S.T., H.I., A.K.Y., and K.H.
Manuscript preparation was done by T.K. The design of
experiments and supervising of project were done by T.K. and
A.M.
Notes
The authors declare the following competing financial
interest(s): The authors are employees (for T.K., K.Y., T.N.,
Y.N., H.Y., F.S., H.I., A.K.Y., K.H., and A.M.) or ex-employees
(for T.E., N.Y., and S.T.) of Kyowa Hakko Kirin, Co., Ltd.
ACKNOWLEDGMENTS
■
The authors acknowledge Mr. Takayuki Atago and Mr.
Shintaro Hosoe for discussion on the synthesis of SST-01
and SST-02; Ms. Yoshimi Akioka for support with LNP
preparation; and Dr. Toshihiko Ishii, Ms. Kana Kurihara, Ms.
Eri Okita, and the animal laboratory members for animal
experiments. The authors also acknowledge Dr. Junichi
Enokizono, Dr. Toshiyuki Atsumi, Ms. Hiroko Sugishita, and
Dr. Motoo Yamasaki for scientific discussion and support.
ABBREVIATIONS
■
ApoB, apolipoprotein B; DSPC, 1,2-distearoyl-sn-glycero-3-
phosphocholine; FVII, coagulation factor VII; siRNA, short
interfering RNA; linoleyl, (Z, Z)-9,12-octadecadien-1-yl; LNP,
lipid nanoparticles; oleyl, (Z)-9-octadecen-1-yl; palmitoleyl,
(Z)-9-hexadecen-1-yl; PEG-DMPE, 1,2-dimiristoyl-sn-glycero-
3-phosphoethanolamine-N-(polyethylene glycol-2000).
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D
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DOI: 10.1021/acsmedchemlett.8b00652
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX