Chem. Pharm. Bull.

69, 1141–1159 (2021)

Vol. 69, No. 121141

Review

Development of Lipid Nanoparticles for the Delivery

of Macromolecules Based on the Molecular Design
of pH-Sensitive Cationic Lipids
Yusuke Sato
Faculty of Pharmaceutical Sciences, Hokkaido University; Kita-12, Nishi-6, Kita-ku,
Sapporo 060–0812, Japan.
Received August 18, 2021

Considerable efforts have been made on the development of lipid nanoparticles (LNPs) for delivering of
nucleic acids in LNP-based medicines, including a first-ever short interfering RNA (siRNA) medicine, Onpat-
tro, and the mRNA vaccines against the coronavirus disease 2019 (COVID-19), which have been approved
and are currently in use worldwide. The successful rational design of ionizable cationic lipids was a major
breakthrough that dramatically increased delivery efficiency in this field. The LNPs would be expected to
be useful as a platform technology for the delivery of various therapeutic modalities for genome editing and
even for undiscovered therapeutic mechanisms. In this review, the current progress of my research, including
the molecular design of pH-sensitive cationic lipids, their applications for various tissues and cell types, and
for delivering various macromolecules, including siRNA, antisense oligonucleotide, mRNA, and the clustered
regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system will be described. Mecha-
nistic studies regarding relationships between the physicochemical properties of LNPs, drug delivery, and
biosafety are also summarized. Furthermore, current issues that need to be addressed for next generation
drug delivery systems are discussed.
Key words lipid nanoparticle; pH-sensitive cationic lipid; endosomal escape; macromolecule delivery;
nucleic acid; ribonucleoprotein

1. Introduction loid polyneuropathy caused by mutations in the transthyletin

Abnormal genetic status, including mutations and dele-
tions in genomes and the mis regulation of gene expressions,
causes a variety of diseases.1–4) These therapeutic targets are
sometimes difficult to treat using conventional small molecule
drugs due to limitations regarding their selectivity, affin-
ity, and untraslated RNAs.5) Therefore, alternative approaches
using (modified) DNAs/RNAs, including short interfering
RNAs (siRNAs), antisense oligonucleotides (ASOs), aptamers,
decoys, and mRNAs, and genome editing, including clustered
regularly palindromic repeat (CRISPR)/Cas, have been consid-
ered for use as novel therapeutics against various refractory
diseases. Among these, some nucleic acid therapeutics have
already been approved by the Ministry of Health, Labour and
Welfare (MHLW) in Japan, including pegaptanib (Macugen®;
aptamer), nusinersen (Spinraza®; ASO), patisiran (Onpattro®;
siRNA), givosiran (Givlaari®; siRNA), viltolarsen (Viltepso®;
ASO), BNT162b2 (Comirnaty®; mRNA), and mRNA-1273
(Spikevax®; mRNA) as of the writing of this review. In par-
ticular, Onpattro® is a first-ever RNA interference (RNAi)
medicine that can be used in the treatment of familial amy-
This review of the author’s work was written by the author upon receiving
the 2021 Pharmaceutical Society of Japan Award for Young Scientists.
 e-mail: y_sato@pharm.hokudai.ac.jp
gene.6) In addition, both Comirnaty® (Pfizer/BionTech) and
Spikevax® (Moderna) are the first mRNA vaccines against the
corona virus diseases 2019 (COVID-19) that were developed
for treatment of infectins caused by the severe acute respira-
tory syndrome coronavirus 2 (SARS-CoV-2).7) Both siRNA
medicines and mRNA vaccines are lipid nanoparticle (LNP)
preparations. The LNP can encapsulate RNAs in its core thus
protecting the cargo from enzymatic degradation by ribo-
nucleases (RNases) present in biological environments and to
efficiently deliver the cargo to the cytosol of target cells (Fig.
1A). The LNPs are typically composed of 4 types of lipids, in-
cluding a cationic lipid (CL), a phospholipid (PL), cholesterol,
and a pegylated (PEG)-lipid (Fig. 1A). Among them, CL is
the major component (approx. 50 mol% of the total lipid) and
is essential for successfully encapsulating negatively charged
nucleic acids through electrostatic interactions and to achieve
the efficient cytosolic delivery of nucleic acids through mem-
brane fusion-mediated endosomal escape. Therefore, as of this
writing, numerous attempts have been made to develop origi-
nal potent CLs. Quaternary ammonium-based permanently
CLs were classically used for nucleic acid delivery because
their highly positive charge facilitates the loading of nucleic
acids on LNPs as well as uptake by cells in vitro.8,9) However,
© 2021 The Pharmaceutical Society of Japan
1142 Chem. Pharm. Bull. Vol. 69, No. 12 (2021)

Fig. 1. Typical Structures of RNA-Loaded LNPs and pH-Sensitive CLs

(A) RNA-loaded LNPs are typically composed of CL, PL, cholesterol and PEG-lipid. The LNPs composed of permanently CLs show highly cationic properties in the
blood stream causing poor biodistribution. The LNPs composed of pH-sensitive CLs shows electrostatically near neutral in blood stream, leading to improved biodistribu-
tion, and convert to cationic property in acidic endosomes, facilitating endosomal escape. (B–F) Chemical structures of DLinDMA (B), DLin-KC2-DMA (C), DLin-MC3-
DMA (D), ALC-0315, and SM-102 (F). (Color figure can be accessed in the online version.)

the positive charge greatly hinders the control of the blood
circulation, biodistribution, and activation of complement sys-
tems due to non-specific interactions with biocomponents10,11)
(Fig. 1A). Therefore, tertiary amine-based pH-sensitive CLs,
which are near neutral at physiological pH (e.g. blood stream)
but develop cationic properties in a weakly acidic environment
(e.g. endosomes/lysosomes), were first reported by Bailey and
Cullis in 199412) (Fig. 1A). After reports on the efficient en-
capsulation of ASOs in LNPs composed of the pH-sensitive
CLs,13,14) these pH-sensitive CLs have now become the current
gold standard.15,16) Hayes et al. clearly demonstrated that un-
saturation in the scaffolds in CLs is critical for inducing mem-
brane fusion for endosomal escape, and found that DLinDMA,
which is composed of 2 scaffolds derived from linoleic acid
(C18:2), showed the best endosomal escape and cytosolic de-
livery of siRNA17) (Fig. 1B). In 2006, Zimmermann et al. first
demonstrated successful induction of hepatic apolipoprotein
B (ApoB) gene silencing in mice and in non-human primates
after the intravenous injection of siApoB-loaded DLinDMA-
LNPs (referred to as a stable nucleic acid lipid particle;
SNALP) at a clinically relevant dose (50% effective dose; ED50
of approx. 1 mg siRNA/kg.18) In 2010, Semple et al. reported
that the linker structure between the tertiary amino group and
the scaffolds was critical for maximizing fusogenic activity.19)
Specifically, the efficacy of a ketal ring linker was found to
be superior to a 1,2-diol-derived linker, which was adopted
in DLinDMA. The best performing CL, DLin-KC2-DMA,
showed an ED50 of approx. 0.02 mg siRNA/kg in a mouse
factor VII (FVII) model, which was a 50-fold improvement
in gene silencing efficiency in hepatocytes (Fig. 1C). Further
structural optimizations resulted in the identification of the
optimal CL, DLin-MC3-DMA (MC3)20) (Fig. 1D), which ex-
hibited an ED50 of approx. 0.005 mg siRNA/kg in the FVII
model and is used as a main ingredient in Onpattro®. Based
on this structure, numerous potent CLs, including ALC-0315
and SM-102 used for COVID-19 mRNA vaccines, have been
developed so far21–31) (Figs. 1E, F). Based on the above find-
ings, the development of potent CLs is one of the major break-
throughs in the field of LNPs.
I began work on developing LNPs for the in vivo delivery

Biography
Yusuke SATO is an Assistant Professor in the Faculty of Pharmaceutical Sciences, Hokkaido Uni-
versity. He joined Laboratory for Molecular Design of Pharmaceutics organized by Prof. Hideyoshi
HARASHIMA when he was a 4th degree of undergraduate, and received the B. S., M. S. and Ph.
D. degrees from Hokkaido University in 2009, 11 and 14, respectively. After completing a Research
Fellowship for Young Scientists from Japan Society for Promotion of Science (JSPS), he was ap-
pointed as an Assistant Professor in the Faculty of Pharmaceutical Sciences, Hokkaido University
in 2014. His main research interest is the development of potent lipid nanoparticles for the in vivo
delivery of macromolecules, including short interfering RNAs (siRNAs), mRNAs, and clustered regu-
Yusuke Sato
larly interspaced short palindromic repeats (CRISPR)-associated (Cas) ribonucleoproteins (RNPs),
based on the molecular design of functional lipids. He promotes industry-academia collaboration activities with the aim
of implementing the use of his lipids and formulation technologies in collaboration with the Institute for the Promotion of
Business-Regional Collaboration at Hokkaido University and private sector companies.
Vol. 69, No. 12 (2021)1143
Chem. Pharm. Bull.

of macromolecules based on the molecular design of origi-
nal pH-sensitive cationic lipids in 2010 in the laboratory for
the molecular design of pharmaceutics organized by Prof.
Hideyoshi Harashima. In this review, current progress of my
research, including the molecular design of pH-sensitive cat-
ionic lipids, their applications for liver, extra-hepatic tissues
and cell types, and for the delivery of various macromol-
ecules, including siRNA, antisense oligonucleotides, mRNA,
and clustered regularly interspaced short palindromic repeats
(CRISPR)-associated (Cas) system are described. Mechanistic
studies regarding the relationship between the physicochemi-
cal properties of LNPs, drug delivery, and biosafety will be
also summarized. Current limitations that should be addressed
for next generation drug delivery systems are also discussed.
2. Development of pH-Sensitive CLs and Their Appli-
cations for Liver
In 2012, a pH-sensitive CL, YSK05, was first synthesized
as the 1st generation CL32,33) (Fig. 2A). YSK05 can be syn-
thesized in a one-step condensation reaction of N-methyl-
4-piperidone and linoleyl alcohols. The structure that two
linoleyl alcohol-derived scaffolds that are attached to one
carbon atom leads to an increase in the angle between the two
scaffolds (109.5°) (Fig. 2A). Based on theory of critical pack-
ing parameter (CPP),34) a framework for determining the type
of aggregates formed by surfactants, this design emphasizes
the formation of a CL with a cone shape. The cone shape of
the CLs is important for inducing membrane fusion-mediated
endosomal escape.35,36) Membrane fusion can be achieved
through the phase transition of the lipid bilayer of biologi-
cal membranes from a lamellar (L) to an inverted hexagonal
(HII) phase. The pH-sensitive CLs acquire a cationic charge
in weakly acidified endosomes, bind to anionic lipids in the
endosomal membranes and form ion pairs (Fig. 2B). When the
ion pairs adopt a cone shape that is sufficient to destabilize
lipid bilayers and to induce a phase transition into non-bilayer
structures (e.g. HII phase), efficient endosomal escape can be
achieved. 31 Phosphorus (31P) NMR can be utilized to under-
stand lipid polymorphism of lipid dispersion. Due to the an-
isotropy of the chemical shift of the 31P of PLs that is derived
from different limitations in molecular motion between lipid
polymorphism, it is known that L and HII phases show a dis-
criminative broad signal with a high-field peak and a low-field
peak, respectively35) (Fig. 2B). A 31P-NMR analysis using lipid
dispersions composed of equimolar amounts of a pH-sensitive
CL, zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphol-
choline (POPC), and negatively charged 1,2-distearoyl-sn-
glycero-3-phosphatidylserine (DSPS) suspended in an acidic
buffer revealed that the phase transition temperature from L to
HII phase (TH) for YSK05 was significantly lower (21–25 °C)
than that for a conventional pH-sensitive CL, 1,2-dioleoyloxy-
3-dimethylaminopropane (DODAP) (35–40 °C) (Fig. 2C). This
result indicates that YSK05 is more fusogenic than DODAP.
A hemolysis assay which can be used to examine the fusoge-
nicity of LNPs using red blood cells as model biomembranes
also revealed that YSK05-containing LNPs (YSK05-LNPs)
are more fusogenic. Indeed, the siRNA-loaded YSK05-LNPs
showed much higher gene silencing activity compared to
DODAP or 1,2-dioleoyloxy-3-trimethylammonium propane
(DOTAP)-LNPs in HeLa cells despite the fact that the cellular
uptake of the YSK05-LNPs was consistent or lower than the
counterparts. Treatment with chloroquine or ammonium chlo-
ride, endosome buffering reagents,37–40) revealed that the acidi-
fication of endosomes is an essential process for the successful

Fig. 2. Structure and Properties of YSK05

(A) Chemical structure of YSK05. A ketal linkage increases the angle between the two scaffolds and emphasizes a cone shape. (B) Schematic illustration of L to H II
phase transition by ion-pairing between CLs and anionic lipids. Both L and H II phases can be determined by 31P-NMR due to the anisotropy of chemical shifts which are
derived from the specific limitation in molecular motion in each phase. (C) 31P-NMR spectra of a lipid dispersion composed of pH-sensitive CL, DODAP or YSK05 at the
indicated temperature. Blue arrow indicates a peak derived from H II phase. (Color figure can be accessed in the online version.)
1144 Chem. Pharm. Bull. Vol. 69, No. 12 (2021)

cytosolic delivery of siRNAs for YSK05-LNPs.
The YSK05-LNPs were applied to liver tissue. It is known
that size-controlled LNPs with diameters of 100 nm or less can
reach hepatocytes due to the structural features of the liver,
including the presence of fenestrae which are pores with diam-
eters in the range of 100 to 150 nm in liver sinusoidal endothe-
lial cells (LSECs) and a lack of basement membranes between
LSECs and hepatocytes.41,42) In addition to this, the presence of
the apolipoprotein E (ApoE)-low density lipoprotein receptor
(LDLR) pathway as an endogenous uptake mechanism in hepa-
tocytes greatly contributes to the development of liver-targeting
LNPs.43,44) It was found that the bare YSK05-LNPs were cleared
from blood stream very quickly (t1/2 of approx. 1.8 min) after
intravenous administration through the ApoE-LDLR pathway45)
(Fig. 3A). This rate was over 10-fold faster than that for N-
acetyl-D -galactosamine (GalNAc)-modified YSK05-LNPs which
were cleared through the GalNAc-asialoglycoprotein receptor
(ASGPR) pathway (Fig. 3A). It is well known that GalNAc
functions as a ligand of ASGPRs, which are highly and spe-
cifically expressed on hepatocytes, and are used for active
targeting to hepatocytes.46–50) Intrahepatic observations revealed
that bare YSK05-LNPs are adsorbed to microvillar surface of
hepatocytes in wild-type mice but not in ApoE-deficient mice,
indicating that the presence of the ApoE protein is essential
for adsorption to occur. This adsorption was competitively
inhibited by treatment with heparin or heparinases, suggesting
that involvement of heparan sulfate proteoglycans (HSPGs),51,52)
which are abundantly expressed on the surface of hepatocytes
and are known to be a receptor of the ApoE protein.53) These
findings that liver accumulation is a complex process in which
the bare LNPs rapidly adsorb to the microvillar surface of he-
patocytes through ApoE-HSPG interactions following the bind-
ing of ApoE proteins to the LNPs in the blood circulation after
intravenous administration (Fig. 3B).
The YSK05-LNPs showed ED50 values of 0.06 mg
siRNA/kg and a durable gene silencing for up to 2 weeks in
a mouse FVII model through optimizing the lipid composi-
tion.54) To apply this to the treatment of hepatitis C virus
(HCV), mice with chronic HCV infections were produced by
the inoculation of the HCV genotype 1b in chimeric mice with
humanized liver cells and the siHCV-loaded YSK05-LNPs
were then intravenously administered. HCV genomic RNA
levels in serum were suppressed up to 90% for periods of up
to 2 weeks (Fig. 3C). A significant decrease in the levels of
HCV core proteins in liver tissue was also observed (Fig. 3D).
The optimized YSK05-LNPs were also applied to the in
vivo validation of candidate genes as therapeutic targets.55) A
comparative transcriptome analysis in liver between diabetic
and normal mice identified an elevated expression of mono-
acylglycerol O-acyltransferase 1 (Mogat-1), an enzyme that
is involved in triglyceride synthesis and its storage.56–58) Long
term Mogat-1 gene silencing by the repeated administration
of siMogat-1-loaded YSK05-LNPs in diabetic mice resulted in
preventive effects of type 2 diabetes, including reduced blood
glucose levels, triglycerides and cholesterol, and increased lev-
els of adiponectin. Related to this study, the durable silencing
of Lipin1, which regulates fatty acid utilization in the triglyc-
eride biosynthesis pathway,59) by YSK05-LNPs successfully
revealed that Lipin1 silencing in liver tissue led to a marked
decrease in adipose tissue mass and adipocyte diameters.60)
These results suggest that the established LNPs could be ap-
plied not only for the treatment of a specific disease but also
for the validation and investigation of in vivo functions of
genes of interest.
PEGylation of the LNP surface is a widely used strategy for
inhibiting non-specific interactions with biocomponents and to
maximize the efficiency of active targeting.61–63) However, the
PEGylation is known to severely inhibit the endosomal escape

Fig. 3. Application of YSK05-LNPs for the Delivery of siRNAs to Hepatocytes

(A) Comparison of blood clearance rate between bare and GalNAc-LNPs which can be cleared via the ApoE/LDLR and GalNAc/ASGPR pathways, respectively. (B)
Schematic illustration of the process of hepatic accumulation of the LNPs. ApoE bind rapidly to bare LNPs trapped by HSPGs on hepatocytes followed by LDLR-mediated
endocytosis. The GalNAc-LNPs gradually accumulate to hepatocytes via GalNAc-ASGPR interactions. (C) Inhibition of HCV infection by intravenous administration of
siHCV-loaded YSK05-LNPs. (D) Microscoppic observation of intrahepatic HCV core proteins after treatment with the YSK05-LNPs. These figures are reproduced, in
part, with permission from Elsevier and Springer Nature. (Color figure can be accessed in the online version.)
Vol. 69, No. 12 (2021)1145
Chem. Pharm. Bull.
process, a phenomenon known as the ‘PEG dilemma.’64,65)
Although pH-sensitive CLs with an acid dissociation constant
(pKa) around 6.4 can be used to reduce the concentration of
PEG on the surface of LNPs needed for long blood circulation
compared to permanently CLs, even such a lower concentra-
tion of surface PEG can inhibit both cellular uptake and endo-
somal escape.32) PEGylation of a LNP surface via a pH-labile
linkage is one of the more useful strategies for overcoming
this dilemma.66–70) Maleic anhydride reacts with amino groups
and forms a labile amide bond in an acidic environment,
and the pH-sensitive degradability of the amide bond can be
Fig. 4. Improvement of Active Targeting Using pH-Labile PEGylation
(A) Maleic anhydride molecules react with primary amino groups and form an
amide bond. The amino bond can be cleaved under weakly acidic conditions. The
cleavage rate can be modulated by the substituents on the cis-double bond of the
maleic anhydride. CDM shows more rapid cleavage rate than CEM. (B) Schematic
illustration of improved active targeting using pH-labile PEGylation. The pH-labile
PEGylation of the GalNAc-LNPs inhibits non-specific interaction in blood stream,
facilitating GalNAc-mediated accumulation to hepatocytes. After being endocy-
tosed, the pH-labile linkages are rapidly cleaved and the LNPs are re-activated,
promoting endosomal escape. These figures are reproduced, in part, with permis-
sion from Elsevier. (Color figure can be accessed in the online version.)
Fig. 5. Development of the 2nd Generation pH-Sensitive CLs, YSK13 and YSK15
(A) Chemical structures of the YSK13 and YSK15. Apparent pKa value can be modulated by carbon length between a tertiary amine and an ester bond. (B) Inhibition
of HBV infection by an intravenous administration of siHBV-loaded YSK05-LNPs. Robust and durable suppression of HBV antigens and HBV DNA was observed. (Color
figure can be accessed in the online version.)
modulated by the substituents on the cis-double bond of the
maleic anhydride71–74) (Fig. 4A). The siRNA-loaded GalNAc-
modified YSK05-LNPs were PEGylated though carboxylated
dimethylmaleic anhydride (CDM) or carboxylated ethylmaleic
anhydride (CEM), a maleic anhydride derivative, linker.75) The
use of a maleic anhydride linker promoted the detachment of
the modified PEG and the re-activation of the fusogenisity of
the LNPs under endosomal pH mimetic conditions. The ef-
ficiency of hepatocyte-targeting and gene silencing activity
were observed to be enhanced in vivo. Taken together, the pH-
labile PEGylation of GalNAc-modified LNPs through a maleic
anhydride linkage successfully shielded the LNPs, enhanced
active targeting to hepatocytes, and re-activated the LNPs by
the detachment of PEG in response to the acidic environment
in endosomes (Fig. 4B).
MicroRNAs (miRNAs) are a class of small non-coding
RNAs that are related to homeostasis and diseases and
function by regulating gene expression by binding to the
3′-untranslated region (3′-UTR) of target mRNAs, thus trig-
gering the degradation of the mRNAs or the suppression of
translation.76) miRNA-122 (miR-122) is a liver-specific miRNA
and is known to play important roles in lipid metabolism and
to facilitate the replication of HCV RNA.77–79) Therefore, the
inhibition of miR-122 by the anti-miRNA oligonucleotide
(AMO) is a promising strategy for the treatment of liver
diseases.80) Consistent with siRNAs, LNPs can increase the
bioavailability of AMOs. Three separate intravenous ad-
ministrations of AMO122-loaded YSK05-LNPs at doses of
1 mg AMO/kg in mice resulted in a nearly 90% inhibition
of miR-122, a value that was significantly higher than free
AMO122 (<40% inhibition).81) This resulted in an elevated ex-
pression of miR-122 target genes (AldoA, Bckdk, and Ndrg3)
in liver tissue and a reduced plasma cholesterol concentration.
The YSK05-LNPs also succeeded in inhibiting miR-122 by
delivering 2′-OMe-4′-thioribonucleoside-modified AMO122.82)
Taken together, these results demonstrate that the use of drug
delivery technologies presents a practical and valuable alterna-
tive to anti-miRNA therapeutics.
In 2016, the 2nd generation pH-sensitive CLs, YSK13 and
YSK15, were designed with the objective of overcoming the
limitations of YSK05, including insufficient fusogenisity and
potential chemical instability under acidic conditions due to
1146 Chem. Pharm. Bull. Vol. 69, No. 12 (2021)

an acid-labile ketal linker.83) The YSK13 and YSK15 contain
α,β-unsaturated ester and allyl ester linkers, respectively, and
two linoleic acid-derived scaffolds are attached to one sp2 car-
bon atom of the linker (Fig. 5A). This design makes the angle
between the two scaffolds be 120°, which is higher than that of
YSK05, and therefore would be expected to emphasize a cone
shape and to increase fusogenicity based on CPP theory.34)
A 31P-NMR analysis demonstrated that YSK13 show clearly
a lower TH compared to that of YSK05, suggesting a higher
fusogenicity. Both the YSK13 and YSK15 contain some de-
rivatives with different spacer lengths between an ester bond
and a tertiary amino group. A longer spacer length resulted
in LNPs with an increased apparent pKa value, which can be
determined by a 6-(p-toluidino)-2-naphthalenesulfonic acid
(TNS) assay. It is known that the optimal pKa range for de-
livering siRNA to hepatocytes is 6.2–6.5.20) A YSK13 deriva-
tive, YSK13-C3, with a pKa of 6.45 and ED50 of 0.015 mg/kg
in a mouse FVII model, was selected as the optimal CL for
hepatocytes. The impact of the apparent pKa value on cell-
specificity is described below. The YSK13-C3 was used in
the treatment of chronic hepatitis B virus (HBV) infections.84)
Mice that are persistently infected with HBV were intrave-
nously administered with the siHBV-loaded YSK13-C3-LNPs
at a dose of 5 mg siRNA/kg. An approximately 80–90% sup-
pression of HBV antigens (HBsAg and HBeAg) and HBV
genomic DNA was observed for periods of up to 10 d (Fig.
5B). The oral administration of a clinically used nucleoside
analogue of the reverse transcription (RT) inhibitor, enteca-
vir,85) strongly suppressed HBV DNA in sera but failed to
reduce the levels of HBV antigens in the sera. The elimination
or suppression of HBV antigens is important for the treatment
of hepatitis because several HBV antigens downregulate the
adaptive immune response to HBV in patients.86–88) Therefore,
the above findings indicate that a gene silencing approach
could be the basis for a novel class of anti-HBV drugs which
would be superior to RT inhibitors.
More recently, a pH-sensitive CL library was developed
from a benchmark CL, YSK12-C4.89) The YSK12-C4, which
shows a high fusogenicity and a high pKa (approx. 8.2),
has been used for treating hard-to-transfect immune cells

Fig. 6. Development of an Original pH-Sensitive CL Library

(A) Overall picture of the pH-sensitive CL library. (B) A plot of calculated pKa by ChemDraw versus the apparent pKa measured by a TNS assay. (C) Structure of one
of the best pH-sensitive CLs, CL4H6, for the delivery of siRNAs to hepatocytes. (D) Dose-dependent FVII silencing activity of the siFVII-loaded CL4H6-LNPs. (E) Cy-
tosolic delivery of siRNAs by the CL4H6-LNPs. The siRNAs are observed as diffused patterns in the cytosol of hepatocytes. These figures are reproduced, in part, with
permission from Elsevier. (Color figure can be accessed in the online version.)
Vol. 69, No. 12 (2021)1147
Chem. Pharm. Bull.
(this subject is described below). To develop the library, the
YSK12-C4 was divided into 3 parts, including the distal and
proximal sides of the scaffolds and the hydrophobic region,
and each region was derivatized independently to develop a
systematic library of pH-sensitive CLs (Fig. 6A). The pH-
sensitive CLs with different structures around tertiary amine
showed various apparent pKa values. A plot of the cLog p
value of the hydrophilic region versus the experimentally
determined apparent pKa value revealed that the apparent pKa
value decreased with increasing hydrophobicity of the hydro-
philic region. On the other hand, the pKa values estimated
by the ChemDraw software were significantly lower than the
apparent pKa values and predicting the precise apparent pKa
value clearly failed (Fig. 6B). This could be explained by the
fact that a tertiary amino group, a proton donor, is present on
the interface of the LNPs where the proton is relatively hard to
access and an increased cLog p value leads to the preferential
localization of the tertiary amine in the hydrophobic layer
of scaffolds, resulting in resistance to protonation. Moreover,
extremely high concentrations of the pH-sensitive CLs (ap-
prox. 50 mol% of total lipid) in the LNPs results in reduced
pKa values due to an increased charge repulsion between the
CLs that are in close proximity with each other.90) In contrast
to the hydrophilic region, the structures of hydrophobic scaf-
folds had relatively minor effects on apparent pKa values and
unexpected interactions between the hydrophobic tails and the
hydrophilic head group on apparent pKa values was limited.
After several examinations regarding structure–activity re-
lationships, a novel pH-sensitive CL, CL4H6, was identified
as one of the best CLs for delivering siRNA into hepatocytes
and was determined to be a 3rd generation CL (Fig. 6C). The
lipid composition-optimized CL4H6-LNPs delivered a dose
of 0.0025 mg siRNA/kg in a mouse FVII model, which was
a 6- and 2-fold higher efficiency compared to 2nd generation
YSK13-C3-LNPs and MC3-LNPs, respectively (Fig. 6D). Flu-
orescent microscopic observations in liver tissue revealed that
AlexaFluor647-labeled siRNAs had escaped from endosomes
and diffused into the cytosol (Fig. 6E). A quantitative PCR
analysis revealed that 4.2% of the siRNAs that had accumu-
lated in liver tissue was successfully loaded in RNA-induced
silencing complex (RISC), which indicates a higher bioavail-
ability of siRNAs compared to previous reports regarding
MC3 and its biodegradable analogue, L319.91,92)
Introducing exogenous mRNAs into target cells is a promis-
ing strategy for expressing therapeutic proteins of interest. For
vaccinations, the introduction of antigen-encoding mRNAs
leads to the expression of antigen proteins and the activation
of antigen presenting cells, resulting in the activation of both
innate and adoptive immunity in attempts to eliminate patho-
gens or cancer cells.93–95) Current progress in mRNA optimi-
zation (including cap, codon, UTRs, poly(A) tail, chemically
modified nucleosides, purification) have substantially improved
the translatability of the RNAs with reduced immunogenicity,
resulted in realization of and successful development of the
current COVID-19 mRNA vaccines.96,97) LNP technology was
also naturally critical for this realization.7,98) However, the
composition of the optimal formulation and the physicochemi-
cal properties of the LNPs for mRNA remain unclear. The
design of experiment (DOE), an experimental design method
that rationally allocates experiments by using mathematical
statistics and reaches reliable conclusions with a considerably
reduced number of experiments, is a useful methodology for
determining the optimal formulation from a large number of
manufacturing factors, including the choice of lipids, lipid
composition, nitrogen per phosphate (N/P) ratio, and appropri-
ate conditions for synthesis (e.g. buffer pH, flow rate ratio and
total flow rate during microfluidic synthesis). However, the
conventional DOE used for formulation optimization involves
the analysis of only one response such as gene expression in
a target tissue.24,99,100) Therefore, a detailed understanding of
the relationship between a main response and physicochemi-
cal properties was very limited. Therefore, DOE with mul-
tiple responses were conducted to optimize liver-targeting
mRNA-loaded LNPs and to identify the important properties
(e.g. physicochemical properties) needed for gene expression.
In addition to the factors examined by DOE (e.g. a type of
lipid and lipid composition), it was found that LNP size and
PEG-to-PL ratio were other key factors for liver-specific gene
expression. The optimized mRNA-loaded CL4H6-LNPs de-
livered over 8000 ng/mL of human erythropoietin protein in
serum at 6 h after the intravenous administration of a 0.5 mg
mRNA/kg dose. The optimized mRNA-loaded CL4H6-LNPs
also showed 2.5-fold higher nanoluciferase expression in liver
tissue compared to MC3-LNPs with no observable toxicity.
Since the discovery of the CRISPR/Cas system,101) targeted
genome editing through engineered nucleases has become
a major focus of attention in both biological research and
therapeutics.102,103) Intended genome editing can be achieved
through several approaches, including gene disruption by non-
homologous end joining and the insertion of a target sequence
by homology-directed repair. Moreover, the rapid generation
of re-engineered Cas nucleases, including high-fidelity Cas9,
protospacer adjacent motif (PAM)-modified Cas9, base edi-
tor, transcription activator or repressor (CRISPRa/CRISPRi),
a programmable epigenetic memory writer (CRISPRoff),
a prime editor, and type I Cas system, has dramatically
amplified application diversity and the significance of the
CRISPR/Cas system for therapeutics.104–111) The introduction
of CRISPR/Cas machinery as a ribonucleoprotein (RNP; a
complex of Cas nuclease and guide RNA) is known to be
preferable for both the efficiency of genome editing and for
achieving a lower off-target effect.112,113) However, the technol-
ogy for formulating RNP delivery has been not established
yet. To overcome this limitation, RNP-loaded LNPs were
synthesized by means of a clinically relevant manufactur-
ing process using a micromixer-equipped microfluidic mixer,
iLiNP114,115) (Fig. 7A). A detailed examination of manufactur-
ing conditions revealed that both a higher pH (5.5 or over)
and a higher flow rate ratio (7 or over) compared to standard
conditions for the synthesis of nucleic acid-loaded LNPs were
required to avoid aggregation and the loss of DNA cleavage
activity of the RNPs.116) Severe aggregation and the loss of
DNA cleavage activity of RNPs were observed under standard
conditions used for the synthesis of nucleic acid-loaded LNPs.
Furthermore, by using an iLiNP device with 3 inlets, the
introduction of a buffer solution from a center inlet between
two inlets used for RNP and lipid solutions resulted in the ag-
gregation of RNPs at the junction to be completely inhibited
by preventing the RNPs from coming into direct contact with
high concentrations of ethanol (Fig. 7A). These optimizations
in manufacturing parameters and the structure of the micro-
fluidic device enabled us to synthesize RNP-loaded LNPs
1148 Chem. Pharm. Bull. Vol. 69, No. 12 (2021)

Fig. 7. Application of the CL4H6-LNPs for Cas RNP Delivery

(A) Strategy for the synthesis of the Cas RNP-loaded LNPs using a microfluidic device, iLiNP. The use of the iLiNP device with 3 inlets resulted in the complete inhibi-
tion of aggregation of RNPs at the junction by avoiding the direct contact of RNPs with high concentrations of ethanol. (B, C) Impact of complexation of ssONs and length
of crRNA on functional delivery of Cpf1 RNPs. KO activity was maximized by combination of ssRNA with 120 nt and elongated crRNA (B) and the KO activity was
well correlated with the total RNA length (C). (D) Inhibition of HBV infection by Cas9 RNP-loaded CL4H6-LNPs in HBV-infected cells. The RNP-loaded LNPs showed
a superior inhibition of HBV infection compared to the AAV2 vector. These figures are reproduced, in part, with permission from Elsevier. (Color figure can be accessed
in the online version.)

reproducibly. The optimization of Streptococcus pyogenes
Cas9 (spCas9)-loaded LNPs was conducted through 2 cycles
of DOE using a flowcytometric analysis of enhanced green
fluorescent protein (EGFP) knockout (KO) in HeLa stably
expressing EGFP (HeLa-GFP) cells. The optimized CL4H6-
LNPs showed a IC50 of approx. 0.2 nM RNP, which was a 10-
to 100-fold lower concentration compared to previous reports
regarding non-viral spCas9 RNP delivery systems. To expand
the applicability of the optimized LNPs, Acidaminococcussp.
BV3L6 Cas12a (Cpf1) RNP delivery was examined. The Cpf1
has a unique PAM sequence that is different from that of
spCas9, exhibits robust genome editing activity in mammalian
cells and potentially shows lower off-target effects compared
to spCas9.117) The Cpf1 utilizes a shorter single crispr RNA
(crRNA) compared to the guide RNA (approx. 100 nt length)
of spCas9, making the loading of this cargo in LNPs dif-
ficult due to the poor electrostatic interactions.118) Therefore,
both the complexation of single strand oligonucleotide (ssON)
with RNPs and the elongation of crRNA were examined in
attempts to increase the negative charge density in the Cpf1
RNPs. Interestingly, while the original Cpf1 RNP-loaded
LNPs failed to induce a significant EGFP KO (<10% at 2 nM
RNP), the elongation of crRNA or the complexation of ssON
dramatically increased EGFP KO activity (Fig. 7B). A combi-
nation of the two approaches further increased the EGFP KO,
providing an IC50 less than 0.5 nM RNP (Fig. 7B). The total
RNA length (crRNA + ssON) was well correlated with the KO
activity (Fig. 7C), indicating the significance of the density of
negative charges of RNPs on functional delivery. Although an
increase in EGFP KO activity was also observed for spCas9
RNP, the impact of ssON complexation was much less than
that for Cpf1 RNP due to the longer guide RNA for spCas9
RNP. To confirm that the optimized RNP-loaded LNPs have
therapeutic potential, the inhibitory effect of HBV in HBV-
infected cells was examined. HBV-infected HepG2-hNTCP-30
cells were consecutively treated with spCas9 RNP-loaded
LNPs for 12 d, resulting in 60 and 80% inhibition of HBV
DNA and covalently closed circular DNA, respectively, a sig-
nificantly higher inhibitory effect than the adeno-associated
virus 2 (AAV2) vectors used for comparison (Fig. 7D). These
data suggest that using RNP-loaded LNPs for gene editing
would be a novel strategy for the treatment of chronic HBV
infections. Optimization of RNP-loaded LNPs targeting liver
tissue for gene KO and knock-in is currently an on-going
effort.
Vol. 69, No. 12 (2021)1149
Chem. Pharm. Bull.

3. Application of LNPs to Extra-Hepatic Tissues and
Cell Types
Macrophages are one of the most important contributors
to homeostasis and various inflammatory diseases.119) Macro-
phages in different tissues play various roles in inflammatory
diseases and are distinguished by different names, including
peritoneal macrophages (PEMs), Kupffer cells, microglia,
and tumor-associated macrophages (TAMs) in the peritoneal
cavity, liver, brain, and tumor tissue, respectively.120–123) Ma-
nipulation of macrophages at the genetic level represents a
promising approach for the treatment of a number of diseases
in which macrophages are involved.
Gene silencing in PEMs was examined by focusing on the
size and route of administration used for the YSK05-LNPs.124)
The diameter of the YSK05-LNPs was modulated from 75 to
460 nm by changing the amount of PEG-lipid. Cellular uptake
in PEMs (defined as F4/80+ cells in the peritoneal cavity) was
clearly size-dependent and reached a maximum at a diameter
of 340 nm after intravenous administration. An intravenous
administration of siCD45-loaded YSK05-LNPs in mice at a
dose of 2 mg siRNA/kg resulted in approx. 80% CD45 silenc-
ing at the protein level for particles with diameters of 200 and
340 nm, which was significantly higher than that for 75 nm
(approx. 40% silencing). On the other hand, no significant dif-
ference in gene silencing activity in PEMs was observed after
an intraperitoneal administration of the same LNPs in mice at
a dose of 0.003 mg siRNA/kg, suggesting that impact of size
on intravenous administration involves translocation from the
blood circulation to the peritoneal cavity. The YSK05-LNPs
showed an ED50 value of 6 µg siRNA/kg for CD45 gene si-
lencing in PEMs after intraperitoneal administration. This
single digit µg/kg range enabled us to easily analyze multiple
genes simultaneously without any obvious adverse effect.
The tumor-microenvironment is known to contain large
numbers of macrophages, which are referred to as TAMs and
are mostly the pro-tumorous (M2) phenotype.125–128) Modify-
ing the functions of TAMs or the manipulation of TAMs from
M2 to the anti-tumorous M1 phenotype by siRNAs could be
a novel immunotherapy for the treatment of cancer. To target
TAMs, the CL4H6-LNPs were modified with distearoylg-
lycerol PEG, which has longer scaffolds (C18:0), dissociates
more slowly from the LNPs, and contributes to longer blood
circulation.129) The CL4H6-LNPs accumulated in subcuta-
neously inoculated human renal cell carcinoma (OS-RC-2)
tumor tissues. Flowcytometric analysis revealed that high
levels of the CL4H6-LNPs were taken up by TAMs (defined
as CD45+CD11b+F4/80+ cells) compared to other cell popula-
tions, including neutrophils (defined as CD45+CD11b+F4/80−
cells), other leukocytes (defined as CD45+CD11b− cells), and
non-leukocytes (defined as CD45− cells, which include tumor
cells, tumor-associated fibroblasts, and endothelial cells). A
significant (approx. 70%) gene silencing of CD45 in TAMs
was obtained after the intravenous administrations of the
CL4H6-LNPs. Motivated by these findings, we examined the
manipulation of TAMs from M2 to M1 phenotype by the gene
silencing of M2-related genes, signal transducer and activator
of transcription 3 (STAT3) and hypoxia-inducible factor 1α
(HIF-1α), which are elevated in TAMs and involved in various
pro-tumorous functions, including immunosuppression, the
promotion of tumor growth, and angiogenesis.130–132) The re-
peated administration of the siSTAT3/siHIF1α-loaded CL4H6-
LNPs in OS-RC-2 tumor-bearing mice induced a significant
anti-tumor effect. An analysis of the gene expression in tumor
tissues revealed the elevation of CD11b (a macrophage mark-

Fig. 8. Development of a pH-Sensitive CL, YSK12-C4

(A) Chemical structure of the YSK12-C4. (B) Efficient Socs1 silencing by siSOCS1-loaded YSK12-C4-LNPs in hard-to-transfect murine BMDCs. (C) Therapeutic anti-
tumor effect of SOCS1-silenced BMDCs in an E.G7-OVA tumor model. The SOCS1-silenced BMDCs by YSK12-C4-LNPs showed superior anti-tumor effect compared
to that by RNAiMAX. (D) Gene silencing activity of the YSK12-C4-LNPs in hard-to-transfect human immune cell lines, Jurkat, THP-1, KG-1, and NK92 cells. These
figures are reproduced, in part, with permission from Elsevier and Springer Nature. (Color figure can be accessed in the online version.)
1150 Chem. Pharm. Bull.
er), CD169 (an M1 macrophage marker), and tumor necrosis
factor-α (TNF-α) and the suppression of CD31 (an endothelial
cell marker) and transforming growth factor-β (TGF-β) in ad-
dition to target genes (STAT3 and HIF-1α). These findings
suggest that the use of CL4H6-LNPs leads to the infiltration
of M1 macrophages in the tumor microenvironment, the
suppression of angiogenesis, and the release of immunosup-
pression. Importantly, these reactions were derived from gene
silencing in TAMs but not in human tumor cells because the
siRNAs utilized only target murine genes.
The delivery of siRNAs to dendritic cells (DCs) is a prom-
ising strategy for improving the efficacy of DC-based cancer
immunotherapy. However, the efficient delivery of siRNAs to
DCs is somewhat challenging.133–135) A fusogenic CL, YSK12-
C4, was originally designed to overcome this hurdle.136)
YSK12-C4 contains two linoleic acid-derived scaffolds that
are attached to one carbon atom, which emphasizes the adop-
tion of a cone shape and enhances fusogenisity (Fig. 8A). A
hydrophilic tertiary hydroxy group that is present between
a tertiary amine and the scaffolds contributes to the higher
apparent pKa (approx. 8.0) (Fig. 8A), which leads to strong
cationic properties in the cell culture medium and promotes
cellular uptake in DCs. The optimized YSK12-C4-LNPs
showed IC50 values of 1.5 nM siRNA and achieved up to
90% gene silencing at higher concentrations in mouse bone
marrow-derived DCs (BMDCs), while a commercially
available transfection reagent, Lipofectamine RNAiMAX,
achieved less than 60% gene silencing at a maximum and
showed an IC50 of 25 nM, which was much higher than that
for YSK12-C4-LNPs (Fig. 8B). The efficient gene silencing
was expected to enhance the efficacy of DC-based immuno-
therapy by targeting the suppressor of cytokine signaling 1
(SOCS1), which blocks the janus kinase (JAK)–STAT sig-
naling pathways.137) The siSOCS1-loaded YSK12-C4-LNPs
achieved approx. 80% silencing of the SOCS1 gene, resulting
in an enhanced production of TNF-α, including interleukin-6
(IL-6) in response to interferon (IFN)-γ stimulation. Immuni-
zation of the SOCS1-silenced DCs by the YSK12-C4-LNPs in
mice bearing the EL4 murine lymphoma cell line expressing
chicken ovalbumin (E.G7-OVA) resulted in a significant anti-
tumor effect, while the SOCS1-silenced DCs by Lipofectamine
RNAiMAX failed (Fig. 8C). These results clearly suggest that
the YSK12-C4-LNPs are a promising technology for use in
enhancing DC-based immunotherapy. The silencing of the
indoleamine 2,3-dioxygenase 1 gene by the YSK12-C4-LNPs
in BMDCs also led to an enhanced antitumor effect against
E.G7-OVA tumors.138) Motivated by these results, the gene
silencing activity of the YSK12-C4-LNPs in several hard-to-
transfect human immune cell lines, including Jurkat, THP-1,
KG-1, and NK92, was examined.139) Although substantial cy-
totoxicity was observed at higher concentrations in the cases
of KG-1 and NK92 cells, the YSK12-C4-LNPs showed an ef-
ficient gene silencing activity of up to 80 to 90% (Fig. 8D). It
was suggested that small-size and colloidal stability in the cell
culture medium of the YSK12-C4-LNPs contributed to the
enhanced cellular uptake followed by efficient gene silencing
in these cells.
The stimulator of the interferon gene (STING) pathway
recognizes cytosolic DNA and cyclic dinucleotides (CDNs) to
induce type I IFN and inflammatory cytokine responses and
appears to be essential for the innate sensing of tumor cells.140)
Vol. 69, No. 12 (2021)
STING agonists such as CDNs are expected to function as
potent cancer adjuvants. However, the delivery of the CDNs
into cytosol where the STING pathway is located is some-
what challenging due to hydrophilic nature of the CDNs.140)
A CDN, cyclic di-GMP (c-di-GMP)-loaded YSK05-LNPs
were developed for cancer immunotherapy.141) The optimized
c-di-GMP-loaded YSK05-LNPs induced a significantly higher
level of IFN-β production in a murine Raw264.7 macrophage
cell line compared to Lipofectamine 2000 and DOTAP-based
LNPs. The subcutaneous administration of the c-di-GMP-
loaded YSK05-LNPs enhanced in vivo antigen-specific cyto-
toxic T cell (CTL) activities and showed preventive antitumor
effects in major histocompatibility complex class I (MHC-
I)-expressing E.G7-OVA-bearing mice, suggesting that the
induction of MHC-I restricted antitumor immunity. The loss
or down regulation of the MHC-I in tumors is a well-known
mechanism for escaping from immunosurveillance by CTL,
which is observed in malignant melanomas. In this situation,
the activation of natural killer (NK) cells would be desirable
for inducing an antitumor effect against malignant melano-
mas.142,143) The intravenous administration of c-di-GMP-loaded
YSK05-LNPs resulted in the production of TNF-α and IFN-γ,
which are secreted from activated NK cells.144) The activation
of NK cells was confirmed by the observation of a significant
increase in the population of NKG2D- and CD69-positive NK
cells. Significant antitumor effects in a lung metastatic mouse
model with a B16-F10 melanoma was observed after the in-
travenous administration of c-di-GMP-loaded YSK05-LNPs
and was cancelled by the depletion of NK cells, thus confirm-
ing that the c-di-GMP-loaded YSK05-LNPs are capable of
activating NK cells and inducing antitumor effects against
tumors with the loss or downregulation of MHC-I. It was re-
cently demonstrated that c-di-GMP-loaded YSK12-C4-LNPs
overcome anti-programmed cell death 1 (PD-1) resistance and
show a synergistic antitumor effect by activating NK cells
followed by inducing the formation of PD ligand 1 on tumor
cells in the same melanoma model.145)
A technology for the treatment of brain disorders, including
traumatic brain injury, Parkinson’s disease, and Alzheimer’s
disease, is still an unmet medical need. Nucleic acid and
gene therapy is a promising strategy for realizing efficacious
therapeutics for the hard-to-treat brain disorders, as dem-
onstrated by the clinically approved zolgensma (wild-type
AAV9-based gene therapy) and spinraza (ASO for exon inclu-
sion) for the treatment of spinal muscular atrophy, intractable
genetic diseases caused by the deletion of a responsible gene,
the survival motor neuron 1.146) A disorder of the brain endo-
thelium is known to be associated with the pathophysiology
of brain disorders.147) Therefore, targeting brain endothelial
cells (BECs) would be a useful approach for the treatment of
brain disorders. The gene silencing activity of siRNA-loaded
YSK05-LNPs in BECs was examined as a possible approach.
YSK05-LNPs were modified with recombinant ApoE proteins
as ligands against LDLR family-expressing BECs.148) An
ApoE-dependent increase in both cellular uptake and gene si-
lencing activity was clearly obtained. Motivated by the impact
of ApoE modification on delivery efficiency, ApoE-modified
YSK05-LNPs containing pDNA were intracerebroventricularly
administered to mice for transgene expression in brain.149)
A significant increase in transgene expression was observed
after the ApoE modification in vivo. Immunostaining analysis
Vol. 69, No. 12 (2021)1151
Chem. Pharm. Bull.
revealed transgene (mCherry) expression in neural stem cells
and/or neural progenitor cells, suggesting usefulness of this
system for the treatment of neurodegenerative diseases.
Targeting the lung endothelium is promising approach
for the treatment of various diseases, including sepsis, pul-
monary hypertension, and lung cancer.150) Previously, our
laboratory coincidentally found that a pH-sensitive fusogenic
GALA peptide, functions as a specific ligand for the lung,
and, using it, we were able to successfully induce gene si-
lencing of an endothelial cell marker, CD31, in lungs with an
ED50 of 0.21–0.4 mg siRNA/kg.151,152) Lung-targeting LNPs
composed of YSK05 were recently finely optimized, and an
ED50 of 0.01 mg siRNA/kg was achieved, a value that was
20- to 40-fold lower than that for previous formulations.153) A
clinically relevant dose (0.5 mg siRNA/kg) was sufficient to
eradicate a metastatic lung cancer model without any signs of
toxicity. Development of novel lung targeting formulations for
mRNA delivery is currently an on-going project.
4. Elucidation of Structure–Activity Relationships
The particle diameter of LNPs is a critical factor that af-
fects their biodistribution and infiltration into deep tissue. It
is possible for small-sized nanoparticles to be translocated
from the lung to lymph nodes where they then drain into
the lymphatic system and penetrate deeply into stromal-rich
tumor tissues.154–158) It is known that subcutaneously admin-
istered small-sized LNPs show an excellent transitivity to and
distribution within the lymph nodes.159) Furthermore, the sub-
cutaneous administration of small-sized siRNA-loaded LNPs
modified with GalNAc successfully reached hepatocytes and
induced substantial gene silencing while a larger counterpart
failed.160) Despite the fact that small-sized LNPs are desired
for efficient drug delivery due to its great potential for tissue
penetration, Efforts to develop small-sized LNPs that can be
used for efficient drug delivery have been limited. The fact
that it is difficult to reproducibly produce small-sized LNPs
with low polydispersity represents a fundamental limita-
tion.115) The recent development of mixer-equipped microflu-
Fig. 9. Impact of Particle Size of the LNPs on the Delivery of siRNAs
(A) Diameter of the LNPs decreases depending on the amount of PEG-lipid. (B) Gene silencing activity of the LNPs with varied diameter in the presence or absence
of FBS in HeLa-dluc cells. (C) The measured total surface area of the LNPs using TNS. (D) Theoretically calculated total surface area of the LNPs. (E) Measurement of
hydration level on the interface of the LNPs using laurdan. (F) Schematic illustration of packing stress derived from the substantial difference between the actual positive
curvature of the YSK05 on the interface of the LNPs and the spontaneous negative curvature of the YSK05. These figures are reproduced, in part, with permission from
Elsevier. (Color figure can be accessed in the online version.)
idic devices that can achieve fast and reproducible mixing of
two or more miscible solutions (e.g. ethanol and water) have
greatly contributed to overcoming this limitation.114,161,162)
However, small-sized LNPs show a lower integrity and drug
delivery efficiency. Chen et al. reported on the rapid disso-
ciation of lipid components, including CL, PL, and PEG-lipid,
from small-sized LNPs after incubation in mouse plasma
compared to a larger counterpart, resulting in poor gene
silencing activity in hepatocytes.163) However, the detailed
mechanism responsible for this poor delivery that is associated
with downsizing remains unknown. Therefore, siRNA-loaded
LNPs with different diameters, approx. 35 and approx. 65 nm,
were synthesized by changing amount of PEG-lipid to 3% and
1%, respectively, to examine the impact of particle size164)
(Fig. 9A). The 3%PEG-LNPs showed a 4-fold lower FVII gene
silencing activity compared to the 1%PEG-LNPs. Fluorescent
microscopic observations of intrahepatic siRNA distribution
revealed that most of the siRNAs showed a punctate pattern
in the case of the 3%PEG-LNPs, indicating failure of endo-
somal escape, while substantial amounts of siRNAs showed
a diffused pattern in the case of 1%PEG-LNPs, indicating the
successful cytosolic delivery of siRNAs. The quantification
of siRNAs also permitted the amount of siRNAs delivered
by 3%PEG-LNPs to be measured and the findings indicated
that the amount was significantly lower than that delivered by
1%PEG-LNPs, suggesting the premature release of siRNAs
from the 3%PEG-LNPs in the blood stream. An in vitro study
confirmed that the siRNAs released from the 3%PEG-LNPs
showed a lower gene silencing activity only in the presence of
fetal bovine serum (FBS) but not in the absence of FBS (Fig.
9B), suggesting that serum components promote the destabi-
lization and inactivation of small-sized LNPs. Measurement
of the relative surface area using a TNS probe revealed that
the actual relative surface area of 3%PEG-LNPs was 2.99-fold
higher than that of the 1%PEG-LNPs (Fig. 9C), which was
significantly higher than the theoretically calculated relative
surface area (1.72-fold) (Fig. 9D), indicating that lower lipid
packing on the interface may have occurred, possible due to
1152 Chem. Pharm. Bull. Vol. 69, No. 12 (2021)

packing stress derived from strain between the actual positive
curvature on the interface of the LNPs and the spontaneous
negative curvature of the fusogenic CLs (Fig. 9E). Measure-
ment of the hydration level of the interface of the LNPs using
laurdan supported the above finding (Fig. 9F). The interface
of the small-sized LNPs can cause instability, thus facilitating
the dissociation of lipid components and the premature release
of siRNAs, and the adsorption of large quantities of serum
proteins on the interface, resulting in the inhibition of fu-
sogenisity, a process that was confirmed by a hemolysis assay
and an siRNA release assay established by the Merck research
group.165) Cholesterol is known to contribute to the stabil-
ity of LNPs.166,167) The premature release of siRNAs from
small-sized LNPs was successfully inhibited by increasing the
molar ratio of cholesterol in the lipid composition. However,
the gene silencing activity of the small-sized LNPs was still
significantly lower than that of the larger counterparts, sug-
gesting that reduced endosomal escape needs to be addressed
if small-sized LNPs with efficient nucleic acid delivery charac-
teristics are to be developed.
The apparent pKa value is another key factor that can af-
fect the biodistribution and endosomal escape of LNPs. As
described above, the optimal pKa range for hepatic siRNA
delivery is known to be from 6.0 to 6.5.20) Therefore, the
original pH-sensitive CLs, YSK05, YSK13-C3, and CL4H6,
that can deliver nucleic acids into hepatocytes efficiently
also show pKa values in the range from 6.35 to 6.45, which
are within the desired optimal range.32,54,83,84,89) However, the
impact of the apparent pKa on intrahepatic distribution has
not yet been examined. The effect of the apparent pKa on the
intrahepatic distribution of the LNPs was examined using the
2nd generation pH-sensitive CLs, YSK13 and YSK15, with
varied apparent pKa values ranging from 5.70 to 7.2583) (Fig.
5A). The intrahepatic distribution of the LNPs clearly changed
from hepatocytes to LSECs. The LNPs with lower pKa values
were optimal from the viewpoint of hepatocyte-specificity.
The LNPs with optimal pKa (approx. 6.4) values for gene
silencing activity in hepatocytes naturally accumulated in he-
patocytes but also were substantially co-localized with LSECs.
The LNPs with pKa values of 6.8 specifically accumulated
in LSECs. Gene silencing activity in LSECs was examined
using siRNA targeting CD31, and a plot of CD31 expression
in the liver versus the apparent pKa value of the LNPs clearly
showed that gene silencing activity in LSECs was clearly pKa-
dependent and became maximum at pKa values in excess of
7.0 (Fig. 10A). A similar experiment was conducted using a
pH-sensitive CL library with different hydrophilic regions, and
it was revealed that the optimal pKa range for gene silencing
activity in LSECs was from 7.0 to 7.5 (Fig. 10B), which was
just 1 unit higher than that in hepatocytes. Although the ap-
parent pKa value of the LNPs can be controlled by the design
of chemical structure of the CL as examples of the 2nd gen-
eration CLs and CL library,83,89) precise adjustment of appar-
ent pKa value is somewhat challenging due to time-consuming
and labor-intensive processes and is sometimes impossible
due to limitations in the permissibility of chemical structure.
Therefore, the manipulation of the apparent pKa value of the
LNPs using a combination of two pH-sensitive CLs with dif-
ferent apparent pKa values would be an alternative strategy for
precisely adjusting apparent pKa to the target values. LNPs
composed of both YSK05 and YSK12-C4 (YSK05/12-LNPs)
were developed in attempts to achieve new pKa values.168) In-
terestingly, a plot of pKa of the final LNP versus the YSK12-
C4 ratio in total CLs indicated that contribution of YSK12-C4
was higher than that of YSK05 for the final pKa pf the LNPs
(Fig. 10C). For example, the actual final pKa of the LNPs com-
posed of equal molar amount of YSK05 and YSK12-C4 was
clearly higher than the pKa value that was simply calculated
as an average of the apparent pKa value of each lipid (Fig.
10C). Similar results were obtained when mixtures of YSK13,

Fig. 10. Manipulation of the Apparent pKa Value for Cell-Specific Delivery of siRNAs
(A, B) The pKa-dependency of gene silencing activity in LSECs. The consistent optimal pKa range for gene silencing in LSECs was observed for the 2nd generation
YSK13/YSK15 (A) and CL library (B). (C) Higher contribution of YSK12-C4 for final pKa was observed in the YSK05/YSK12-C4-LNP system. (D) Higher contribution of
YSK13-C4 for the final pKa was observed in the YSK13-C2/YSK13-C4-LNP system. (E) Mixture of YSK13-C2 and YSK13-C4 resulted in a new pKa that superimposes
with the theoretical curve obtained by the Henderson-Hasselbalch equation. These figures are reproduced, in part, with permission from Elsevier and Dovepress. (Color
figure can be accessed in the online version.)
Vol. 69, No. 12 (2021)1153
Chem. Pharm. Bull.
YSK13-C2 and YSK13-C4 lipids were used (Fig. 10D). Impor-
tantly, the contribution of the pH of LNPs composed of two
CLs with different pKa values was superimposed on the theo-
retical curve for the new pKa but not on the averaged curve of
original two pKa (Fig. 10E), strongly suggesting that the new
pKa value was contributed by the mixing of two CLs.
Neutral zwitterionic lipids with a bulky phosphochline, ph-
sophatidylcholine (PC) and sphingomyelin (SM) are known to
stabilize the L phase and inhibit the phase transition to an HII
phase due to their cylindrical structure.35) As described above,
a phase transition is essential for achieving membrane fusion-
mediated endosomal escape. Therefore, a high amount of PCs
or SMs in siRNA-loaded LNPs greatly inhibits endosomal
escape followed by the cytosolic release of siRNAs. Indeed,
the YSK05-LNPs showed robust gene silencing activity, but
only when phosphatidylethanolamines and not PCs were used
as a helper lipid.32) Interestingly, siRNA-loaded LNPs com-
posed of CL15H6, which has much longer scaffolds (C24 + O1)
compared to the conventional linoleic acid-derived scaffolds
(C18), and 50 mol% egg-derived SM showed excellent gene
silencing in HeLa-dluc cells, while that with CL15A6, a coun-
terpart with conventional scaffolds, failed.169) Gene silencing
activity was also clearly observed when any PCs with C16 or
C18 scaffolds were used but not when brain- and milk-derived
SMs which contain longer scaffolds (C22 to C24) were used.
It is known that lipids with different scaffold lengths have a
tendency to be segregated from each other due to the higher
Gibbs free energy for mixing.170) The inhibitory effect of PCs
and SMs on the L-to-HII phase transition should be affected
only when fusogenic CLs are miscible with the PCs and SMs
during the phase transition. Taking the above into consider-
ation, CLs with much longer scaffolds would be immiscible
with typical PCs and SMs consisting of C16 to C18 scaffolds
and would be resistant to the inhibitory effect of PCs and SMs
on membrane fusion (Fig. 11A). CL4H6, which is made up of
scaffolds with CL15H6, formed well-dispersed lipid suspen-
sions composed of equal molar ratios of CL4H6, distearoyl PC
(DSPC), and DSPS in an acidic buffer even at higher tempera-
ture (50 °C), and therefore, a 31P-NMR analysis failed to detect
a phase transition because the anisotropic 31P spectra from
the L or HII phases were averaged as the result of the rapid
rotation of relatively small-sized dispersed particles (Fig. 11B,
insertion). A lipid suspension consisting of CLs with conven-
tional scaffolds, including YSK05, adopts a completely aggre-
gated form at temperatures higher than TH due to the highly
Fig. 11. Impact of Scaffold Length of CLs on Immiscibility with Neutral PLs
(A) Schematic illustration of the impact of scaffold length of CLs on the segregation of neutral PLs from H II structures of CL-anionic PL complexes. (B) SAXS analysis
of CL4H6/DSPC/DSPS dispersion that the inserted isotropic 31P-NMR spectra were obtained. These figures are reproduced, in part, with permission from Elsevier. (Color
figure can be accessed in the online version.)
hydrophobic nature of the HII phase. These findings lead to
the hypothesis that PCs (L phase) are segregated from the HII
structures formed by CH4H6-DSPS ion pairs and cover the
HII structure, which leads to a dispersion of small vesicles
that are well-suspended, even at high temperatures (Fig. 11A).
A small-angle X-ray scattering (SAXS) analysis of the same
lipid dispersion revealed the existence of a mixture of L and
HII phases at higher temperature (Fig. 11B), supporting the hy-
pothesis that CL4H6 is immiscible with PCs when in the HII
structure. The immiscible property of the CLs would greatly
contribute to membrane fusion-mediated endosomal escape
because the main lipid component of endosomal membranes is
PCs that can inhibit phase transition. This hypothesis is also
supported by the fact that mRNA-loaded CL4H6-LNPs with
high amounts (25 mol%) of DSPC showed consistent gene ex-
pression in liver tissue compared to a lower amount (5 mol%)
in liver tissue.171)
5. Improving the Biosafety of LNPs
While the administration of nucleic acid-loaded LNPs
sometimes causes toxicity,172–176) our knowledge regarding
LNP-associated inflammatory toxicities remains limited and
is somewhat controversial. Two reports demonstrated that a
co-treatment of dexamethasone (Dex), a glucocorticoid recep-
tor agonist, and a JAK 2 inhibitor with siRNA-loaded LNPs
dramatically inhibited LNP-associated toxicities including
elevated serum chemistry parameters and inflammatory cyto-
kines.177,178) Another study reported on the controversial find-
ing that a pro-inflammatory cytokine response was observed
after the administration of LNPs even with pre-medication
with Dex and other drugs.179) Differences in the components,
characteristics, and biodistribution of LNPs, and in the animal
model being used likely explain the difficulties associated with
the interpretation of these findings. Elucidation of the mecha-
nism regarding LNP-associated toxicity would contribute to
the development of new strategies for improving the safety of
LNPs.
To elucidate mechanism responsible for LNP-associated
hepatotoxicity, elevated doses of siRNA-loaded YSK13-C3-
LNPs were intravenously administered to mice.180) Interesting-
ly, dose-dependent hepatotoxicity was observed with changes
in the intrahepatic distribution of the LNPs from hepatocytes
to LSECs (Figs. 12A, B). Hepatotoxicity was also observed
at all dose range regardless of the siRNA-loading, indicating
that the toxicity is derived from the LNP itself and appears
1154 Chem. Pharm. Bull. Vol. 69, No. 12 (2021)

Fig. 12. Strategies for Improving the Safety of the LNPs

(A) Dose-dependency of intrahepatic distribution of the LNPs. The LNPs accumulated to LSECs at a higher dose. (B) Dose-dependency of the hepatotoxicity of the
LNPs. The hepatotoxicity was correlated with the change in intrahepatic distribution. (C) Schematic illustration of the estimated mechanism of LNP-associated hepato-
toxicity. The LNPs partially accumulate in LSECs, leading to the expression of adhesion molecules and cytokines followed by neutrophilic inflammation that aggravates
hepato- and systemic toxicity. (D) GalNAc modification significantly decreased the accumulation of the LNPs in LSECs. (E) Impact of surface modification of the LNPs
on hepatotoxicity. GalNAc modification significantly decreased the LNP-associated hepatotoxicity. Further PEG modification completely suppressed the hepatotoxicity.
(F) Schematic illustration of the development of the LLC-NPs. (G) Weight of each component. The amount of lipids for the LLC-NPs is significantly lower than that for
conventional HL-NP formulations. The amount of protamine is much lower than that for lipids or siRNAs. (H) Estimated cleavage of the CL4H6 by endogenous lipases.
(I) Biodegradation of the CL4H6 in both the liver and spleen. These figures are reproduced, in part, with permission from Elsevier. (Color figure can be accessed in the
online version.)

to be associated with changes in intrahepatic distribution. In
toxic conditions, adhesion molecules, including the vascular
cell adhesion molecule-1, E-selectin, and intercellular adhe-
sion molecule-1, are upregulated in liver tissue. A multiplex
suspension assay revealed the production of neutrophil-related
cytokines, including keratinocyte-derived cytokines and
granulocyte colony stimulating factor, in addition to some
inflammatory cytokines, the IL-6, IFN-γ-induced protein 10.
Neutrophil depletion resulted in the significant suppression
of LNP-associated toxicity, suggesting the involvement of
neutrophilic inflammation on hepato- and systemic toxicity. It
should be noted here that Kupffer cells did not contribute to
the toxicity due to rapid depletion of these cells after the ad-
ministration a high dose of the LNP. Taken together, it thought
that the unintended accumulation of LNPs to LSECs cause
the upregulation in adhesion molecules and pro-inflammatory
Vol. 69, No. 12 (2021)1155
Chem. Pharm. Bull.
cytokines including neutrophil-related cytokines followed by
the induction of neutrophilic inflammation (Fig. 12C). Based
on this supposition, the LNPs were modified with GalNAc to
improve hepatocyte-specific accumulation and to avoid the
unintended accumulation to LSECs. Hepatocyte-specificity
was substantially improved by the GalNAc modification (Fig.
12D). Surprisingly, LNP-associated hepatotoxicity was dra-
matically suppressed by GalNAc modification (Fig. 12E).
Further modification of GalNAc-LNPs with PEG to minimize
the ApoE-mediated unintended accumulation to LSECs com-
pletely inhibited hepatotoxicity (Fig. 12E). Importantly, gene
silencing activity in hepatocytes was fully maintained (ED50
of 0.015 mg siRNA/kg in a mouse FVII model), even after
surface modification. A single intravenous administration of
the modified YSK13-C3-LNPs in mice persistently infected
with HBV resulted in the suppression of HBV antigens by ap-
prox. 90% without any signs of toxicity.
It is known that high doses or high concentrations of CLs
can cause undesired toxicity through multiple mechanisms,
including the production of reactive oxygen species, the in-
duction of apoptosis and pro-inflammatory cytokines.172–176)
Therefore, the reduction of CLs for functional delivery would
be a useful and straightforward strategy for improving safety.
However, the reduction of CLs by reducing the molar ratio or
reducing the total lipids against nucleic acids typically causes
a dramatic decrease in delivery efficiency because the CLs
are critical components for endosomal escape, which is a late-
limiting step in nucleic acid delivery181,182) (Fig. 12F). To ad-
dress this dilemma, the negative charges of siRNAs were neu-
tralized by protamines, cationic proteins, in an effort to reduce
the consumption of CLs for siRNA-loading and to reduce the
net dose of CLs183) (Fig. 12F). The siRNA/protamine core-
loaded LNPs with low lipid/siRNA charge ratios are referred
to as low lipid core-nanoparticles (LLC-NPs) (Fig. 12F). The
LLC-NPs showed consistent gene silencing activity (IC50 of
approx. 1 nM siRNA) when conventional siRNA-loaded LNPs
with high lipid/siRNA charge ratios (high lipid-nanoparticles;
HL-NPs) were used in HeLa cells stably expressing dual-
luciferase (HeLa-dluc). On the other hand, the gene silencing
activity of the siRNA-loaded LNPs with low lipid/siRNA
charge ratios (low lipid-nanoparticles; LL-NPs) were dramati-
cally reduced (approx. 10% gene silencing at 30 nM siRNA).
These data suggest that the neutralization of the negative
charges of the siRNAs by protamines significantly restored
the gene silencing activity of the LNPs with low lipid/siRNA
ratios. A liposome fusion assay revealed that the neutraliza-
tion restored fusogenic activity, indicating that the amount of
CLs available for endosomal escape was increased by the re-
duced consumption of CLs for siRNA loading by protamines.
Importantly, while the total amount of lipid was significantly
decreased from 152.3 µg (HL-NPs) to 38.1 µg (LLC-NPs), only
7.9 µg of protamines were added against 10 µg siRNA (Fig.
12G). Moreover, protamines are biodegradable and were elim-
inated from liver tissue with a half time (t1/2) of less than 4 h.
The restoration of gene silencing activity in liver tissue and
the reduced hepatotoxicity for LLC-NPs was also confirmed.
Furthermore, a similar strategy was also successful in the case
of human immune cells.184)
Because CLs cause toxicity, the addition of biodegradability
into CLs would be also promising strategy in addition to the
above strategy for reducing the amount of CLs used.27,28,185)
The 3rd generation CL, CL4H6, contains biodegradable ester
bonds in its hydrophobic scaffolds. Cleavage of the ester bonds
produces a water-soluble alkanol amine and two oleic acids89)
(Fig. 12H). The amount of CL4H6 in both the liver and spleen
tissues rapidly dropped with time (Fig. 12I). The expected
alkanol amine was detected at 30 min in both tissues, indicat-
ing that the CL4H6 was rapidly hydrolyzed after the delivery
of siRNAs, as expected. The incubation of CL4H6-LNPs in
90% mouse plasma revealed that the extent of degradation
of CL4H6 was negligible for periods of up to 2 h, suggesting
that CL4H6, when contained within the LNPs is protected
from enzymatic degradation and is rapidly degraded once
the integrity of the LNPs is lost by membrane fusion with
endosomal membranes. A single dose toxicity test revealed
that the CL4H6-LNPs were tolerated for at least up to levels
of 5 mg siRNA/kg while the undegradable MC3-LNPs showed
a dose-dependent elevation in both alanine transaminase
and aspartate transaminase and a decrease in body weight.
Furthermore, repeated dose toxicity also confirmed that the
CL4H6-LNPs were well tolerated.
These strategies for improving the safety of LNPs, includ-
ing maximizing hepatocyte-specificity, reduction of the CL
dose, and the addition of biodegradability to CLs, are not ex-
clusive and could be combined with each other. It is expected
that these findings will contribute to the further development
of LNPs with excellent safety profiles.
6. Perspectives
LNP technology has been enjoyed dramatic progress in the
last 10 years, which in part, was driven by a breakthrough
of development of potential CLs. However, the limitations
that yet need to be addressed to realize ideal drug delivery
systems and to overcome widespread refractory diseases still
remain. There is still much room for the development of the
efficient extra-hepatic targeting of therapeutically important
tissues, including heart, muscles, and brain, and cell types,
including specific subpopulations of immune cells. In addi-
tion, payloads that can be loaded and delivered by LNPs are
still limited. Developing general loading/delivery strategies for
various classes of payloads (e.g. proteins, peptides, and others)
remains an important issue. It is also important to understand
the fundamental reasons for in vivo fate of the LNPs (e.g. bio-
distribution, stability, endosomal escape, toxicity, and so on)
at the molecular and particle levels. Therefore, the importance
of related technologies such as synthetic devices (e.g. sophis-
ticated and flexible microfluidic devices) and analytical meth-
odologies (e.g. NMR, SAXS, small-angle neutron scattering,
thermal analyses, transmission electron microscopy, robotics,
machine learning and so on) promise to become more and
more important.186–192) I sincerely hope that the LNP technol-
ogy will continue to develop and contribute to the provision of
advanced and safe medical care in the future.
Acknowledgments The author wishes to acknowledge the
financial support by, in part, a Grant-in-Aid for JSPS Fellows,
a Grant-in-Aid for Research Activity Start-up, Grant-in-Aid
for Young Scientists (B) (Grant Number: 15K20831), a Grant-
in-Aid for Young Scientists (A) (Grant Number: 17H05052),
Grant-in-Aid for Challenging Research (Exploratory) (Grant
Number: 18K19889), Grant-in-Aid for Scientific Research
(A) (Grant Number: 19H01170) from the Japan Society for
1156 Chem. Pharm. Bull.
the Promotion of Science (JSPS), the Special Education and
Research Expenses from the Ministry of Education, Culture,
Sports, Science and Technology (MEXT), The Uehara Memo-
rial Foundation, The Mochida Memorial Foundation for Medi-
cal and Pharmaceutical Research, TERUMO LIFE SCIENCE
FOUNDATION, and Creative Research Institute (CRIS,
Hokkaido University). I also wish to thank Dr. Milton Feather
for his helpful advice in preparing this manuscript. I would
like to thank Prof. Hideyoshi Harashima, Prof. Hidetaka
Akita, Dr. Hiroto Hatakeyama, Dr. Mamoru Hyodo, Dr. Yuma
Yamada, and Dr. Takashi Nakamura for critical advice, tech-
niques and other supports, and all the students who worked
diligently with me.
Conflict of Interest The author declares no conflict of
interest.
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