TCIMAIL No.

187 l Summer 2021

Research Article

Nucleic Acid Delivery System with DOP-DEDA,

a Charge-Reversible Lipid Derivative

Tomohiro Asai
University of Shizuoka, School of Pharmaceutical Sciences

Abstract
We have developed dioleoylglycerophosphate-diethylenediamine (DOP-DEDA) for nucleic acid delivery using
lipid nanoparticles (LNPs). DOP-DEDA is a charge-reversible lipid derivative in which the net charge of the head group
changes from -1 to +2 depending on pH, and can be applied to the delivery of small interfering RNA (siRNA) and
mRNA. Here, we introduce siRNA delivery using DOP-DEDA-based LNP (DOP-DEDA LNP). siRNA-encapsulated
DOP-DEDA LNP prepared by micromixing were shown to be uniform spherical particles having a particle size of about
100 nm. DOP-DEDA LNP showed high dispersibility and formed uniform particles without the use of polyethylene
glycol (PEG) lipids because of the amphipathic property of DOP-DEDA. The surface charge of DOP-DEDA LNP
was almost neutral at physiological pH and cationic under acidic conditions. DOP-DEDA LNP showed high pH
responsiveness, suggesting high endosomal escapability. Potent gene silencing effects were observed in cancer cells
transfected with siRNA encapsulated in DOP-DEDA LNP, even at low siRNA concentration. Our findings indicate that
DOP-DEDA is a pH-responsive lipid derivative having characteristics different from those of general ionizable lipids,
and is expected to contribute the development of nucleic acid drugs and mRNA vaccines.

Keywords: dioleoylglycerophosphate-diethylenediamine (DOP-DEDA), lipid nanoparticle (LNP), DOP-DEDA LNP,

charge-reversible lipid derivative, nucleic acid delivery system

Introduction
In recent years, nucleic acid drugs have received cells, and their endosomal escape. DLin-MC3-DMA,
a great deal of attention as a new modality in drug discovery. SM-102, and ALC-0315 are pH-responsive lipid
With the progress of chemical modification of nucleic derivatives formulated in ONPATTRO, mRNA-1273, and
acid and drug delivery system (DDS) technologies, RNA COMIRNATY, respectively (1). The common structure
interference drugs and mRNA vaccines have come into of these lipid derivatives is that they have a tertiary
practical use. ONPATTRO®, the first RNA interference amine on their head group, and the head group is ionized
drug in the world, developed by Alnylam Pharmaceuticals and positively charged only under acidic conditions.
Inc. is a lipid nanoparticle (LNP) formulation of a small Therefore, these lipid derivatives are called an ionizable
interfering RNA (siRNA). LNP technology is also used lipid. The performance of LNP in nucleic acid delivery
in the mRNA vaccine “mRNA-1273” for coronavirus depends on the performance of the pH-responsive lipid
disease 2019 (COVID-19) developed by Moderna, derivative. To achieve safe and highly efficient nucleic
Inc. and the vaccine “COMIRNATY ®” developed by acid delivery, it is necessary to appropriately design
BioNTech SE and Pfizer Inc. Encapsulation of siRNA or the pH-responsive lipid derivative and then optimize
mRNA in LNP significantly improves the stability and the LNP formulation such as the lipid composition.
the transfection efficiency of RNA in vivo, resulting in the We have studied the nucleic acid delivery system and
effects of RNA interference drugs and mRNA vaccines, developed original pH-responsive lipid derivatives for
respectively. In general, the lipid component of LNP LNP formulation (2, 3). Recently, in collaboration with
includes a pH-responsive lipid derivative that is essential Nippon Fine Chemical Co., Ltd., we have newly designed
for encapsulation of nucleic acids, their delivery into dioleoylglycerophosphate-diethylenediamine (DOP-DEDA),

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an amphipathic charge-reversible lipid derivative with acid delivery (4, 5). Here, we introduce the nucleic acid
high pH responsiveness and prepared DOP-DEDA- delivery system using DOP-DEDA LNP.
based LNP (DOP-DEDA LNP) for efficient nucleic

Structural features of DOP-DEDA

The structural formula of DOP-DEDA is shown in charged under acidic conditions. In addition, since it
Fig. 1. The basic skeleton of DOP-DEDA is very similar is negatively charged under alkaline conditions, DOP-
to that of natural glycerophospholipids. In DOP-DEDA, DEDA LNP is an LNP that has the property of charge
two oleic acids and phosphoric acid are bound to the inversion depending on pH. DOP-DEDA is a state in
glycerol skeleton, and DEDA is bound via phosphoric which the head group is ionized even at physiological pH,
acid. DOP-DEDA is a lipid derivative in which the net and the positive and negative charges are offset so that the
charge of the head group changes from -1 to +2 depending net charge is almost zero (Fig. 3). Since the head group of
on pH. Under acidic conditions, the head group of DOP- general ionizable lipids containing a tertiary amine is not
DEDA is positively charged and therefore electrostatically ionized at physiological pH, the authors consider that it
interacts with negatively charged nucleic acids (siRNA, is not appropriate to classify DOP-DEDA as an ionizable
mRNA, etc.). Therefore, by mixing the component lipid. Therefore, we refer it to as a charge-reversible
lipids including DOP-DEDA and nucleic acids under lipid derivative or pH-responsive lipid derivative.
acidic condition in a microchannel, DOP-DEDA LNP The properties of DOP-DEDA, which does not show
encapsulating nucleic acids can be prepared (Fig. 2). The cationicity under physiological conditions and behaves as
surface charge of DOP-DEDA LNP encapsulating nucleic a neutral lipid, are considered to be advantageous in terms
acids is almost neutral at physiological pH and positively of safety in the same manner as general ionizable lipids.

Figure 1. Structure of DOP-DEDA

Figure 2. Schematic image of DOP-DEDA LNP

Figure 3. Change in the net charge of DOP-DEDA depending on pH

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DOP-DEDA has the characteristic of having commercially available ionizable lipids without PEG-
physicochemical properties similar to amphipathic lipids, we could not obtain uniform particles due to lipid
glycerophospholipids at physiological pH, which is aggregation. In addition, it was reported that ionizable
different from tertiary amine-containing ionizable lipid-based LNPs were not formed due to aggregation
lipids. Since the head group of the ionizable lipid at unless PEG-lipid was added (6). A cryogenic transmission
physiological pH is not ionized, the polarity of the electron microscopy (cryo-TEM) image of DOP-DEDA
head group is lower than that of general amphipathic LNPs encapsulating siRNA is shown in Fig. 4. The
phospholipids. On the other hand, since DOP-DEDA LNPs are formed with a composition of DOP-DEDA /
behaves as an amphipathic phospholipid at physiological dipalmitoylphosphatidylcholine (DPPC)/cholesterol =
pH, DOP-DEDA LNP has excellent dispersibility and 45/10/45 (molar ratio). This LNP formulation contains
stability in an aqueous solution. Therefore, even if no PEG-lipids. We believe that DOP-DEDA can be
the lipid composition of DOP-DEDA LNP does not differentiated from conventional ionizable lipids in that
contain polyethylene glycol (PEG)-lipids, it is stable in it can form highly dispersible LNPs without the use of
the aqueous solution. LNPs containing ionizable lipids PEG-lipids. Concerns about PEG-lipids have been raised
generally contain less than a few mole percent of PEG- in the side effects of mRNA vaccines (LNP preparations)
lipids relative to total lipids, and it is known that these (7). The fact that DOP-DEDA does not require PEG-
PEG-lipids contribute to stable particle formation. lipids for LNP formation may be an important advantage.
In fact, the authors tried to prepare LNPs containing

Figure 4. Cryo-TEM image of DOP-DEDA LNPs encapsulating siRNA

DOP-DEDA/DPPC/cholesterol = 45/10/45 (molar ratio), total lipids/siRNA = 7000/1 (molar ratio)

Preparation of DOP-DEDA LNP

DOP-DEDA LNP can be prepared by a method using We use 1 mM citric acid solution (pH 4.5) as the
a microchannel like LNP containing an ionizable lipid. aqueous solvent for dissolving siRNA or mRNA. The
It can be prepared by mixing a mixed lipid dissolved in concentration of citric acid is one of the factors that affect
alcohol and RNA dissolved in an aqueous solvent in a the formation of LNP. When mixed lipids containing
microchannel, and then removing the alcohol by dialysis. DOP-DEDA dissolved in ethanol and siRNA or mRNA
We first prepared a freeze-dried mixture of DOP-DEDA dissolved in an aqueous citric acid solution are mixed
and helper lipids, and added alcohol to the lyophilized in the microchannel, intermediate particles of DOP-
mixture to dissolve the mixed lipids. The alcohol we used DEDA LNP are formed. The intermediate particles are
is tert-butanol or ethanol. Because the solubility of mixed transferred in a dialysis membrane and dialyzed in water
lipids in alcohol is higher with tert-butanol than with to remove ethanol. The final DOP-DEDA LNPs are
ethanol, tert-butanol is useful when a high concentration formed by dialysis.
lipid solution needs to be prepared. However, it is The particle size and polydispersity index (PdI)
considered preferable to dissolve the mixed lipids with of DOP-DEDA LNP can be controlled by adjusting
ethanol for practical use. preparation conditions such as alcohol/water solvent

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ratio and lipid concentration. The optimal conditions ζ- potential, and siRNA encapsulation efficiency of DOP-
for adjusting the LNP to the desired particle size should DEDA LNP. The encapsulation efficiency of siRNA and
be determined using the microchannels that you use. mRNA in DOP-DEDA LNP can be calculated using the
We usually prepare DOP-DEDA LNP with the average RiboGreen® reagent. When the encapsulation efficiency
particle size of about 100 nm and the PdI of less than of siRNA or mRNA into DOP-DEDA LNP was examined
0.100. Table 1 shows our results of the particle size, PdI, by this method, it was more than 95%.

Table 1. Particle size, PDI, ζ-potential, and siRNA encapsulation efficiency of DOP-DEDA LNP

Particle size (nm) PdI ζ-Potential (mV) Encapsulation efficiency (%)

98.3 ± 7.20 0.08 ± 0.02 +0.8 ± 0.2 97.8

DOP-DEDA/DPPC/cholesterol = 45/10/45 (molar ratio), total lipids/siRNA = 7000/1 (molar ratio)

DOP-DEDA LNP was diluted with 10 mM Tris-HCl buffer (pH 7.4) for the measurement of ζ-potential.

pH responsiveness of DOP-DEDA LNP

To deliver nucleic acids into the cytoplasm using DEDA LNPs become more positive with decreasing
LNPs, it is necessary for LNPs taken up by the cell via pH. As mentioned above, the net charge of DOP-DEDA
endocytosis and escape from the endosome. LNPs with varies from -1 to +2. On the other hand, the LNPs used
high escapability from endosomes efficiently deliver as a control in the TNS assay contain ionizable lipids,
nucleic acids into the cytoplasm, resulting in beneficial the net charge change of which is 0 to +1. Reflecting
effects. After pH-responsive LNPs are endocytosed into this difference, DOP-DEDA LNPs have a greater change
cells, the pH-responsive lipids of LNPs are protonated in fluorescence intensity in response to pH changes.
under acidic condition in endosomes (pH 5-6), and LNPs The apparent pKa of DOP-DEDA was calculated from
become positively charged. As protons are absorbed the titration curve to be about 6.5. When DOP-DEDA
by LNP, protons and anions flow into the endosome LNPs were incubated with bovine erythrocytes, they
from the outside, increasing the salt concentration and induced hemolysis at pH 5.5, endosome pH, but did not
osmotic pressure in the endosome, and destabilizing the induce hemolysis at pH 7.4 (5). This result suggests that
endosome. In addition, because positively charged LNPs DOP-DEDA LNPs do not affect the barrier capacity of
easily interact with the inner membrane of endosomes, the membrane under physiological pH condition, but
the escape of LNPs from endosomes is promoted. destabilize the membrane under endosomal pH condition.
Therefore, pH responsiveness of LNPs is a determinant of The time-lapse imaging using a confocal laser scanning
the efficiency of nucleic acid delivery. microscope for analyzing the intracellular dynamics of the
One of the methods for evaluating the pH siRNA showed that siRNA transfected with DOP-DEDA
responsiveness is an assay using 2-(p-Toluidino)- LNPs diffused throughout the cytoplasm (5). From these
naphthalene-6-sulfonic acid (TNS). Figure 5 shows the results, it is considered that DOP-DEDA LNP having high
results of evaluating the pH responsiveness of DOP- pH responsiveness has excellent endosomal escapability
DEDA LNP encapsulating siRNA by the TNS assay. and is useful as a carrier for delivering nucleic acids into
The fluorescence intensity of TNS increases as the pH the cytoplasm.
decreases, indicating that the surface charge of DOP-

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Figure 5. Titration of DOP-DEDA LNP by the TNS assay

DOP-DEDA/DPPC/cholesterol = 45/10/45 (molar ratio)
Control LNP: control ionizable lipid /DSPC/cholesterol/DMG-PEG2000 = 50/10/38.5/1.5 (molar ratio)
Concentration of DOP-DEDA and control LNPs: 20 mM as a total lipid concentration
The figure is reused and modified from (5) with permission of Elsevier.

siRNA delivery system using DOP-DEDA LNP

DOP-DEDA LNPs encapsulating siRNA targeting polo- depending on the cells used, but the reason has not been
like kinase 1 (siPLK1), a cell cycle control protein, were clarified at present. If we can clarify the kind of cells that
prepared to evaluate their gene silencing effects on MDA- are targetable by DOP-DEDA LNPs and the mechanism
MB-231 human breast cancer cells. The expression of PLK1 of delivery with them in future studies, it may lead to
mRNA was measured by RT-PCR 72 h after the transfection cell-selective treatment. In the case of LNPs containing
of MDA-MB-231 cells with siPLK1. DOP-DEDA ionizable lipids, the cellular uptake mechanism mediated
LNPs encapsulating siPLK1 significantly induced gene by the apolipoprotein E (ApoE) receptor is well known (8).
silencing at a low siRNA concentration of 3 to 15 nM In addition, the enhanced expression of ApoE receptor
in a dose dependent manner (Fig. 6a). In contrast, DOP- on MDA-MB-231 cells was reported (9). Therefore, we
DEDA LNP encapsulating scrambled siRNA did not investigated whether DOP-DEDA LNPs were also taken
induce any gene silencing effect. The amount of PLK1 up by the same mechanism. It was shown that DOP-
protein was examined by western blotting 72 h after DEDA LNPs were taken up by MDA-MB-231 cells in an
the transfection of MDA-MB-231 cells with siPLK1. ApoE dose-dependent manner (5). However, there are still
Consistent with the results of RT-PCR, western blotting many unclear points about the cellular uptake mechanism
data showed that DOP-DEDA LNPs encapsulating of DOP-DEDA LNP.
siPLK1 suppressed the expression of PLK1 protein at One of the features of DOP-DEDA LNP is that it
a low siRNA concentration (Fig. 6b). Damage of DOP- forms a stable LNP even in the absence of PEG-lipids.
DEDA LNPs to cell membrane was examined by lactate However, it is also possible to modify DOP-DEDA LNP
dehydrogenase (LDH) assay. The results showed that with PEG to improve blood retention. In fact, PEGylated
DOP-DEDA LNPs encapsulating siPLK1 can induce DOP-DEDA LNPs injected intravenously accumulated in
significant gene silencing without membrane damage solid tumors due to enhanced permeability and retention
(data not shown). Gene silencing using DOP-DEDA (EPR) effects in tumor-bearing mice (4). In addition,
LNP encapsulating siRNA was also confirmed with other the silencing of the target gene was confirmed in the
cancer cell lines such as HT1080 human fibrosarcoma solid tumors after the intravenous injection of siRNA
cells and immune cells such as macrophages. The encapsulated in DOP-DEDA LNPs (4).
transfection efficiency of DOP-DEDA LNPs differs

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Figure 6. Gene silencing in MDA-MB-231 cells transfected with siRNA-encapsulated DOP-DEDA LNPs
The figure is reused and modified from (5) with permission of Elsevier.

Conclusion
The medical application of lipid-based nanoparticles is considered to be a promising approach for innovative
began with the liposome formulations such as drug development. Lipid-based DDS is an old but new
AmBisome ® and Doxil ® , which were developed in technology.
the 1990s. In recent years, LNP formulations such Here, we introduced a siRNA delivery system using
as ONPATTRO and COVID-19 vaccine (mRNA- DOP-DEDA LNP, but our study also showed that DOP-
1273, COMIRNATY) had a great impact on drug and DEDA LNP can also be applied to mRNA delivery. In
vaccine developments. It has been proven by history addition, it may be applicable to protein delivery. Since
that lipid-based nanoparticles are useful and practical DOP-DEDA is a glycerophospholipid derivative having
in the medical application of nano-DDS. Since small characteristics different from those of ionizable lipids, it is
vesicles (exosomes, organelles, etc.) surrounded by lipid considered that there may be applications that utilize their
membranes have various functions in living organisms, own characteristics and advantages. We hope that our
the range of applications of artificially created lipid DOP-DEDA technology contributes to the development
vesicles is expected to expand more. In particular, nano- of nucleic acid drugs and mRNA vaccines.
DDS, which mimics the physiological transport system,

ONPATTRO® is a registered trademark of Alnylam Pharmaceuticals, Inc.

COMIRNATY® is a registered trademark of BioNTech SE.
RiboGreen® is a registered trademark of Molecular Probes, Inc.
AmBisome® is a registered trademark of GILEAD SCIENCES, INC.
Doxil® is a registered trademark of BAXTER HEALTHCARE CORPORATION.

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Acknowledgments
We would like to express our sincere gratitude Professor, University of Shizuoka) for his great
to Professor Naoto Oku (Professor, Faculty of contribution to this research.
Pharmaceutical Sciences, Teikyo University, Emeritus

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Author Information

Tomohiro Asai

Apr. 1993 to Mar. 1997 University of Shizuoka School of Pharmaceutical Sciences, Shizuoka,
Japan. (B. Sc.)
Apr. 1997 to Mar. 1999 University of Shizuoka Graduate School of Pharmaceutical Sciences,
Shizuoka, Japan. (M. Sc.)
Apr. 1999 to Mar. 2002 University of Shizuoka Graduate School of Pharmaceutical Sciences,
Shizuoka, Japan. (Ph. D.)
Research Fellowships of the Japan Society for the Promotion of
Science
Apr. 2002 to Jan. 2004 Researcher, Mitsubishi Pharma Corporation
Feb. 2004 to Mar. 2013 Assistant Professor, Department of Medical Biochemistry, University of
Shizuoka School of Pharmaceutical Sciences
Oct. 2005 to Dec. 2005 Visiting Scholar, Division of Drug Delivery and Disposition, Eshelman
School of Pharmacy, University of North Carolina
Jul. 2011 to Jun. 2012 Visiting Scholar, Division of Molecular Pharmaceutics, Eshelman
School of Pharmacy, University of North Carolina
Apr. 2013 to Mar. 2018 Associate Professor, Department of Medical Biochemistry, University
of Shizuoka School of Pharmaceutical Sciences
Apr. 2018 to present Professor, Department of Medical Biochemistry, University of Shizuoka
School of Pharmaceutical Sciences