Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo

Article abstract of DOI:10.1002/anie.201203263

Special (lipid) delivery: The role of the ionizable lipid pKa in the in?vivo delivery of siRNA by lipid nanoparticles has been studied with a large number of head group modifications to the lipids. A tight correlation between the lipid pKa?value and silencing of the mouse FVII gene (FVII ED50) was found, with an optimal pKa range of 6.2-6.5 (see graph). The most potent cationic lipid from this study has ED50 levels around 0.005?mg?kg?1 in mice and less than 0.03?mg?kg?1 in non-human primates.

Full text of DOI:10.1002/anie.201203263

Angewandte
Chemie
DOI: 10.1002/anie.201203263
siRNA Delivery
Maximizing the Potency of siRNA Lipid Nanoparticles for Hepatic
Gene Silencing In Vivo**
Muthusamy Jayaraman,* Steven M. Ansell, Barbara L. Mui, Ying K. Tam, Jianxin Chen,
Xinyao Du, David Butler, Laxman Eltepu, Shigeo Matsuda, Jayaprakash K. Narayanannair,
Kallanthottathil G. Rajeev, Ismail M. Hafez, Akin Akinc, Martin A. Maier, Mark A. Tracy,
Pieter R. Cullis, Thomas D. Madden, Muthiah Manoharan, and Michael J. Hope*
RNA interference (RNAi) is a ubiquitous and highly effective
biological mechanism to control gene expression. Over the
last few years it has been established that synthetic double-
stranded, small interfering RNA (siRNA) molecules can be
applied as therapeutics that potently harness RNAi in humans
and animal models, by demonstrating silencing of target
mRNA transcripts in rodents and non-human primates
(NHP).[1] Because of their high molecular weight and
polyanionic nature, synthetic siRNAs fail to cross biological
membranes by passive diffusion and therefore, generally
require transmembrane drug delivery technologies to access
the cytoplasm of target cells.[2,3] Once in the cytoplasm,
however, siRNAs readily load into the RNA-induced silenc-
ing complex (RISC) and direct sequence-specific cleavage of
target mRNA. The resulting reduction in expression of
protein, for example, can be profound and durable, lasting
several weeks after administration of a single dose.[4] This new
class of therapeutics, with its unique mechanism of action and
ability to specifically inhibit previously “undruggable” dis-
ease-causing proteins, has prompted research into intracellu-
lar delivery technologies suitable for parenteral administra-
tion.
recent years to maximize efficacy and to achieve a favorable
therapeutic index for human applications.[2,4] The ionizable
amino lipid plays a dual role in the drug delivery process.
First, through electrostatic interaction with polyanionic
nucleic acids, it promotes the self-assembly of formulation
components into macromolecular nanoparticles encapsulat-
ing the siRNA. Second, following endocytosis of LNPs by
target cells, it enables siRNA to escape the endosomal
compartment and access the cell cytoplasm. In a recently
published analysis of the structure–activity relationship of
cationic lipid molecules we noted that permanently charged
quaternary amine moieties in the hydrophilic head group
region were consistently less effective in RNAi-mediated
gene knockdown in vivo as compared to ionizable lipids.[5]
Herein, we report on a detailed study aimed at further
investigating the role of the amine head group in the delivery
of siRNA in vivo. Through systematic structural modifica-
tions to the hydrophilic head group we have modulated the
apparent acid dissociation constant (pKa) of the ionizable
amino lipids present in the LNPs and investigated the
relationship between the pKa value and activity, that is, the
ability of the LNPs to deliver functionally active siRNAs into
hepatocytes for eliciting gene silencing in vivo.
Lipid nanoparticles (LNPs) containing ionizable amino
lipids that self-assemble into approximately 100 nm particles
when mixed with polyanionic oligonucleotides, represent the
most advanced delivery platforms for systemic administration
of RNAi therapeutics. Substantial progress has been made in
Amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-
dioxolane (DLin-KC2-DMA, 1), has been identified as
a highly potent cationic lipid (Figure 1, Table 1)[5] when
[*] Dr. S. M. Ansell, Dr. B. L. Mui, Dr. Y. K. Tam, Dr. J. Chen, Dr. X. Du,
Dr. T. D. Madden, Dr. M. J. Hope
AlCana Technologies, Vancouver, BC (Canada)
E-mail: mhope@alcanatech.com
Homepage: http://www.alnylam.com
Dr. M. Jayaraman, Dr. D. Butler, Dr. L. Eltepu, Dr. S. Matsuda,
Dr. J. K. Narayanannair, Dr. K. G. Rajeev, Dr. A. Akinc,
Dr. M. A. Maier, Dr. M. A. Tracy, Dr. M. Manoharan
Alnylam Pharmaceuticals, Cambridge, MA (USA)
E-mail: mjayaraman@alnylam.com
Figure 1. 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-
DMA, 1). No difference in activity between enantiomers (4,5) or the
racemic mixture was observed (Table 1).
Dr. I. M. Hafez, Dr. P. R. Cullis
Department of Biochemistry and Molecular Biology, University of
British Columbia, Vancouver, BC (Canada)
incorporated into LNPs encapsulating siRNA to target
murine clotting factor VII (FVII) mRNA.[6] The hydrophobic
dilinoleyl chain is known to be optimal for activity,[5,7] hence
this unsaturated alkyl chain configuration was maintained in
all the lipids investigated herein and structural modifications
were added to the head group region of the molecule. The
formulations were prepared by a preformed vesicle method,
[**] This work was supported in part by the Canadian Institutes for
Health Research (CIHR) through U/I grant FRN 59836. We would
like to thank John Maraganore for helpful comments.
Supporting information for this article (experimental details) is
available on the WWW under http://dx.doi.org/10.1002/anie.
201203263.
Angew. Chem. Int. Ed. 2012, 51, 1 – 6
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!

.
Angewandte
Communications
as described in the Supporting Information (page 38), with
the following lipid composition: amino lipid, distearoylphos-
phatidylcholine (DSPC), cholesterol and (R)-2,3-bis(octade-
cyloxy)propyl-1-(methoxy poly(ethylene glycol)2000)propyl-
carbamate (PEG-lipid) in the molar ratio 40/10/40/10, respec-
tively, and a FVII siRNA/total lipid ratio of approximately
0.05 (w/w).[5] To ensure a narrow particle size distribution in
the range of 70–90 nm and a low polydispersity index of
0.11 Æ 0.04 (n = 56), the particles were extruded up to three
times through 80 nm membranes prior to adding the siRNA.[5]
Amino lipid pKa values were determined for each LNP by
measuring the fluorescence of 2-(p-toluidino)-6-napthalene
sulfonic acid (TNS) during titration from pH 3 to 10 in
increments of 0.5 pH units (Supporting Information,
page 40).[8–10] The anionic TNS molecule fluoresces when
associated with the surface of positively charged membranes,
but is not fluorescent when free in solution, and this property
has been used extensively to probe positive charge at
membrane surfaces.[8–10]
philic region. This is important because having a small head
group is a characteristic that enables amino lipids to adopt
inverted, non-bilayer structures, which are hypothesized to be
responsible for destabilizing the endosomal membrane,
resulting in efficient release of siRNA into the cytoplasm of
target cells.[5,11]
A plot of pKa value versus ED50 (median effective dose
for FVII gene silencing in female C57BL/6 mice by i.v.
administration) for all 56 amino lipids tested reveals a strong
correlation between the in vivo activity and the acid dissoci-
ation constant of the amine, with a pKa optimum seen
between 6.2 and 6.5, on either side of which LNP potency
rapidly decreases (Figure 3). On the other hand, significant
From the resulting fluorescence titration curve a pH value,
at which the charge on the LNP is 50% of maximum (pKa),
can be calculated using curve-fit analysis (Figure 2). Impor-
Figure 3. Plot of in vivo hepatic gene silencing activity vs. pKa in mice.
The 56 amino lipids were formulated in LNPs and subjected to an
ED50 analysis (described in Supporting Information, page 39) and
plotted against their pKa. For 15 lipids the ED50 dose was not achieved,
these are indicated by the open diamonds representing the highest
dose tested for that lipid. For the remaining lipids (closed diamonds),
a polynomial best-fit curve highlights the most active compounds,
which exhibit an optimal pKa between 6.2 and 6.5. Each data point is
derived from a four-dose response curve with groups of n=4 mice per
dose.
Figure 2. Determination of pKa by in situ TNS fluorescence titration.
The four amino lipids shown are examples illustrating the full pKa
range evaluated. Sigmoidal best-fit analyses were applied with pKa
defined as the pH at half-maximal fluorescence intensity. A) 14,
pKa =4.17; B) 52, pKa =5.73; C) 19, pKa =6.95; D) 48, pKa =8.12 (see
Supporting Information page 40 for details).
potency differences were found for lipids with similar pKa
values. These results indicate that an optimal pKa value is
a necessary but not sufficient requirement for good in vivo
activity and other structural features, such as the nature of the
linker between the head group and the lipid tails, can also
contribute substantially to the in vivo activity.
To further investigate the pKa–activity relationship,
a selected group of lipids were studied in more detail. These
novel lipids were designed around the structure of dilinoleyl-
methyl-4-dimethylaminobutyrate (16, ED50 = 0.03 mgkgÀ1,
pKa = 6.44) also referred to as DLin-MC3-DMA (Figure 4),
one of the most active cationic lipids from the group of 56
lipids. In the first group of lipids (31, 30, 38, and 37), the
distance between ester and amine is maintained at three
methylene units and the substitution on the N-atom altered to
create a pKa range between 5.44 and 7.62.
tantly, this method enables a pKa determination of amino
lipids in situ, which accounts for potential influences of
membrane structure and neighboring lipids on the dissocia-
tion properties of the amino group. Similar pKa values were
obtained for select formulations using zeta potential meas-
urements over the same pH titration range, but TNS was
found to be the more sensitive method (data not shown).
The hydrophilic head-group region of 1 was extensively
modified, producing 53 novel amino lipids with pKa values
ranging from 4.17 to 8.12 (Table 1; see the Supporting
Information for full experimental details on lipid synthesis).
Lipids 1, 3, and 53 have been reported earlier and were used
as references in this study.[5,7] The structural modifications
introduced were sufficient to affect the pKa value without
significantly altering the molecular dimensions of the hydro-
In the second group (14, 15, 17, and 18), the dimethyl-
amino moiety is maintained but the distance between the
2
www.angewandte.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 1 – 6
These are not the final page numbers!

Angewandte
Chemie
Table 1: Structures of the 56 amino lipids tested for gene silencing activity in vivo. ED50 is for FVII gene silencing in mice (mg siRNA kgÀ1) and the pKa
of the lipid was measured in situ. Lipids 1, 3, and 53 were reported earlier and used as references in this study.[5, 7]
Angew. Chem. Int. Ed. 2012, 51, 1 – 6
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

.
Angewandte
Communications
adopt inverted, non-bilayer configurations that disrupt the
integrity of the endosome membrane and the siRNA is
subsequently released into the cytoplasm.[5] The ED50–pKa
profile may, therefore, represent a balance between two
opposing delivery requirements, namely minimum charge at
pH 7.4 to maximize hepatocyte uptake by ApoE-mediated
endocytosis and maximum positive charge in the acidified
lumen of endosomes to promote membrane disruption.
If potency differences of structurally similar lipids are
predominantly a function of pKa value, we hypothesized that
the activity of LNPs containing two or more cationic lipids
should reflect the average pKa of the particle and not the
average activity measured for the individual amino lipids. To
test this, LNP formulations encapsulating FVII siRNA were
prepared in which the 40 mole% amino lipid component
consisted of different ratios of lipids 15, 16, and 17 thus
producing LNPs with average surface pKa values that
increased incrementally from 5.64 (15) to 6.93 (17). The
measured pKa value for mixed lipid LNPs was shown to be the
same (within experimental error) as that calculated from the
mole ratio of the mixed amino lipids (Supporting Informa-
tion, page 41). Each LNP was administered at the same dose
(0.1 mgkgÀ1 siRNA) and the concentration of murine plasma
FVII protein was determined 24 h later and expressed as
a percentage of that measured in a saline-treated control
(Figure 6).
Figure 4. (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-tetraen-19-yl
4-(dimethylamino)butanoate (16, Table 1).
ester and the amine is varied from one to five methylene units
to generate lipids with pKa values ranging from 4.17 to 7.16.
The plot of ED50 as a function of pKa value shows that the
highest potency is achieved at a pKa of 6.44 for the siRNA
LNP formulation containing lipid 16 (Figure 5, blue line).
Figure 5. Plot of hepatic gene silencing activity in mice, pKa and
calculated protonated/unprotonated amino lipid (% mole fractions)
for the set of novel lipids between pKa 4 and 8.5. Each FVII ED50 data
point (blue) is derived from a four-dose response (n=4 mice per dose
level). Open diamonds represent lipids for which the ED50 was not
achieved and are shown at the highest dose tested. The mole% of
each amino lipid estimated to carry a positive charge in blood,
assuming pH 7.4, is shown in red and the mole% amino lipid
estimated to be uncharged in acidified endosomes at pH 5.5 is shown
in green.
Activity substantially decreases as the pKa value shifts to
either the acidic or basic side, underlining the importance of
the ionization behavior of LNPs in the functional delivery of
siRNA to hepatocytes in mice.
Figure 6. Lipid mixing supports pKa value as the dominant factor in
determining in vivo hepatic gene-silencing activity for a given set of
amino lipids. Mixtures of amino lipids 15, 16, and 17 were made to
incrementally shift the average surface pKa value of the resulting LNP
between 5.64 and 6.93. Numbers in parenthesis indicate the amount
of each lipid in the mixture to make up the 40 mole% amino lipid
component of the LNP. Bars represent group mean (n=4)Æs.d.
The surprisingly sharp pKa value dependence of LNP
activity provides more insight into the complex in vivo
mechanism for this highly potent class of LNPs for siRNA
delivery. In brief, it has been proposed that intravenous
administration of LNPs with low to zero surface charge will
minimize nonspecific binding of LNPs to tissues and proteins
thus increasing the likelihood of particles reaching hepato-
cytes.[5] In addition, we recently demonstrated that uptake of
ionizable LNPs by hepatocytes in vitro and in vivo is medi-
ated by apolipoprotein E (ApoE),[12] which is also known to
preferentially associate with neutral membrane surfaces.[13]
On the other hand, LNPs must acquire sufficient positive
surface charge following endocytosis to promote an electro-
static interaction with the negative surface of the endosome
lumen. The resulting close proximity of LNP and membrane
allows lipid mixing to occur, leading to the formation of ion
pairs between the exogenous amphipathic amine and endog-
enous anionic membrane lipids.[11,14] The ion pairs prefer to
As the average pKa value increases from 5.64 (15) or
decreases from 6.93 (17) by mixing with 16, LNP activity
increases. We did not identify a mixture with greater activity
than 16 alone, but importantly, LNPs containing an equimolar
mixture of 15 (ED50 = 0.6) and 17 (ED50 = 0.15), lipids that
individually are 20- and 5-fold less potent than 16, respec-
tively, showed an average apparent pKa value similar to 16
and equivalent activity. These data further confirm that for
amino lipids of similar structure, in vivo hepatic gene silenc-
ing is driven by pKa rather than specific head group structure
or linker length (the distance between the ester and the
amine). We have subsequently shown this to be true for
4
www.angewandte.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 1 – 6
These are not the final page numbers!

Angewandte
Chemie
mixtures of other cationic lipids with different head group
structures (data not shown).
polyneuropathy.[16] Animals received a 15 min intravenous
infusion at siRNA doses of 0.03, 0.1, or 0.3 mgkgÀ1 and from
the dose response curve an ED50 less than 0.03 mgkgÀ1 was
obtained (Figure 7).
The pH-dependent surface charge for each LNP in the
blood or endosomal compartment can be calculated from
their pKa, using the Henderson–Hasselbach equation, assum-
ing blood pH 7.4 and acidified endosome pH 5.5.[15] The
amount of fully or partially charged LNPs carrying positive
charge for each chosen lipid (14–18, 30, 31, 37, and 38) is
expressed as a fraction of charged amino lipids plotted red in
Figure 5 along with the fraction of amino lipids that would be
uncharged at pH 5.5 in the endosome, plotted green. This
demonstrates that the optimal pKa value for in vivo hepatic
gene silencing coincides with the pKa giving rise to LNPs that
exhibit minimum surface charge in the blood at pH 7.4 and
maximum charge in acidified endosomes at pH 5.5. It should
be noted, however, that the pKa-value trend found for this
class of ionizable lipids with apoE-dependent hepatic delivery
may not apply to other cationic and potentially ApoE-
independent delivery systems.[6,12]
In conclusion, we have identified the lipid pKa value as an
important molecular characteristic of amino lipids, which
determines their ability to mediate potent hepatic gene
silencing in vivo. Combined with our current understanding
of the optimal hydrocarbon chain length, lipid-chain unsatu-
ration and linker chemistry for these molecules, the tight
correlation between activity and ionization behavior, with
a sharp pKa optimum between 6.2–6.5, serves as a guide for
the design of LNPs for efficient delivery of therapeutic
siRNAs. In fact, a LNP formulation based on DLin-MC3-
DMA (16), one of the most potent lipids identified in this
work, is currently being utilized in a clinical trial for treating
hypercholesterolemia; the target is PCSK9 and the data (not
shown) indicate decreases in LDL-C after a single low
dose.[17,18]
To identify potent and safe siRNA delivery systems for
clinical development, it is important that the potency in mice
translates to non-human primates. For the following experi-
ments LNPs containing the highly potent amino lipid 16 were
used, in which the molar ratio of the four lipid components 16,
DSPC, cholesterol and PEG-lipid (50/10/38.5/1.5) had been
further optimized to enhance in vivo activity. In the murine
FVII model, an ED50 of 0.005 mgkgÀ1 siRNA was achieved in
the optimized composition, which represented a sixfold
improvement relative to the 40/10/40/10 molar ratio compo-
sition (Figure 7, inset). The same lipid composition was
administered to cynomolgus monkeys but with an encapsu-
lated siRNA targeting hepatic transthyretin (TTR), a gene of
therapeutic interest in the treatment of familial amyloidotic
Received: April 27, 2012
Published online: && &&, &&&&
Keywords: drug delivery · gene silencing ·
.
ionizable amino lipids · liposomes · siRNA
[1] A. De Fougerolles, H. P. Vornlocher, J. Maraganore, J. Lieber-
man, Nat. Rev. Drug Discovery 2007, 6, 443 – 453.
[2] S. Akhtar, I. F. Benter, J. Clin. Invest. 2007, 117, 3623 – 3632.
[3] K. A. Whitehead, R. Langer, D. G. Anderson, Nat. Rev. Drug
Discovery 2009, 8, 129 – 138.
[4] T. S. Zimmermann, et al., Nature 2006, 441, 111 – 114.
[5] S. C. Semple, et al., Nat. Biotechnol. 2010, 28, 172 – 176.
[6] A. Akinc, M. Goldberg, J. Qin, J. R. Dorkin, C. Gamba-Vitalo,
M. Maier, K. N. Jayaprakash, M. Jayaraman, K. G. Rajeev, M.
Manoharan, V. Koteliansky, I. Rohl, E. S. Leshchiner, R. Langer,
Mol. Ther. 2009, 17, 872 – 879.
[7] J. Heyes, L. Palmer, K. Bremner, I. MacLachlan, J. Controlled
Release 2005, 107, 276 – 287.
[8] A. L. Bailey, P. R. Cullis, Biochemistry 1994, 33, 12573 – 12580.
[9] S. J. Eastman, M. J. Hope, P. R. Cullis, Biochemistry 1991, 30,
1740 – 1745.
[10] J. Zhang, H. Fan, D. A. Levorse, L. S. Crocker, Langmuir 2011,
27, 1907 – 1914.
[11] I. M. Hafez, N. Maurer, P. R. Cullis, Gene Ther. 2001, 8, 1188 –
1196.
[12] A. Akinc, et al., Mol. Ther. 2010, 18, 1357 – 1364.
[13] X. Yan, F. Kuipers, L. M. Havekes, R. Havinga, B. Dontje, K.
Poelstra, G. L. Scherphof, J. A. Kamps, Biochem. Biophys. Res.
Commun. 2005, 328, 57 – 62.
[14] O. Zelphati, F. C. Szoka Jr., J. Liposome Res. 1997, 7, 31 – 49.
[15] N. Demaurex, News Physiol. Sci. 2002, 17, 1 – 5.
[16] T. Kurosawa, S. Igarashi, M. Nishizawa, O. Onodera, Biochem.
Biophys. Res. Commun. 2005, 337, 1012 – 1018.
[17] Trial to Evaluate Safety and Tolerability of ALN-PCS02 in
Subjects With Elevated LDL-Cholesterol (LDL-C). (Registry of
Federally and Privately Supported Clinical Trials, U.S. National
Institutes of Health, 2011).
Figure 7. Efficacy of siRNA-LNP containing amino lipid 16 in mice and
non-human primates. An optimized formulation composition contain-
ing 50 mole% of 16 was employed in this study (see text and
Supporting Information page 41 for details). Inset: residual FVII
activity in mouse serum relative to a saline control group with
a median effective siRNA dose (ED50) of about 0.005 mgkgÀ1; data
points represent group mean (n=5)Æs.d. Cynomolgus monkeys were
administered siRNA targeting the TTR gene at doses of 0.03, 0.1 and
0.3 mgkgÀ1 (bar graph). Data represent group mean (n=3)Æs.d.,
expressed as TTR mRNA relative to GAPDH mRNA levels determined
[18] A. Simon, Phase I Safety, Pharmacokinetic and Pharmacody-
namic Results for ALN-PCS, Preliminary Study Results. (Alny-
lam Pharmaceuticals, Inc, 2012).
in liver samples, with the ED50 estimated to be <0.03 mgkgÀ1
.
Angew. Chem. Int. Ed. 2012, 51, 1 – 6
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

.
Angewandte
Communications
Communications
siRNA Delivery
Special (lipid) delivery: The role of the
ionizable lipid pKa in the in vivo delivery
of siRNA by lipid nanoparticles has been
studied with a large number of head
group modifications to the lipids. A tight
correlation between the lipid pKa value
and silencing of the mouse FVII gene
(FVII ED50) was found, with an optimal
pKa range of 6.2–6.5 (see graph). The
most potent cationic lipid from this study
has ED50 levels around 0.005 mgkgÀ1 in
mice and less than 0.03 mgkgÀ1 in non-
human primates.
M. Jayaraman,* S. M. Ansell, B. L. Mui,
Y. K. Tam, J. Chen, X. Du, D. Butler,
L. Eltepu, S. Matsuda,
J. K. Narayanannair, K. G. Rajeev,
I. M. Hafez, A. Akinc, M. A. Maier,
M. A. Tracy, P. R. Cullis, T. D. Madden,
M. Manoharan,
M. J. Hope*
&&&& — &&&&
Maximizing the Potency of siRNA Lipid
Nanoparticles for Hepatic Gene Silencing
In Vivo
6
www.angewandte.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 1 – 6
These are not the final page numbers!