A Single Methylene Group in Oligoalkylamine-Based Cationic Polymers and Lipids Promotes Enhanced mRNA Delivery

Article abstract of DOI:10.1002/anie.201603648

The development of chemically modified mRNA holds great promise as a new class of biologic therapeutics. However, the intracellular delivery and endosomal escape of mRNA encapsulated in nanoparticles has not been systematically investigated. Here, we synthesized a diverse set of cationic polymers and lipids from a series of oligoalkylamines and subsequently characterized their mRNA delivery capability. Notably, a structure with an alternating alkyl chain length between amines showed the highest transfection efficiency, which was linked to a high buffering capacity in a narrow range of pH 6.2 to 6.5. Variation in only one methylene group resulted in enhanced mRNA delivery to both the murine liver as well as porcine lungs after systemic or aerosol administration, respectively. These findings reveal a novel fundamental structure–activity relationship for the delivery of mRNA that is independent of the class of mRNA carrier and define a promising new path of exploration in the field of mRNA therapeutics.

Full text of DOI:10.1002/anie.201603648

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201603648
German Edition: DOI: 10.1002/ange.201603648
mRNA Delivery
A Single Methylene Group in Oligoalkylamine-Based Cationic Poly-
mers and Lipids Promotes Enhanced mRNA Delivery
´
Anita Jarze˛binska, Tamara Pasewald, Jana Lambrecht, Olga Mykhaylyk, Linda Kꢀmmerling,
Philipp Beck, Gꢀnther Hasenpusch, Carsten Rudolph, Christian Plank, and Christian Dohmen*
Dedicated to the memory of Braydon (Brad) Guild
Abstract: The development of chemically modified mRNA
holds great promise as a new class of biologic therapeutics.
However, the intracellular delivery and endosomal escape of
mRNA encapsulated in nanoparticles has not been systemati-
cally investigated. Here, we synthesized a diverse set of cationic
polymers and lipids from a series of oligoalkylamines and
subsequently characterized their mRNA delivery capability.
Notably, a structure with an alternating alkyl chain length
between amines showed the highest transfection efficiency,
which was linked to a high buffering capacity in a narrow
range of pH 6.2 to 6.5. Variation in only one methylene group
resulted in enhanced mRNA delivery to both the murine liver
as well as porcine lungs after systemic or aerosol administra-
tion, respectively. These findings reveal a novel fundamental
structure–activity relationship for the delivery of mRNA that is
independent of the class of mRNA carrier and define
a promising new path of exploration in the field of mRNA
therapeutics.
by its highly anionic character that prevents a passage across
cell membrane unaided and the ubiquitous presence of
RNases. Therefore, a delivery system that is designed for
the efficient encapsulation of mRNA using carriers that
possess effective cell penetrating properties is required.
Although a variety of carriers for siRNA and pDNA have
been investigated,[3–5] a tailored approach is required for
mRNA due to its low stability, different size, and single-
stranded nature. Thus, the de novo development of a delivery
system is needed that can be adjusted to this nucleic acid.[2]
The aim of this study was to develop efficient mRNA
carriers that can be used in vivo for both aerosol and
intravenous application to face the challenge of diseases
associated with the airways as well as metabolic disorders of
the liver, respectively. Cationic polymers have proven to
efficiently transport pDNA to the lung tissue after aerosol
application,[6] while lipid nanoparticles are extremely capable
in pDNA/siRNA delivery to hepatocytes via systemic admin-
istration. Since no carriers for mRNA-based therapies in vivo
exist on the market so far, we chose these two distinct delivery
systems and application routes as a starting point.
A key parameter that determines the transfection effi-
ciency of any nucleic acid delivery agent is an acid dissoci-
ation constant (pKa) in a pH range of 6.2 to 6.5, which is
hypothesized to enhance endosomal escape of the carrier
complex into the cytoplasm.[2,7,8] We expected this step to be
critical for successful mRNA delivery as well. Since oligoal-
kylamine-based carriers are known effective delivery systems
for various nucleic acids,[9–14] we chose a small diverse set of
tri- (2-2, 3-3) and tetramines bearing ethylene (2-2-2) and/or
propylene spacers (2-3-2, 3-3-3, Figure 1a) and determined
the buffering capacity for each structure by potentiometric
titration to predict their potency in mRNA delivery (Fig-
ure 1b).
R
ecently the field of transcript therapy by treating diseases
with messenger RNA (mRNA) has gained momentum and
raised the interest in both academia and industry for wide-
spread application in a variety of therapeutic areas.[1,2] In
contrast to gene therapy, mRNA-based drugs do not bear the
inherent risk of insertional mutagenesis. Moreover, they
provide a transient protein production platform for in situ
generation of the therapeutic protein due to the relatively
short half-life of mRNA.[1] Additionally, chemical modifica-
tions of RNA nucleotides have been shown to decrease
immune responses and improve the nucleic acid stability.[1]
However, the direct delivery of mRNA to cells is hampered
[*] Dr. T. Pasewald, Dr. P. Beck, Dr. G. Hasenpusch,
Priv.-Doz. Dr. C. Rudolph, Prof. Dr. C. Plank, Dr. C. Dohmen
ethris GmbH
To enable an accurate comparison in a non-polymeric
system, the oligoalkylamines were succinylated (Supporting
Information, Scheme S1), which imitates the occupation of
one primary amine after chemical coupling to the polymer
scaffold by formation of amide bonds. The modified oligoal-
kylamines were titrated with NaOH, revealing that diethyle-
netriamine (2-2) and the (2-3-2) tetramine bearing an
alternating alkylamine chain length exhibited the highest
buffering capacity in the endosomal pH range (Figure 1b).
Hence, both were expected to transfect cells efficiently when
incorporated in a polymeric delivery system.
Semmelweisstrasse 3, 82152 Planegg (Germany)
E-mail: dohmen@ethris.com
´
A. Jarze˛binska, Priv.-Doz. Dr. C. Rudolph
Department of Pediatrics, Ludwig Maximilians University
Lindwurmstrasse 2a, 80337 Mꢀnchen (Germany)
J. Lambrecht, O. Mykhaylyk, L. Kꢀmmerling, Prof. Dr. C. Plank
Institute of Molecular Immunology & Experimental Oncology
Technische Universitꢁt Mꢀnchen
Ismaningerstrasse 22, 81675 Mꢀnchen (Germany)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under
http://dx.doi.org/10.1002/anie.201603648.
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Figure 1. a) Structures of oligoalkylamines. b) Plots of buffering capacities of succinylated oligoalkylamines. The area in between the dotted lines
indicates the ideal buffering range for nucleic acid carriers, enhancing endosomal escape. Buffering capacity b was defined as volume of 50 mm
NaOH added to the solution containing 72 mmoles protonable amines resulting in a pH change of 0.1. c) Synthesis of oligoalkylamine-modified
poly(acrylic acid) carriers. MES=2-(N-morpholino)ethanesulfonic acid, EDC=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. d),e) Transfection
efficiency of polyplexes carrying mRNA-FLuc on d) murine fibroblasts (NIH 3T3) and e) target tissue-derived human alveolar type II-like cells
(A549). The reporter protein (firefly luciferase) level was determined 24 h post transfection by measuring bioluminescence expressed in counts
per second (cps). f) Influence of polyplexes on cell viability of NIH 3T3 cells 24 h after transfection was evaluated using an MTT assay.
g) Influence of poly(acrylic acid) molecular weight on transfection efficiency of (2-3-2)-based carriers. PAA20k-(2-3-2) exhibits superior transfection
efficiency over both PAA70k-(2-3-2) and PAA8k-(2-3-2). The reporter protein (firefly luciferase) level was determined 24 h post transfection by
measuring bioluminescence expressed in counts per second (cps). *p<0.05 and ****p<0.0001.
For this purpose, oligoalkylamines were grafted to an
8000 Da poly(acrylic acid) (PAA8k) scaffold (Figure 1c)
resulting in polymers that maintained the previously observed
pattern in buffering capacity (Supporting Information, Fig-
ure S4). When complexed with chemically modified mRNA
at nitrogen to phosphate (N/P) ratio 20 in water (Aqua ad
iniectabilia, B. Braun), all resulting polyplexes formed decent
monodisperse particles with hydrodynamic diameters of 65 to
236 nm and overall positive surface charge (Supporting
Information, Table ST1). Next, the polymeric carriers were
screened on murine fibroblasts (NIH 3T3) for their ability to
transfect cells using chemically modified mRNA encoding for
firefly luciferase (mRNA-FLuc) as a reporter (Figure 1d).
Suprisingly, the activity of PAA8k-(2-2) was at the back-
ground level, whereas PAA8k-(2-3-2) transfected cells up to
85-fold more efficiently (at 250 ng mRNA per well) compared
to the second best PAA8k-(3-3) at similar cell viability (ca.
90%). Since the polymeric carrier was intended for aerosol
application,[1] human alveolar type II like cells (A549) were
transfected with the polymeric particles, resulting in highest
levels of the reporter enzyme in PAA8k-(2-3-2)-treated cells
as observed during the screening on NIH 3T3 cells (Fig-
ure 1e). Interestingly, irrespective of the applied N/P ratio
(Supporting Information, Figure S1), the alternating structure
(2-3-2) provided superior mRNA transfection compared to all
other compounds including (2-2), which exhibited similar
buffering capacity.
mRNA in NIH 3T3 cells treated with polyplexes (ca. 3 pg
mRNA per cell) via qPCR. Briefly, cells were incubated 0.5, 1,
2, or 4 h with polyplexes and the amounts of the reporter
sequence were quantified after each time point. The lowest
uptake was detected for PAA8k-(2-2), which led to even 4-
fold lower mRNA levels compared to PAA8k-(2-3-2) (0.04–
0.09 pg per cell; Supporting Information, Figure S2a). This
explains the finding that PAA8k-(2-2)-based carriers did not
result in efficient transfection although they showed similar
buffering capacity in the critical pH range. In contrast, all
other polyplexes provided higher cellular uptake of reporter
mRNA (PAA8k-(3-3) being most efficient resulting in 0.17–
0.25 pg per cell). These findings support the notion that the
increased uptake of complexes does not correlate with higher
transfection levels,[15] although for mRNA delivery a certain
threshold needs to be exceeded.
Next, MTTassays were performed to exclude the effect of
cell viability on polyplex efficacy (Figure 1 f). The highest
toxicity was observed in the case of PAA8k-(3-3-3), which led
to only 63% viable cells 24 h after transfection with 500 ng of
mRNA. In contrast, the same dose of mRNA in complex with
tetramines PAA8k-(2-3-2) or PAA8k-(2-2-2) resulted in
a significantly higher amount of viable cells (89 or 86%,
respectively), whereas only the alternating (2-3-2) scaffold led
to reasonable reporter enzyme activity. Therefore, cell
viability after incubation with polyplexes is independent of
transfection efficiency, implying that after sufficient particle
uptake, buffering capacity is a driving force for augmented
mRNA translation.
Since sufficient particle uptake is a prerequesite for high
transfection efficiency, we evaluated the amounts of reporter
2
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Figure 2. a) Ex vivo enzyme activity of firefly luciferase in murine lungs after treatment with 12.5 mg mRNA-FLuc complexed with PAA8k-(2-3-2),
PAA8k-(2-2-2) or PAA8k-(3-3-3); n=3. b) Bioluminescence imaging of porcine lung tissue after aerosol application of PAA20k-(2-3-2) mRNA-FLuc
polyplexes. Applied dose: 2 mg in 8 mL of total volume; Picture 1: L. cranialis sinister, L. caudalis dexter; Picture 2: L. accessorius, L. cranialis dexter,
L. medius; Picture 3: L. caudalis sinister, Trachea; Picture 4: L. caudalis dexter, L. caudalis sinister; L. Lobus.
To further confirm the in vitro findings of best performing
tetramine transfection agents PAA8k-(2-3-2), -(2-2-2), and
-(3-3-3) as mRNA carriers in vivo, we tested mRNA-FLuc
polyplexes under different conditions in both mice and a pig.
After pulmonary application in mice, the order of carrier
efficacy (2-3-2) > (2-2-2) > (3-3-3) observed in A549 cells was
confirmed as a trend in vivo (Figure 2a).
Next, we extended our investigation to a pig model, which
is a more relevant human disease animal model as the
nanoparticles can be applied via aerosol into a ventilated
body as performed in clinical application. For this purpose,
the PAA molecular weight was optimized regarding trans-
fection efficiency in vitro, showing that 20 kDa is noticeably
more effective than 8 kDa and 70 kDa (Figure 1g). Thus,
mRNA-FLuc complexed with (2-3-2)-modified PAA20k was
nebulized into the lungs of a pig, which resulted in firefly
luciferase enzyme activity in well-ventilated cranial parts of
the lung (Figure 2b; images of a water-treated control in the
Supporting Information, Figure S4).
Encouraged by our findings with the polymer-based
delivery system, we investigated whether the correlation
between buffering capacity and transfection efficiency
remains valid in lipid-derivatized alkylamine carriers. Thus,
a small set of oligoalkylamine cationic lipid structures were
generated by applying the method reported by Love et al.
(Figure 3a).[9] Briefly, a ring-opening of CH3(CH2)9-epoxide
by oligoalkylamines resulted in a library of C12-decorated
carrier scaffolds (Supporting Information, Scheme S2). As in
the case of the polymer library, the lipidic compounds were
analysed for their buffering capacity in a presumed optimal
pH range (6.2 to 6.5) for endosomal release. While the lipid-
derivatized triamines did not possess increased buffering
capability in the tested range, the tetramines exhibited clear
peaks (Figure 3b). However, for C12-(3-3-3) and -(2-2-2),
these were above and/or below pH 6.2 and 6.5. Only the C12-
(2-3-2) displayed a peak buffering capacity in the desired
range.
The transfection efficiency of all the lipids was tested by
complexation of mRNA-FLuc, as described in the Supporting
Information. Lipid-based nanoparticles were found to com-
prise uniform spherical particles with hydrodynamic diameter
between 54 and 61 nm and a neutral or slightly positive
surface charge (Figure 3c; Supporting Information,
Table ST2). The transfection of NIH 3T3 cells with lipoplexes
comprised of carriers with low buffering capacity in the
endosomal pH range exhibited only low levels of reporter
enzyme activity (Figure 3d). In contrast, C12-(2-3-2)-based
lipoplexes demonstrated high transfection efficiency with up
to a nearly 7-fold increase compared to the second best C12-
(2-2-2) at a comparable cell viability of about 100%.
Next, lipoplexes were investigated with the aim of protein
production in hepatocytes, targeting the liver via systemic
administration as described for lipidic systems for siRNA
applied intravenously.[7,9] Transfection of human liver carci-
noma cells (HepG2, Figure 3e) with the lipidic particles
confirmed the trend observed during the previous screening
on NIH 3T3 cells, namely, the alternating (2-3-2)-structure
exerts the highest transfection capabilities compared to other
oligoalkylamines. We found that this trend was maintained
when extended to studies using other cell lines (Supporting
Information, Figure S3). Furthermore, transfection efficiency
of lipoplexes proved to be independent of intracellular
reporter mRNA levels or cell viability as determined by
qPCR and MTTassays, respectively (Supporting Information,
Figure S2b; Figure 3 f).
As a final proof of concept, the potency of lipoplexes
consisting of tetramine-based lipid to deliver mRNA-FLuc
was investigated in female Balb/c mice. Six hours post
intravenous injection, d-luciferin was applied intraperito-
neally as a substrate. After 10 min of incubation, biolumines-
cence in animals treated with cationic lipids C12-(2-2-2) and
C12-(3-3-3) was at the background level, whereas a strong
signal in the liver of C12-(2-3-2)-treated mice was observed
(Figure 3g–i).
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Figure 3. a) Synthesis of oligoalkylamine-based lipids. b) Plots of buffering capacities of C12-lipids with oligoalkylamine backbones. The area in
between the dotted lines indicates the ideal buffering range for nucleic acid carriers, enhancing endosomal escape. Buffering capacity b was
defined as volume of 50 mm NaOH added to the solution containing 10 mmoles lipid resulting in a pH change of 0.1. c) Transmission electron
microscopic (TEM) images of C12-(2-3-2) lipoplexes. d),e) Transfection efficiency of lipoplexes carrying mRNA-FLuc on d) murine fibroblasts
(NIH 3T3) and e) target tissue-derived human liver carcinoma cells (HepG2). The protein level was determined 24 h post transfection by
measuring bioluminescence expressed in counts per second (cps). f) Influence of lipoplexes on cell viability of NIH 3T3 cells 24 h after
transfection was evaluated using an MTT assay. g) Quantification of bioluminescence intensity in treated mice (observed in (h)). n=3. **p<0.01,
***p<0.001, and ****p<0.0001. h) Exemplary bioluminescence images of mice treated intraveneously with 18 mg mRNA-FLuc complexed with
C12-(2-3-2), C12-(2-2-2), and C12-(3-3-3). i) Exemplary bioluminescence images of excised livers.
In conclusion, by screening a set of oligoalkylamines for
efficient delivery of mRNA in nanoparticles, we found that
a tetramine with alternating ethyl–propyl–ethyl spacers
exhibited a high ability to mediate robust levels of protein
translation in vitro, although the oligoalkylamines within the
tested carriers hardly varied. A trend towards higher effi-
ciency of the (2-3-2)-based polymer/lipid was further con-
firmed in animal models. The observation of enhanced
transfection properties of mRNA nanoparticles in two
distinct delivery systems indicates a universal mechanism of
action. We hypothesize that the scaffold with an alternating
alkyl chain length between amines combines good buffering
capacity with sufficient complex stability. As a consequence,
endosomal release of (2-3-2) tetramine is facilitated and the
intact complex is able to enter the cytosol. These surprising
findings reveal that even subtle changes in the structure of
carriers hold high potential for carrier improvements. How-
ever, a detailed investigation regarding the underlying
mechanism of this structure–activity relationship has to be
the topic of further studies.
Since the (2-3-2)-motif bridges the gap between in vitro
and in vivo efficiency, this work provides a foundation to
develop mRNA delivery systems that may have future clinical
applications.
Acknowledgements
We thank Prof. Dr. Ernst Wagner and Dr. Ulrich Lꢀchelt
from Faculty of Chemistry and Pharmacy, Ludwig Max-
imillian University of Munich (LMU) for their support and
helpful advice. Financial support by the German Federal
Ministry of Education and Research Go-Bio grant 0315986
and the Excellence Cluster “Nanosystems Initiative Munich
(NIM)” is gratefully acknowledged. The authors also would
like to thank Dr. Rebekka Kubisch-Dohmen for her guidance
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Kempter for taking TEM pictures, Christian Koch for his
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Received: April 14, 2016
Revised: May 17, 2016
Published online: && &&, &&&&
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Communications
mRNA Delivery
Oligoalkylamine-based lipids and poly-
mers were generated and screened for
buffering capacity and mRNA delivery.
Alternating ethylene/propylene/ethylene
spacers in the tetramines improve trans-
fection efficiency (see picture, middle)
over ethylene-only (left) or propylene-only
repeats (right). This trend is valid
´
A. Jarze˛binska, T. Pasewald, J. Lambrecht,
O. Mykhaylyk, L. Kꢁmmerling, P. Beck,
G. Hasenpusch, C. Rudolph, C. Plank,
C. Dohmen*
&&&& — &&&&
A Single Methylene Group in
Oligoalkylamine-Based Cationic Polymers
and Lipids Promotes Enhanced mRNA
Delivery
regardless of delivery platform (lipids or
polymers), tested cell line, targeted
tissue, or application route in vivo.
6
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