Design of Ionizable Lipids to Overcome the Limiting Step of Endosomal Escape: Application in the Intracellular Delivery of mRNA, DNA, and siRNA

Article abstract of DOI:10.1021/acs.jmedchem.5b01679

The intracellular delivery of nucleic acid molecules is a complex process involving several distinct steps; among these the endosomal escape appeared to be of particular importance for an efficient protein production (or inhibition) into host cells. In the present study, a new series of ionizable vectors, derived from naturally occurring aminoglycoside tobramycin, was prepared using improved synthetic procedures that allow structural variations on the linker and hydrophobic domain levels. Complexes formed between the new ionizable lipids and mRNA, DNA, or siRNA were characterized by cryo-TEM experiments and their transfection potency was evaluated using different cell types. We demonstrated that lead molecule 30, bearing a biodegradable diester linker, formed small complexes with nucleic acids and provided very high transfection efficiency with all nucleic acids and cell types tested. The obtained results suggested that the improved and "universal" delivery properties of 30 resulted from an optimized endosomal escape, through the lipid-mixing mechanism.

Full text of DOI:10.1021/acs.jmedchem.5b01679

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Article
Design of ionizable lipids to overcome the limiting step of endosomal
escape - Application in the intracellular delivery of mRNA, DNA and siRNA.
Damien Habrant, Pauline Peuziat, Thibault Colombani, Laurence Dallet, Johan Gehin,
Emilie Goudeau, Bérangère Evrard, Olivier Lambert, Thomas Haudebourg, and Bruno Pitard
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01679 • Publication Date (Web): 04 Mar 2016
Downloaded from http://pubs.acs.org on March 6, 2016
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Design of ionizable lipids to overcome the limiting
step of endosomal escape ꢀ Application in the
intracellular delivery of mRNA, DNA and siRNA.
†, §
‡, ǁ, §
‡, ǁ
#
Damien Habrant, Pauline Peuziat,
Thibault Colombani, Laurence Dallet, Johan
†
†
†
#
‡, ǁ
Gehin, Emilie Goudeau, Bérangère Evrard, Olivier Lambert, Thomas Haudebourg, Bruno
,
†,‡,ǁ,□
Pitard*
†
InꢀCellꢀArt, 21 rue de la Noue Bras de Fer, 44200 Nantes, France
‡
Unité INSERM UMR1087, CNRS UMR 6291, 8 quai Moncousu, 44007 Nantes, France
ǁ
Université de Nantes, L’institut du Thorax, 8 quai Moncousu, 44007 Nantes, France
CBMN UMRꢀCNRS 5248 IECB, Université de Bordeaux 1ꢀIPB, Allée Geoffroy Saint
#
Hilaire, Pessac Fꢀ33600 France
□
Current address: InꢀCellꢀArt, Nantes, France.
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Abstract. The intracellular delivery of nucleic acid molecules is a complex process involving
several distinct steps; among these the endosomal escape appeared to be of particular importance
for an efficient protein production (or inhibition) into host cells. In the present study, a new
series of ionizable vectors, derived from naturallyꢀoccurring aminoglycoside Tobramycin, was
prepared using improved synthetic procedures that allow structural variations on the linker and
hydrophobic domain levels. Complexes formed between the new ionizable lipids and mRNA,
DNA or siRNA were characterized by cryoꢀTEM experiments and their transfection potency was
evaluated using different cell types. We demonstrated that lead molecule 30, bearing a
biodegradable diester linker, formed small complexes with nucleic acids and provided very high
transfection efficiency with all nucleic acids and cell types tested. The obtained results suggested
that the improved and “universal” delivery properties of 30 resulted from an optimized
endosomal escape, through the lipidꢀmixing mechanism.
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ꢀ
Introduction
Messenger RNA (mRNA)ꢀbased drugs have recently emerged as a new promising therapeutic
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,2
class to deliver genetic information. Indeed, mRNA offers several advantages compared to
longꢀstudied plasmid DNA. Unlike DNA, mRNA has the characteristic of being translated
directly in the cytoplasm and being fully metabolized. Moreover, there is no risk of genome
integration for mRNA, which represents one of the main drawbacks of DNAꢀbased therapeutics.
For these reasons, there is currently a growing interest in mRNA and its potential applications for
humans. Therapeutic approaches involving RNA interference (RNAi) are also actively
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investigated. RNAi is a postꢀtranscriptional process triggered by the introduction of smallꢀ
interfering RNA (siRNA) which leads to gene silencing in a sequenceꢀspecific manner.
As unformulated nucleic acids do not promote sufficient levels of gene expression (or
inhibition in the case of siRNA), a critical issue for the efficient application of gene therapy to
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ꢀ7
human diseases are the vectors used to improve the delivery of nucleic acids into host cells.
Viral vectors have proven their high efficiency in DNA transfer; however they can lead to
undesired immunological responses and are still limited for the delivery of more specific nucleic
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ꢀ11
acids such as mRNA or siRNA. These observations have led to the development of nonꢀviral
synthetic vectors. Within the difference classes of nonꢀviral vectors, several SARs have been
performed in order to improve the ability of cationic amphiphilic molecules to formulate and
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deliver nucleic acids.
However, due to the complex process of delivery, no universal
structural feature has been clearly identified and the majority of ionizable lipids described in the
literature require the presence of a helper fusogenic lipid to achieve efficient transfection
efficiency via liposomal formulations.
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The accepted mechanism for the intracellular delivery of nucleic acids mediated by ionizable
lipids consists in several distinct steps: formation of the lipoplex, binding to the cell membrane,
endocytosis, destabilization of the endosomal membrane and release of the nucleic acid. The
endosomal escape pathway for improved delivery of genetic information has been recently
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investigated, but only few reports mention ionizable lipids.
The recent study from Zerial and
coꢀworkers concluded that the endosomal escape “provides the major opportunity to improve the
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delivery system”.
The endosomal escape step has been described to be mediated by the ionizable lipids used as
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delivery vehicles.
Indeed, when lipolexes composed of ionizable lipids and nucleic acids
interact with the endosomal membranes, the anionic phospholipids (usually located on the
cytoplasmicꢀfacing monolayer of the membrane) flip towards the inside of the endosome,
causing the destabilization of the membrane. Neutral ion pairs between the positively charged
lipids used as delivery vehicle and the negatively charged phospholipids of the membrane are
then formed. This ‘lipid mixing’ mechanism leads to the formation of new multiꢀlamellar lipid
structures and thus to the disruption of the integrity of the endosomal membrane. CryoꢀTEM
experiments have demonstrated that the structures of the newly formed lipidic assemblies are
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highly dependent on the nature of the ionizable lipids. After this step, nucleic acid molecules
are then present in the cytosol without being surrounded by a closed endosomal membrane and
are able to diffuse. The interactions between the ionizable lipids and the anionic phospholipids
are therefore of crucial importance to drive the efficient release of the nucleic acids.
If different in their mechanism of action, the different classes of nucleic acids mRNA, DNA
and siRNA share some common features concerning their delivery mechanism. Thus, identifying
and overcoming common limiting step(s) may help for the design of universal delivery vehicles.
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In this purpose, we decided to prepare various ionizable lipids based on the aminoglycoside
Tobramycin, which was used as a model of a 4,6ꢀdisubstitued 2ꢀdeoxystreptamine derivatives.
Aminoglycosides are natural products able to provide a versatile polycationic framework
because several amino groups are present in their structure (together with numerous hydroxyl
functions). The family of aminoglycoside derivatives already showed promising results for the
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delivery of plasmid DNA and siRNA.
Interestingly aminoglycosides are also known to be
natural binders to threeꢀdimensional RNA structures, which make them of particular interest for
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the development of mRNA therapeutics.
Despite of being promising candidates for nucleic acids delivery, this family still lacks a SAR
study to elucidate the key structural features required to provide improved transfection efficiency
through facilitated endosomal escape. Using a rational approach, our objective was to discover
simple and versatile ionizable lipids, able to reach high levels of transfection without the need of
a helper lipid. We herein report on the synthesis and the characterization of a new series of
lipidic aminoglycoside derivatives and their corresponding transfection efficiency in different
cell types using three distinct types of nucleic acids: mRNA, DNA and siRNA.
•
Results
o Synthesis of aminoglycosideꢀderived ionizable lipids
Ionizable lipids are amphiphilic molecules, commonly composed of three distinct parts: an
ionizable headgroup, a linker (also called spacer) and a lipophilic moiety. They interact with
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nucleic acids, forming condensed aggregates named lipoplexes. In the present study, we aimed
at assessing different parameters that may be involved in the lipid mixing mechanism and
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therefore in the nucleic acid delivery: 1) the optimal configuration of the hydrophobic domain
(length, insaturation), 2) the importance of the distance between the ionizable headgroup and the
lipophilic domain (by changing the length of the linker), 3) the behavior of environmentꢀ
sensitive linkers, supposed to be perturbed after reaching the intracellular compartment (reduced
pH, presence of enzymes and reducing agents). The last category is of particular interest because
the presence of biodegradable functions in the linker was described to be associated with higher
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transfection efficiency and with decreased toxicity compared to “classical” linkers.
The starting point of our SAR was the molecule 13 (DOST for DiꢀOleylꢀSuccinyl Tobramycin)
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which is composed of a succinyl linker and two oleyl chains as hydrophobic moiety. The
general strategy for the preparation of 13 followed the synthetic route already described for this
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type of compounds (Scheme 1).
First, oleylamine 1 and oleoyl chloride 2 were coupled to form amide 3, which was then reduced
by LiAlH to dioleylamine 4. Then, addition of succinic anhydride furnished the “DOS” moiety
The linkerꢀhydrophobic tail was prepared in 3 simple steps.
4
5
in good yield. Concerning the aminoglycoside part, the described strategy employing
orthogonal protecting groups was applied. Starting with Tobramycin, selective Cbzꢀprotection of
the primary amine on the 6’ position was achieved using bulky acylating agent Nꢀ
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0
benzyloxycarbonyloxyꢀ5ꢀnorborneneꢀ2,3ꢀdicarboximide (NorꢀCbz 7). The four remaining
secondary amines of 8 (positions 1, 3, 2’ and 3’’) were then protected with a Teoc group by 2ꢀ
(trimethylsilyl)ethyl pꢀnitrophenylcarbonate (TeocꢀONp 9). As an improvement to the described
procedure, we were able to perform these consecutive protection steps in a single pot:
Tobramycin was first dissolved in H O/Et N mixture before addition of NorꢀCbz in DMF. After
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24 h, a solution of TeocꢀONp in DMF was added and the mixture stirred for 48 h at 55 °C. This
sequential addition of protecting groups allowed for the preparation of fully protected
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Tobramycin derivative 10 in a single step and with a good 71% yield. Catalytic hydrogenation of
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1. Then, peptideꢀlike coupling between carboxylic acid 5 and amine 11 was performed using
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HBTU and DMAP in DMF to produce the protected compound 12. Finally, 12 was treated by
TFA in DCM, which allowed for the isolation of the final compound 13 as a TFA salt.
a
Scheme 1: Synthesis of DiꢀOleylꢀSuccinyl Tobramycin (13)
a
Reagents and conditions: (a) Et N, DCM, 20 h, rt. (b) LiAlH , THF, 12 h, 55 °C. (c) Succinic
3
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anhydride, Et N, DCM, 12 h, rt. (d) 7, Et N, DMF, H O, 24 h, rt. (e) 8, Et N, DMF, 48 h, 55 °C.
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(
f) H , 10% Pd/C, MeOH, H O, 12 h, rt. (g) 5, HBTU, DMAP, DMF, 12 h, rt. (h) TFA, DCM, 2
2 2
h, rt.
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A series of new analogues derived from 13 was then synthesized. For their preparation, these
molecules used the common intermediate 11, but the linkerꢀhydrophobic tail conjugate was
different. As described in the introduction, the first parameters we wanted to assess were the
influence of the double bond in the lipophilic chains and the length of the chains themselves. In
this context, we prepared different symmetrical dialkyl amines, inspired by the synthesis of
scaffold 5 (Scheme 2). Myristylamine 14a and palmitylamine 14b respectively reacted with
myristoyl chloride 15a and palmitoyl chloride 15b to give the corresponding amides 16a and
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16b, which were then reduced with LiAlH to the corresponding symmetrical amines 17a and
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17b. The linker part was introduced by reaction of these amines, as well as commercially
available distearylamine 17c, with succinic anhydride to give the conjugates 18aꢀc in overall
good yields. As previously described, these carboxylic acids were coupled with the Tobramycin
amino derivative 11, before removal of the Teoc groups by TFA. This sequence allowed for the
preparation of 3 new molecules: 20a (DMST for DiꢀMyristylꢀSuccinylꢀTobramycin), 20b (DPST
for DiꢀPalmitylꢀSuccinylꢀTobramycin) and 20c (DSST for DiꢀStearylꢀSuccinylꢀTobramycin).
Scheme 2: Synthesis of DiꢀMyristylꢀSuccinylꢀTobramycin (20a), DiꢀPalmitylꢀSuccinylꢀ
a
Tobramycin (20b) and DiꢀStearylꢀSuccinylꢀTobramycin (20c)
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a
Reagents and conditions: (a) Et N, THF, 20 h, rt. (b) LiAlH , THF, 12 h, 55 °C. (c) Succinic
3
4
anhydride, Et N, DCM, 12 h, rt. (d) 11, HBTU, DMAP, DMF, 12 h, rt. (e) TFA, DCM, 2 h, rt.
3
Next, we wanted to assess the influence of the linker on the transfection efficiency. In this
purpose, two types of linkers were considered: the first type was a linker that was simply
elongated, by introducing supplementary methylene units compared to the original succinyl
linker, and for the second type the linker was modified to contain biodegradable functions
(
disulfide bridge or diester).
In this context, dioleylamine 4 reacted with different dicarboxylic acids (adipic 21a, suberic
21b, sebacic 21c and dithioglycolic 21d acids), in the presence of EDC/DMAP to give the
corresponding conjugates 22aꢀd, in moderate yields (Scheme 3). Similarly, these conjugates
were coupled with amine 11, and after Teocꢀremoval gave four new analogues: 24a (DOAT for
DiꢀOleylꢀAdipylꢀTobramycin), 24b (DOSUT for DiꢀOleylꢀSUberylꢀTobramycin), 24c (DOSET
for DiꢀOleylꢀSEbacylꢀTobramycin) and 24d (DODT for DiꢀOleylꢀDithioglycolylꢀTobramycin).
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Scheme 3: Synthesis of DiꢀOleylꢀAdipylꢀTobramycin (24a), DiꢀOleylꢀSuberylꢀTobramycin
a
(24b), DiꢀOleylꢀSebacylꢀTobramycin (24c), DiꢀOleylꢀDithioglycolylꢀTobramycin (24d)
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rt. (c) TFA, DCM, 2 h, rt.
The two most hydrophobic ionizable lipids 24b and 24c could not be assessed in the
transfection experiments, due to solubility issues in water (unlike the other molecules, we
observed for these two compounds a visible precipitate when water was added). Despite being
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bad news for our structureꢀactivity relationship study, this indicates that too lipophilic
compounds cannot be used as efficient nanocarriers for nucleic acids in our tested conditions.
Lastly, the compound 30 (DOSST for DiꢀOleylꢀSuccinylꢀSerinylꢀTobramycin) bearing a
diester function was prepared (Scheme 4). This molecule was inspired by the structure of
DOTAP, a wellꢀstudied commercial cationic lipid, known as the diester equivalent of
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DOTMA.
Scheme 4: Structure of DOTMA and DOTAP
X
O
X = CH : DOTMA
X = CO : DOTAP
2
N+
O
X
Cl-
The synthesis started with NꢀBoc serinol 25 which was diacylated with oleoyl chloride to give
6. After TFA treatment and reaction with succinic anhydride, the conjugate 28 was obtained.
2
Similarly, the carboxylic acid function of 28 reacted with the amino group of 11 and gave access
to the final compound 30 after removal of the protecting groups.
a
Scheme 5: Synthesis of DiꢀOleylꢀSuccinylꢀSerinylꢀTobramycin (30)
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a
Reagents and conditions: (a) Pyridine, CHCl , 12 h, rt. (b) TFA, DCM, 2 h, rt. (c) Succinic
3
anhydride, Et N, DCM, 12 h, rt. (d) 11, HBTU, DMAP, DMF, 12 h, rt. (e) TFA, DCM, 2 h, rt.
3
With these molecules in hands, experiments were carried out to study whether the chemical
structure of the linker and/or the hydrophobic tail had an impact on the transfection efficiency of
different classes of nucleic acids.
o Transfection efficiency evaluation
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Many transfection reagents have been described to lead to effective gene transfer in cell lines
but their transfection potencies are still limited in mammalian primary cells. Yet, transfecting
primary cells is a major concern as these cells are biologically more relevant and mimic in vivo
situation with better accuracy. In this context, we decided to evaluate the transfection efficiency
not only on HeLa cell lines, but also on two types of primary cells: rat aorta muscle and human
fibroblasts cells.
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First, we evaluated the efficiency of the Tobramycin lipidic derivatives for the mRNA
transfection (Figure 1). In this first set of experiments, we observed that all cell types displayed a
similar trend where three compounds provided better transfection efficiency compared to the
reference compound 13. Results indeed showed that the formulation with 30 containing a diester
linker led to a much higher level of luciferase expression (2 to 3 fold) compared to 13.
Complexes of 24a/mRNA (adipic linker) and palmityl derivative 20b/mRNA also led to a
slightly higher luciferase expression compared to 13, on the three cell types. On the other hand,
cells transfected with myristyl and stearyl compounds – 20a and 20c ꢀ showed lower expression
than cells transfected with 13, while compound 24d with a disulfide bridge produced almost no
luciferase signal in the tested conditions. These observations for 24d were similar for the other
nucleic acids. This suggests that the disulfide bridge in the structure of 24d is detrimental for the
transfection efficiency; the peculiar behavior of this compound will be discussed in a following
section.
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Figure 1: In vitro transfection of mRNA encoding Luciferase in different cells. HeLa cells (Aꢀ
B), rat aorta smooth muscle cells (CꢀD) and human fibroblasts cells (EꢀF) were transfected with
0.5 µg of mRNA formulated with each ionizable lipid at a charge ratio of 5. Data represent
mean, +/ꢀ SEM, luciferase activity in count per second (CPS) 24h postꢀtransfection for each
nanocarrier compared to 13 (gray).
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In addition, we have formulated mRNA encoding eGFP with the reference lipid 13 and the
best candidate for mRNA 30 to observe transfected cells by microscopy (Figure 2). HeLa cells
(Figures 2A and 2D) and human fibroblasts cells (Figures 2B and 2E) transfected with 13 or 30
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showed approximately the same percentage of eGFP expressing cells (about 80% for HeLa and
30% for fibroblasts) and the same viability (about 90% in both cases). Thus, both 13 and 30
confirmed their high transfection efficiency, with a low toxicity. Interestingly, the observation at
a higher magnification of rat aorta smooth muscle cells transfected with 30/mRNA complexes
showed higher expression of eGFP protein than cells transfected with 13/mRNA complexes
(Figures 2C and 2F). Compared to reference molecule 13, compound 30 seems to have the
capacity to bring more copies of mRNA inside the cells, which leads to higher expression levels.
HeLa cells
Human fibroblasts
Rat aorta muscle cells
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Figure 2: in vitro transfection of eGFP mRNA formulated with 13 in HeLa (A), human
fibroblasts (B) and rat aorta muscle cells (C) or with 30 in HeLa (D), human fibroblasts (E) and
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rat aorta muscle cells (F). Cells were transfected with 0.5 µg of mRNA formulated with 13 or 30
at a charge ratio of 5. Plates were incubated 24h before microscopy observation.
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After these results with mRNA, we performed experiments with two other types of nucleic
acids, DNA and siRNA, to evaluate the versatile character of these new compounds. In this
context, the transfection activities of lipoplexes formed by the formulation of plasmid DNA with
either compound 13 or one of its derivatives were measured by a luciferase assay on different
cell lines (Figure 3). For the cell line HeLa (Figures 3A and 3B), no significant difference was
observed, as all molecules displayed high luciferase expression, with the exception of disulfideꢀ
compound 24d, where no luciferase was produced. On the other hand, the more difficult to
transfect aorta muscle cells and the human fibroblasts cells showed some differences between the
molecules and both cell types displayed similar trends (Figures 3Cꢀ3F). Indeed, in both cases, the
best transfection efficiency was again obtained with diester 30, which led to higher expression
values than the reference molecule 13. Concerning the molecule with an adipic linker 24a, the
expression was comparable to the results obtained with 13. However, we could clearly see that
changing the hydrophobic tail had a negative impact on the transfection of these two types of
cells. Indeed, myristyl 20a, palmityl 20b and stearyl 20c derivatives all displayed lower
expression compared to 13.
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Figure 3: In vitro transfection of plasmid DNA encoding Luciferase in different cells. HeLa
cells (AꢀB), rat aorta smooth muscle cells (CꢀD) and human fibroblasts cells (EꢀF) were
transfected with 0.5 µg of DNA formulated with each ionizable lipid at a charge ratio of 5. Data
represent mean, +/ꢀ SEM, luciferase activity in count per second (CPS) 24h postꢀtransfection for
each nanocarrier compared to 13 (gray).
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These experiments led us to the conclusion that both the linker and the hydrophobic tail had an
impact on the DNA transfection efficiency in primary cells, with the dioleyl chains showing the
best results compared to smaller and/or nonꢀsaturated chains. The linker as well proved to be an
important part, as either lower transfection (disulfide 24d) or better transfection (diester 30)
compared to the reference molecule 13 were obtained.
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In addition, we have evaluated the transfection efficiency by measuring the percentage of
transfected HeLa cells by flow cytometry. For this purpose, DNA plasmids encoding eGFP were
formulated with the lipid 13 or one of its analogues. The results confirmed the trends observed in
the DNA luciferase assay and in the eGFP mRNA experiment. Indeed, 24a (adipic linker) and 30
(diester linker) both displayed similar or slightly higher expression levels (75ꢀ80% of transfected
cells) compared to 13 (70% of transfected cells), while myristyl, palmityl and stearyl derivatives
were less efficient than 13 (with approximately 50% of transfected cells for 20a, 20b and 20c).
As in previous experiments, low transfection was observed with disulfideꢀcontaining lipid 24d.
We next assessed the gene silencing potential after transfection with the various ionizable
lipids. We have performed RNAi experiments with siRNA targeting the endogenous lamin A/C
expression in murine or human cells (Figure 4). Here, RTꢀqPCR results indicated that cells
transfected with 13/siRNA, 24a/siRNA and 30/siRNA complexes had very little residual lamin
A/C expression, with less than 15% in HeLa and human fibroblasts cells and around 20ꢀ25 % in
C2C12 cells, compared to cells transfected with nonꢀformulated siRNA (which resulted in more
than 95% of expression, data not shown). These results clearly demonstrate that these complexes
allow for the efficient knockdown of the expression of an endogenous gene in different cell
types. On the other hand, modifying the hydrophobic tail to myristyl, palmityl or stearyl chains
had a negative impact on the transfection, as 20a, 20b and 20c all displayed less efficient gene
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silencing properties with higher residual lamin A/C expression measured compared to 13.
Finally, as observed for mRNA and DNA, compound 24d with a disulfide linker did not lead to
any inhibition of expression compared to negative control (unformulated siRNA).
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Figure 4: Inhibition of LaminA/C expression. HeLa cells (AꢀB), C2C12 cells (CꢀD) or human
fibroblast cells (EꢀF) were transfected with 150 ng (A, B) or 300 ng (CꢀF) of siRNA, formulated
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with each ionizable lipid at a charge ratio of 5 and compared to 13 (gray). The gene expression
levels of LaminA/C were measured by qPCR analysis, normalized against the expression levels
of HPRT (housekeeping gene). The results are expressed as the 2ꢀ∆∆CT formula. Bars represent
mean values ± SEM
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The versatile potency of 30 and 13 were evaluated in headꢀtoꢀhead comparison experiments
with commercially available Lipofectamine 3000 (Thermo Fisher Scientific). In the tested
conditions, 30 appeared to be more potent than Lipofectamine 3000 for the inꢀvitro transfection
of mRNA and DNA, when siRNA experiments provided similar activities for both compounds
(see Supporting Information, Figures S19ꢀ21). In addition, the cell survival was much higher
with 30 (with close to 100% survival) compared to Lipofectamine 3000 (around 20% survival) in
our experiments (see Supporting Information, Figure S22).
o
Characterization of the lipoplexes by CryoTEM
Morphologies of complexes made with selected candidates 13, 24a (adipic linker) and 30
(diester linker) and nucleic acids, either mRNA (Figure 5AꢀC), plasmid DNA (Figure 5DꢀF) or
siRNA (Figure 5GꢀI) were then observed by cryoTEM.
In the case of mRNA, the complexes exhibited various sizes, depending on the molecule.
Complexes made with 13 exhibited sizes in a range of 200ꢀ500 nm while those formed with 24a
appeared of about 200ꢀ300 nm (Figures 5A,B). Interestingly, very small complexes (about 70
nm in diameter) were observed with 30, in addition to larger complexes (200 to 500 nm) (Figure
5C). The morphology of these ARN complexes contained small globular features with sizes
ranging about 20 nm in diameter.
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Plasmid DNA mixed with 24a formed very large complexes, larger than 1µm (Figure 5E). In
contrast, the sizes of DNA complexes formed with 13 and 30 were in the range of 200 and 500
nm (Figure 5D and 5F). As in the case of mRNA, small globular features (about 40 nm)
comprising of lipid and DNA appeared to assemble together leading to larger structures.
Finally, complexes formed with siRNA are similar to those formed with mRNA and DNA.
Complexes made with 13 and 24a had a size in the range of 80 to 150 nm (Figure 5G,H) while
those formed with 30 were of about 100 nm (Figure 5I). Like mRNA complexes, the structures
of these siRNA complexes contained small globular features about 20 nm in diameter.
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1
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24a
30
mRNA
DNA
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siRNA
Figure 5: CryoTEM images of complexes made of mRNA mixed with 13 (A), 24a (B) or 30
C), plasmid DNA mixed with 13 (D), 24a (E) or 30 (F), and siRNA mixed with 13 (G), 24a (H)
(
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or 30 (I). Scale bars 50 nm (AꢀI) and 10 nm (inset). Complexes were prepared by mixing 0.5 µg
of nucleic acids with the corresponding lipid at charge ration (+/ꢀ) of 5.
o Complexation study of disulfide compound 24d
Regardless of what type of nucleic acid we have used, the previous experiments showed that
molecule 24d, containing a disulfide bridge in the linker, was a very poor transfection agent. In
order to better understand the mechanism and the reasons for such poor performance, we
analyzed the formulations formed between DNA and 24d by monitoring the size of the
lipoplexes and the DNA complexation.
The size of the lipoplex, which is linked to the colloidal stability, is a key parameter for nucleic
acid delivery. To assess the mean diameter of the system, dynamic light scattering was used.
Figure 6A illustrates the evolution of the mean diameter as a function of the charge ratio; we
could observe that 24d follows the classical threeꢀzone colloidal stability observed for many
43
ionizable lipids. Zone A (CR 0.2 to 1) was comprised of colloidally stable complexes with a
negative charge ratio and uncondensed DNA surrounding the lipoplexes. Zone B complexes
were essentially neutrally charged (CR 1.5 to 2) with condensed DNA contained within
colloidally unstable aggregates. Lipoplexes from zone B exhibited a visible precipitate. Thus, an
arbitrary value of 700 nm was attributed to those complexes, since the diameter of the
precipitated lipolexes could not be measured by dynamic light scattering. Finally, zone C (CR >
2
.5) lipolexes were composed of colloidally stable particles with condensed DNA and positive
charges, thus strongly repelling each other.
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Agarose gel electrophoresis experiments on a 0.8% agarose gel stained with ethidium bromide
were used to follow DNA complexation, by assessing the relative amounts of DNA that were
free or incorporated into the lipoplex. Figure 6B shows that the amount of free DNA decreased
as the charge ratio (+/ꢀ) increased. For charge ratios between 0 and 1, free DNA molecules were
present and migrated out of the well. For charge ratios above 1, no free DNA was observed and
aggregates were formed, that did not migrate out of the well. Again, this behavior is classical for
ionizable lipids used as transfection reagents.
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The same electrophoresis experiment was repeated after 30 min of incubation of the 24d/DNA
lipoplexes in the presence of reducing agent DTT (Figure 6C). What was observed is a
quantitative release of the DNA after incubation with DTT, independently of the charge ratio (+/ꢀ
). This suggests that the DTT treatment of the 24d/DNA complexes led to the cleavage of the SꢀS
bond, the perturbation of the lipoplexes, and the DNA molecules that were entrapped in the
lipolexes became free and migrated out of the well. This was in good agreement with what was
expected and confirmed the ability of DTT to reduce cleavable functions as the disulfide bridge.
A.
B.
C.
Figure 6: Physicoꢀchemical characterization of 24d/DNA lipids complexes. (A) Colloidal
stability of lipoplexes, as a function of the charge ratio and determined by dynamic light
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scattering. The analysis was done after 10 min of complexation at room temperature. Lipolexes
from zone A and C were stable; whereas those from zone B exhibited a visible precipitate (thus
an arbitrary value of 700 nm was attributed). (B,C) Agarose gel electrophoresis of lipoplexes of
DNA formulated with 24d in the absence (B) or in the presence (C) of 10 mM DTT.
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These two experiments illustrated that 24d is able to complex DNA in the same way as other
ionizable lipids in nonꢀreducing environment (the other molecules prepared previously were also
tested for comparison and showed similar behaviors, data not shown). The experimental
observations were different in the presence of reducing agent DTT, as 24d entirely lost his ability
to form lipoplex. For the other molecules of the series, the DTT treatment had no impact on the
lipoplexes (data not shown).
o Liposomal formulations
Next, we decided to investigate the behavior of disulfide 24d and diester 30 (our best
candidate) lipids used as liposomal form, where a different delivery mechanism takes place. In
this context, liposomes were prepared by mixing 24d or 30 with neutral coꢀlipid DOPE, in a 1:1
3
1
molar ratio, as previously described. The liposome preparations were then formulated with
DNA, at a charge ratio of 5, and the transfection efficiency compared to micellar formulations
(Figure 7). The results obtained showed that, as seen in Figure 3, micellar preparations of 24d
were not able to transfect DNA molecules. The results were different when the liposomal
formulation was used, as high transfection efficiency was observed with 24d:DOPE liposomes,
that reached almost the same level as the diester 30 under the micellar form. On the other hand,
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results showed that for molecule 30, formulations as micellar or liposomal form provided
approximatively the same transfection efficiency.
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Figure 7: In vitro transfection of plasmid DNA encoding Luciferase in different cells comparing
micellar (24a, 30) and liposomal (24a:DOPE, 30:DOPE) formulations. HeLa cells were
transfected with 0.5 µg of DNA formulated with each formulation at a charge ratio of 5. Data
represent mean, +/ꢀ SEM, luciferase activity in count per second (CPS) 24h postꢀtransfection for
each nanocarrier.
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•
Discussion
These sets of results coming from the study of the transfection efficiency with three different
types of nucleic acids gave us some useful information about the properties of the
aminoglycoside lipidic derivatives and their mechanism of action.
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It was observed that “parent” molecule 13 is a good and versatile transfecting agent. Indeed, it
provided good transfection potency in the case of mRNA and DNA, and was also among the best
molecules tested for gene silencing using siRNA. This strengthens the data available in the
literature about 13 and related aminoglycoside family members as efficient vectors for nucleic
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acid delivery.
In addition, the new Tobramycin lipidic derivatives described in this study allowed us to draw
some conclusions on the key structural features of this type of molecules.
Concerning the hydrophobic domain, we could observe that all modifications (with the
exception of palmityl derivative 20b in the mRNA experiments, Figure 1) led to detrimental
effects compared to the dioleyl chain present in the reference molecule 13. It can therefore be
concluded that saturated and/or shorter than C18 chains are less effective for nucleic acid
delivery. This observation is rather in good agreement with what was previously described in the
literature about other families of ionizable lipids and could be attributed to less favorable
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conditions for the endosomal escape.
The linker and hydrophobic portions of compound 30 were designed using encouraging results
from the literature with ionizable lipid DOTAP. Indeed, DOTAP, which contains ester functions
in the linker and two oleyl chains as hydrophobic part, showed higher transfection efficiency and
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reduced toxicity compared to DOTMA, its analog bearing ether functions.
In our
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experiments, the introduction of such ester functions on the linker (with similar hydrophobic
domain than in the case of 13) also led to improved transfection efficiency. This was especially
true in the case of mRNA, where 30 appeared to be 2 to 3 fold more efficient than reference
molecule 13, on the three cell types tested, leading to very high transfection.
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The fluorescence experiments using eGFP mRNA and DNA as reporter genes provided some
insights on the mechanism of this improved delivery. Indeed, the percentage of transfected cells
obtained with HeLa and fibroblasts cells for 13 and 30 were very similar. The pictures in Figure
2 clearly show that 13 was as efficient as 30 in terms of numbers of transfected cells (with
approximately 80% of transfected HeLa cells and 30% of human fibroblasts) and that both
compounds display very low cytotoxicity, with around 90% of viable cells. The improved
efficacy of 30 is therefore not related to a lower toxicity (as we could expect with results
comparing DOTMA and DOTAP). More surprisingly, it does not seem to be correlated to a
better biodegradability either. Indeed, we have performed stability studies where 13 and 30 were
mixed with porcine liver esterases and we have followed by HPLC the degradation of the
molecules. We were expecting that 30 would degrade faster than 13, due to the presence of the
ester bonds that should be cleavable by these enzymes and that the improved features of 30 could
be explained by a better biodegradability. Unfortunately, no difference could be detected in
terms of stability towards these esterases (data not shown).
The better efficiency of 30 could be explained by the fluorescence experiment on rat aorta
muscle cells. The observations of single transfected cells clearly showed that the transfection
with 30 led to a higher expression of eGFP protein compared to 13 (Figure 2C and 2F). This
seems to demonstrate that, for the same number of transfected cells, 30 is able to deliver more
copies of mRNA encoding eGFP inside the cell cytoplasm. We believe that this could result from
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an improved release mechanism of nucleic acids in the cytosol. Indeed, the fact that 30 was the
best molecule of the series both for mRNA and DNA transfection, and one of the best for
siRNA, tends to indicate that the improved feature of 30 influences the same step of the delivery
mechanism, independently of the nucleic acid transported.
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As stated in the introduction, the mechanism for the intracellular delivery of nucleic acids
mediated by ionizable lipids is composed of several distinct steps and the key endosomal escape
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step is influenced by the ionizable lipids used as delivery vehicles.
In our case, the presence
of ester bonds in the linker portion of 30, which makes it endogenous to natural lipids, may be
responsible for better hydrophobic interactions with the natural anionic phospholipids, when
compared to the other molecules of the series. The better transfection potency of 30 may be a
consequence of these improved interactions, favoring the formation of new bilayer structures
obtained from the association of the anionic lipids from the membrane and the cationic lipids
from the lipoplex. Results tend to suggest that these interactions are able to promote a more
efficient release of mRNA, DNA or siRNA from the lipoplex and from the endosome.
On the other hand, the molecule 24d ꢀ with a disulfide function in the linker structure ꢀ
provided negative results in all types of nucleic acids under micellar form; but has still allowed
us to drive some conclusions. Previous reports have demonstrated that the introduction of a
disulfide bridge in the structure of a given ionizable lipid can lead to the reduction of the lipid in
the intracellular compartment, causing the destabilization of the complex and the release of the
DNA into the cytosol. However, this improved release compared to nonꢀreducible molecules was
not necessarily associated with improved transfection efficacy in micellar complexes, but the
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effect was more pronounced in liposomal formulations.
We have shown in our study that 24d
was able to complex nucleic acids in a similar way than the other molecules of the series.
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However, in the presence of reducing agent DTT, 24d showed a quantitative release of the DNA
molecules in the gel retardation assay (Figure 6). It can therefore be envisioned that such
reduction of the disulfide bridge may also occur in the endosomal compartment, leading to the
separation of the Tobramycin headgroup and the lipophilic tail. This seems to be detrimental for
the lipid mixing mechanism, because both electrostatic and hydrophobic interactions are
necessary to trigger the disruption of the endosomal membrane by the lipid mixing mechanism.
With the lipid 24d, the nucleic acids entrapped in the endosome are therefore not released in the
cytoplasm and are degraded by fusion with lysosomes. To confirm the prevalence of the lipid
mixing mechanism in the previous tests, we performed additional experiments where 24d was
used as a liposomal form, in an equimolar formulation with the neutral fusogenic coꢀlipid DOPE
ꢀ which induces another endosomal escape mechanism. In this experiment, the 24d:DOPE
formulation proved to be effective for the intracellular delivery of plasmid DNA, while the 24d
micellar version did not transfect at all (Figure 7). The mechanism that takes place in the
presence of the coꢀlipid is different, since DOPE is known to lead to a fusion and destabilization
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of the membrane at reduced pH.
These results demonstrated that the endosomal escape is a key step for the effective delivery of
nucleic acids when using a micellar formulation. In the case of lowꢀtransfecting agents like 24d,
the ineffectiveness of the lipid mixing mechanism can be counterbalanced by the addition of a
neutral coꢀlipid able to induce an alternative fusion mechanism for the disruption of the
endosomal membrane.
Formulating a given ionizable synthetic delivery system with a neutral coꢀlipid (in our case,
DOPE) usually led to higher transfection potency compared to the micellar form, but increases
the complexity of the formulation. In our case, the results demonstrated that the micellar 30 was
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