Design, synthesis and transfection efficiency of a novel redox-sensitive polycationic amphiphile

Article abstract of DOI:10.1016/j.bmcl.2016.11.005

A novel redox-sensitive polycationic amphiphile (2S3) with disulphide linkers for nucleic acid delivery was developed. Cationic liposomes formed by 2S3 and the helper lipid DOPE demonstrated effective DNA delivery into HEK293 cells with a maximal transfection activity that is superior than both nonredox-sensitive cationic liposomes and Lipofectamine 2000 at an N/P ratio of 6/1. Redox-sensitivity was tested by experiments with extracellular glutathione which shown the ability of disulphide linker degradation. Our results suggest that polycationic amphiphile 2S3 is a promising candidate for nucleic acid delivery.

Full text of DOI:10.1016/j.bmcl.2016.11.005

Accepted Manuscript
Design, synthesis and transfection efficiency of a novel redox-sensitive poly-
cationic amphiphile
Pavel A. Puchkov, Elena V. Shmendel, Anastasya S. Luneva, Nina G.
Morozova, Marina A. Zenkova, Mikhail A. Maslov
PII:
S0960-894X(16)31145-3
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.11.005
BMCL 24400
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 September 2016
31 October 2016
1 November 2016
Please cite this article as: Puchkov, P.A., Shmendel, E.V., Luneva, A.S., Morozova, N.G., Zenkova, M.A., Maslov,
M.A., Design, synthesis and transfection efficiency of a novel redox-sensitive polycationic amphiphile, Bioorganic
& Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.11.005
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Design, synthesis and transfection efficiency
of a novel redox-sensitive polycationic
amphiphile
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Pavel A. Puchkov, Elena V. Shmendel, Anastasya S. Luneva, Nina G. Morozova, Marina A. Zenkova and
Michael A. Maslov

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Design, synthesis and transfection efficiency of a novel redox-sensitive
polycationic amphiphile
Pavel A. Puchkova , Elena V. Shmendela , Anastasya S. Lunevaa , Nina G. Morozovaa , Marina A. Zenkovab
and Mikhail A. Maslova, *
aInstitute of Fine Chemical Technologies, Moscow Technological University, Vernadsky ave. 86, Moscow 119571, Russia
bInstitute of Chemical Biology and Fundamental Medicine SB RAS, Lavrentiev ave. 8, Novosibirsk 630090, Russia
ARTICLE INFO
ABSTRACT
Article history:
Received
A novel redox-sensitive polycationic amphiphile (2S3) with disulphide linkers for nucleic acid
delivery was developed. Cationic liposomes formed by 2S3 and the helper lipid DOPE
demonstrated effective DNA delivery into HEK293 cells with a maximal transfection activity
that is superior than both nonredox-sensitive cationic liposomes and Lipofectamine® 2000 at an
N/P ratio of 6/1. Redox-sensitivity was tested by experiments with extracellular glutathione
which shown the ability of disulphide linker degradation. Our results suggest that polycationic
amphiphile 2S3 is a promising candidate for nucleic acid delivery.
Revised
Accepted
Available online
Keywords:
Cationic liposomes
Redox-sensitive
Disulphide linker
Gemini amphiphiles
Gene therapy
2009 Elsevier Ltd. All rights reserved.
Gene therapy is a tool for the treatment of both inherited and
acquired diseases by nucleic acid (NA) transfer. For this purpose,
a special vehicle is needed for NA protection against nucleases
and other damaging factors. For example, cationic liposomes
(CLs) based on cationic amphiphiles (CAs) represent a safe and
promising vehicle, which has advantages in terms of lower
immunogenicity, relative ease of production, and high carrying
capacity.1 Nevertheless, it is known that CL/NA complexes can
induce an inflammatory response involving the release of
cytokines into the serum.2,3
approach to improve delivery efficiency is to modify the CA
structure with labile stimuli-responsive groups, degradable after
the internalization of CLs into the cells. Intracellular CA
degradation may lead to NA release from liposomes/NA
complexes and a more effective endosomal escape.8
The most common linker is an acetal-type linker, degradable
under acidic pH during endosomal trafficking.8–13 A disulphide
redox-sensitive linker, degradable by intracellular reducing
agents such as glutathione (GSH)14 is another type of linker.
Recently, Yang et al. demonstrated that disulphide cationic
gemini amphiphile with two lysine polar heads possesed a high
potential for NA delivery into HeLa cells.15 Sheng et al.
synthesised a number of disulphide CAs based on cholesterol
Despite these benefits liposomes do not possess a high enough
efficiency for clinical applications because of a number of
biological barriers that reduce NA transfer into the target cells.4–6
Extracellular barriers include nucleases, serum proteins and cell
membrane while intracellular ones represent endosomal
trafficking, cytosolic transport and transfer into the nucleus.
There are different approaches to overcoming biological barriers,
for example excess positive charge of CLs provides electrostatic
interactions with negatively charged cell membranes contributing
to cellular internalisation.
which revealed
a high transfection efficiency that was
comparable with commercial agents. The most effective CAs
against COS-7 cells were compounds with a polyamine cationic
domain (L-lysine or triethylenetetramine).16 Bajaj et al.
synthesised three redox-sensitive gemini CAs based on
thiocholesterol with tertiary amine groups and various spacers.
Among them, CAs with flexible hydrophilic spacer and ether
linkers were the most effective against hard-to-transfect HaCaT
cells.17 Disulphide groups were also introduced into cationic
lipophosphoramidates, and NA delivery was shown to be
effective compared with that of Lipofectamine®.18 Disulphide
linker insertion in CA molecules may not lead to an increase in
efficiency but can substantially decrease CA toxicity, thus
A typical CA consists of hydrophilic and hydrophobic
domains, a linker connecting them, and a spacer that maintains
the sterical arrangement of domains. There may be various
combinations of the respective components.7,5 Nevertheless, each
C—A—ele—ment contributes to the resulting delivery efficiency. One
* Corresponding author. Tel.: +7-499-600-8202; fax: +7-495-434-9287; e-mail: mamaslov@mail.ru

Figure 1. Structures of polycationic monomeric and gemini amphiphiles discussed.
allowing the use of higher NA-liposome complex
concentrations.19 Dauty et al. used thiol-to-disulphide conversion
to form complexes with thiol-containing monomeric CAs and
DNA. This approach allowed to obtain very small particles that
favored nucleus entry of DNA.20 Poly(disulphide)s may be also
used in delivery of NA and other biologically active compounds
but have different delivery mechanism.21
being the most active structures.24 A disulphide linker was added
to the structure of polycationic monomeric amphiphiles (S1-S3;
Fig. 1) to improve their transfection efficiency. We found that
disulphide monomeric amphiphiles S1-S3 transfected a slightly
lower percentage of the cells but provided a much greater
transgene expression compared with analogs without similar
degradable linkers.25 Unfortunately, monomeric amphiphiles lost
their transfection efficiency in the presence of serum unlike
gemini amphiphiles which show better serum stability.21
Therefore, in this work, we designed and synthesized
polycationic gemini amphiphile 2S3 (Fig. 1) with a structure
similar to the highly effective CA 2X3 but bearing disulphide
groups. Disulphide groups degradable in the presence of reducing
agents should contribute to better NA release into the cytosol.
Synthetic protocol presented below (Fig. 2) permits protonation
of all amino groups in spermine motif that increase CA charge
and favor NA condensation. Moreover, we found appropriate
reagents which provide an effective condensation of hydrophilic
and hydrophobic domains. In addition, we evaluated the
physiochemical properties and transfection efficiency of
liposomes composed of 2S3.
Gemini amphiphiles are molecules with two (at least)
hydrophobic and two hydrophilic domains connected with
various linkers and spacers. They may have symmetrical or
unsymmetrical structure and possess a number of significantly
enhanced surface properties as compared with their monomeric
analogs. Their much lower CMC (critical micelle concentration)
and ability to reduce surface tension make gemini amphiphiles an
attractive tool for biological applications including gene
therapy.22,23 Recently, we developed a number of polycationic
monomeric and gemini amphiphiles based on cholesterol and
natural polyamine spermine. All of the CAs demonstrated high
transfection efficiency on different cell lines.24,25 However,
gemini amphiphiles were transfected more effectively in
comparison with their monomeric counterparts, with CAs bearing
a hexamethylene spacer and a carbamoyl linker (2X3; Fig. 1)
To synthesise amphiphile 2S3, we obtained the spermine
Figure 2. Synthetic route of 2S3. a) CF3COOEt, MeOH, 70 °C, 5 h, next Boc2O, Et3N, 24 °C, 96 h, next NaOH, MeOH, 24 °C, 48
h, next 2-NO2C6H4SO2Cl, Et3N, DCM, 24 °C, 5 h;23 b) BrCH2COOEt, Cs2CO3, DMF, 65 °C, 30 h; c) PhSH, K2CO3, DMF, 24 °C,
1 h; d) Boc2O, Et3N, DCM, 24 °C, 48 h; e) NaOH, MeOH, 24 °C, 96 h; f) NH2(CH2)2SS(CH2)2NH2, Et3N, DCM, for 6a 22 °C, 2 h,
for 6b 40 °C, 45 h; g) 5, EEDQ, DIEA, DCM, 50 °C, 48 h; h) 3 M HCl/dioxane, DCM, 22 °C, 24 h.

derivative (2) by regioselective protection of spermine (1),
previously described.26
Table 1. Optimisation of the synthesis of 8.
Yielda of
8, %
9
Reagents
Solvent
The alkylation of 2 with ethyl bromoacetate in the presence of
cesium carbonate in Fukuyama reaction conditions27,28 produced
a good yield of compound 3 (90 %). The removal of 2-
nitrobenzenesulfonyl protecting group by treatment with
thiophenol in the presence of potassium carbonate gave amine 4,
which was isolated by column chromatography with 63 % yield.
Secondary amino groups were blocked with Boc2O in the
presence of TEA at 0 °С, the resulting fully protected spermine
derivative was treated with NaOH in methanol-water solution to
give compound 5 with two-step yield of 60 %.
1
2
3
4
5
5, TBTU, DIEA
DMF
DMF
5, DMTMM, DIEA
5, DMTMM, DIEA
n.d.b
Et O
22
2
5, DMTMM, NMM MeOH:THF=5:1
5, EEDQ, DIEA CH2Cl2
30
69
aIsolated yield;
bTLC indicates no product forming.
we used NMM as a base. As expected, the additional amount of
NMM increased the reaction yield (Table 1, run 4). EEDQ is
another condensing reagent, which can be used with chlorinated
solvents including CH2Cl2 that made reaction conditions
completely homogeneous. Employment of EEDQ produced
compound 8 with maximal yield (Table 1, run 5). The removal of
Вос protecting groups by 3 М HCl in dioxane produced the
target CA 2S3 with 91 % yield after recrystallisation from
ethanol and ether.
There are different approaches to introduce a disulphide group
into a target molecule, such as the conversion of hydroxyl or
amino groups into thiols, followed by a mild oxidation or using
of bifunctional reagents.29 We used an approach based on the
reaction between cystamine (2,2′-diaminodiethyl disulfide) and
activated target cholesterol precursor. The activation may be
performed by various reagents,
for
example N-
hydroxysuccinimide30 or N,N’-disuccinimidyl carbonate.31,32 We
used two activated derivatives, namely cholesterol chloroformate
6a and (cholest-5-en-3β-yl) imidazole-1-carboxylate 6b33 (Fig.
2). Commercially available cystamine dihydrochloride was
transformed into a free base by treatment with aqueous NaOH to
improve its solubility in organic solvents. The use of the more
active cholesterol chloroformate (6a) led to a rapid and complete
conversion under mild conditions. In this case, disulphide
lipophilic derivative 7 was obtained in 66 % yield, which
exceeded that of reaction with (cholest-5-en-3β-yl) imidazole-1-
carboxylate 6b (55 %), as well as literature data (40-50 %).30–32
Based on the redox-sensitive CA 2S3 and zwitter-ionic helper
lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine)
2S3 CLs were prepared at lipid molar ratio of 1:1 by the thin film
hydration method. The average diameter of CLs studied by
dynamic light scattering was about 90 nm (Table 2).
2S3 CLs were used for the delivery of a pEGFP-C2 plasmid,
which encodes enhanced green fluorescent protein (EGFP), into
HEK293 cells at various N/P ratios (number of polycationic
amino groups of CAs per phosphate group of nucleic acids).
CL/pDNA complexes formed at an N/P ratio of 2/1 had
diameters above 300 nm and a negative ξ-potential (Table 2).
Increasing the amount of CLs in the complexes led to a decrease
in average hydrodynamic diameter and polydispersity index as
well as changing the ξ-potential from negative to positive. These
changes indicated better complex stabilisation due to more
complete DNA condensation at the surface of the CLs through
N/P increasing.39,40
A key step of the synthesis was the condensation of spermine
(5) and cholesterol (7) derivatives. It is known that disulfide
compounds may be obtained with moderate overall yields.18,34
Possible explanations may include their lower solubility in
organic solvents as compared with non-disulfide molecules or
eventual disulfide bond degradation under some conditions.
Nevertheless, disulphide amphiphile syntheses with high yields
were also described,16,35 demonstrating the importance of
optimizing reaction conditions. We used three condensation
The cytotoxicity of cationic liposomes was evaluated by a
MTT assay in HEK293 cells. Cells were incubated with cationic
liposomes at concentrations ranging from 1 to 80 µM for 24 h in
the presence of serum. The results was presented as IC50 values
(concentration of the liposomes providing 50% cell viability), the
CLs of 2X3 and 2S3 had IC50 37 µM and 66 µM, respectively.
We suggest that the lower cytotoxicity of 2S3 CLs can be
explained by better biodegradability caused by the presence of
disulphide groups.. IC50 values for Lipofectamine® 2000 (Lf
2000) was determined in µL since the chemical composition of
this proprietary formulation is not disclosed. IC50 for Lf 2000 was
8.55 µL as shown previously.24
agents:
TBTU
(2-(1H-benzotriazole-1-yl)-1,1,3,3-
tetramethyluronium tetrafluoroborate), DMTMM (4-(4,6-
dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride)
and EEDQ (2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline).
TBTU is commonly used in the peptide synthesis as well as
disulfide amphiphile synthesis;36 however, it provided a very low
yield of 8 (Table 1, run 1) because of heterogeneous reaction
conditions caused by a limited range of appropriate solvents. The
other reagent using in amide and ester syntheses is DMTMM.37
A
disadvantage of DMTMM is its decomposition in chlorinated and
some other solvents. We carried out reactions with DMTMM
under several conditions, and the best yield was 30 % (Table 1,
runs 2-4). According to reaction mechanism, the presence of
NMM normally liberated from DMTMM during reaction pass
leads to rapid and selective amide formation.38 Considering this,
The results of plasmid DNA (pDNA) delivery experiments
monitored by flow cytometry are summarised in Table 3.
Nonredox-sensitive 2X3 CLs and Lf 2000 were used as a positive
Table 2. Physicochemical characterisation of CLs.
2X3
2S3
CL/ pEGFP-C2
Diameter,
mean, nm
92.4
377.4
171.6
130.8
ξ-potential,
mV
22.1
Diameter,
mean, nm
91.5
350.9
98.1
82.5
ξ-potential,
mV
27.9
PIa
PIa
1/0
2/1
4/1
6/1
0.374
0.355
0.200
0.250
0.332
1.0
0.247
0.253
-26.2
7.0
15.4
-6.5
9.4
18.7
aPI: polydispersity index.

In this study, a novel redox-sensitive polycationic gemini
amphiphile 2S3 was synthesised for DNA delivery, and synthetic
procedures were optimised. The transfection activity of 2S3 CLs
was high in comparison with that of both nonredox-sensitive 2X3
CLs and the commercial transfectant Lipofectamine® 2000. The
redox sensitivity of 2S3 was demonstrated by GSH treatment.
Therefore, 2S3 CLs are a potential vehicle for DNA delivery.
DNA release may be triggered by reducing agents (GSH) that
degrade disulphide bonds.
Table 3. Transfection efficacy of CL/pDNA complexes.
2X3
2S3
Lf
2000
N/P
2/1
4/1
6/1
2/1
4/1
6/1
Fluorescent
cells, %
Mean
44.5
14.5
17.6
52.3
78.3
16.0
52.4
64.2
fluorescence
intensity,
RFU
4.0
17.6
18.6
4.0
11.9
22.0
*The standard deviation did not exceed 7%–9%.
Acknowledgments
This work was supported by the President's Program In
Support Of Leading Scientific Schools SS-7946.2016.11.
Supplementary Material
Supplementary data associated with this article can be found,
in the online version, at
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