Cationic lipids bearing succinic-based, acyclic and macrocyclic hydrophobic domains: Synthetic studies and in vitro gene transfer

Article abstract of DOI:10.1016/j.ejmech.2016.09.027

In this communication we describe the construction of four succinic-based cationic lipids, their formulation with plasmid DNA (pDNA), and an evaluation of their in vitro gene delivery into Chinese hamster ovarian (CHO-K1) cells. The cationic lipids employed in this work possess either a dimethylamine or trimethylamine headgroup, and a macrocyclic or an acyclic hydrophobic domain composed of, or derived from two 16-atom, succinic-based acyl chains. The synthesized lipids and a co-lipid of neutral charge, either cholesterol or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), were formulated in an overall 3:2 cationic-to-neutral lipid molar ratio, then complexed with plasmid DNA (pDNA). The relative transfection performance was evaluated via a comparison between matched versus mismatched formulations defined by the rigidity relationship between the lipids employed. Gel electrophoresis was used to characterize the binding of the lipid formulations with plasmid DNA and the relative degree of plasmid degradation using a DNase I degradation assay. Small angle X-ray diffraction (SAXD) was employed to characterize the packing morphology of the lipid-DNA complexes. In general, the succinic unit embedded within the hydrophobic domain of the cationic lipids was found to improve lipid hydration. The transfection assays revealed a general trend in which mismatched formulations that employed a rigid lipid combined with a non-rigid (or flexible) lipid, outperformed the matched formulations. The results from this work suggest that the design of the cationic lipid structure and the composition of the lipoplex formulation play key roles in governing the transfection performance of nonviral gene delivery agents.

Full text of DOI:10.1016/j.ejmech.2016.09.027

Accepted Manuscript
Cationic lipids bearing succinic-based, acyclic and macrocyclic hydrophobic domains:
Synthetic studies and in vitro gene transfer
Emile Jubeli, Amanda B. Maginty, Nada Abdul Khalique, Liji Raju, David G.
Nicholson, Helge Larsen, Michael D. Pungente, William P.D. Goldring
PII:
S0223-5234(16)30757-7
10.1016/j.ejmech.2016.09.027
EJMECH 8893
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 February 2016
Revised Date: 13 May 2016
Accepted Date: 9 September 2016
Please cite this article as: E. Jubeli, A.B. Maginty, N.A. Khalique, L. Raju, D.G. Nicholson, H. Larsen,
M.D. Pungente, W.P.D. Goldring, Cationic lipids bearing succinic-based, acyclic and macrocyclic
hydrophobic domains: Synthetic studies and in vitro gene transfer, European Journal of Medicinal
Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.027.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Cationic lipids bearing succinic-based, acyclic and macrocyclic
hydrophobic domains: synthetic studies and in vitro gene transfer
Emile Jubelia, Amanda B. Magintyb, Nada Abdul Khaliquec, Liji Rajuc, David G.
Nicholsond, Helge Larsene, Michael D. Pungentef,* and William P. D. Goldringb,1,*
aUniversité Paris-Sud, EA 401, IFR 141, Faculté de pharmacie, F-92296 Châtenay Malabry,
France
bSchool of Chemistry and Chemical Engineering, Queen’s University Belfast, David Keir
Building, Stranmillis Road, Belfast, Northern Ireland, BT9 5AG, United Kingdom
cResearch Division, Weill Cornell Medicine in Qatar, Education City, P.O. Box 24144, Doha,
Qatar
dDepartment of Chemistry, Norwegian University of Science and Technology (NTNU), 7491
Trondheim, Norway
eDepartment of Physics, University of Stavanger, 4036 Stavanger, Norway
fPremedical Unit, Weill Cornell Medicine in Qatar, Education City, P.O. Box 24144, Doha,
Qatar
* Corresponding authors. Tel.: +45-4674-2308; e-mail: goldring@ruc.dk (W. P. D. Goldring).
Tel.: +974-4492-8216; fax: +974-4492-8222; e-mail: mdp2001@qatar-med.cornell.edu (M. D.
Pungente).
Keywords: macrocyclic lipids; cationic; succinic; lipoplex formulation; in vitro DNA transfer
1Present address: Department of Science and Environment, Roskilde University, PO Box 260,
Universitetsvej 1, DK-4000 Roskilde, Denmark
Abbreviations: pDNA, plasmid DNA; CHO-K1, Chinese hamster ovarian cells; DMA,
dimethylamine; TMA, trimethylamine; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine;
Chol, cholesterol; EPC, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine; SAXD, small angle
X-ray diffraction; DCC, N,N’-dicyclohexylcarbodiimide; DMAP, 4-(dimethylamino)pyridine;
THF, tetrahydrofuran; DMF, dimethylformamide; DLS, dynamic light scattering; PDI,
polydispersity index; RT, room temperature.
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ABSTRACT
In this communication we describe the construction of four succinic-based cationic lipids,
their formulation with plasmid DNA (pDNA), and an evaluation of their in vitro gene delivery
into Chinese hamster ovarian (CHO-K1) cells. The cationic lipids employed in this work possess
either a dimethylamine or trimethylamine headgroup, and a macrocyclic or an acyclic
hydrophobic domain composed of, or derived from two 16-atom, succinic-based acyl chains. The
synthesized lipids and a co-lipid of neutral charge, either cholesterol or 1,2-dioleoyl-sn-glycero-
3-phosphoethanolamine (DOPE), were formulated in an overall 3:2 cationic-to-neutral lipid
molar ratio, then complexed with plasmid DNA (pDNA). The relative transfection performance
was evaluated via a comparison between matched versus mismatched formulations defined by
the rigidity relationship between the lipids employed. Gel electrophoresis was used to
characterize the binding of the lipid formulations with plasmid DNA and the relative degree of
plasmid degradation using a DNase I degradation assay. Small angle X-ray diffraction (SAXD)
was employed to characterize the packing morphology of the lipid-DNA complexes. In general,
the succinic unit embedded within the hydrophobic domain of the cationic lipids was found to
improve lipid hydration. The transfection assays revealed a general trend in which mismatched
formulations that employed a rigid lipid combined with a non-rigid (or flexible) lipid,
outperformed the matched formulations. The results from this work suggest that the design of the
cationic lipid structure and the composition of the lipoplex formulation play key roles in
governing the transfection performance of nonviral gene delivery agents.
1. Introduction
The application of cationic lipids as nonviral gene delivery vectors continues to be an
area of significant interest. Since the development of the earliest examples of synthetic cationic
lipids as gene transfer reagents some three decades ago [1], many others have since been
reported [2-5]. While some lipid designs focused on modifications to the headgroup structure [6-
10], others investigated the impact of changes to the backbone structure [11-13]. Structure
modifications within the hydrophobic domain have focused mainly on the length of the carbon
chains and the presence or absence of alkene units [14,15]. However, such structural changes
have not had a consistent or predictable influence on transfection efficiency. Furthermore, few
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reports appear in the literature which describe the impact of gene carriers that include polar
functionality in the hydrophobic domain. Notable examples include bile acid-oligopeptide
conjugates [16], cyclodextrins [17], and the synthesis of a macrocyclic polyether that has not yet
been the subject of transfection studies [18]. Therefore, the inclusion of a succinic unit within the
hydrophobic domain of a cationic lipid, previously employed in polymeric vectors [19,20] and
sterol-modified phospholipids [21], presents a unique opportunity to study the impact this polar
group has, when formulated with plasmid DNA (pDNA), on the safe and efficient delivery of
genetic material into cells.
As part of our ongoing research program towards the development and screening of novel
nonviral vectors for gene delivery, we set out here to expand on our repertoire of previously
reported cationic lipids characterized by non-rigid and rigid hydrophobic domain structures.
Attempts to formulate our previous library of synthetic cationic lipids, based on acyclic or
macrocyclic hydrophobic domains, into liposomes required a co-cationic lipid, 1,2-dimyristoyl-
sn-glycero-3-ethylphosphocholine (EPC) to aid hydration [22]. The novel lipids reported here
were designed with polar succinic units embedded within the hydrophobic domain, near to the
glycerol backbone, in an attempt to improve lipid hydration and thus remove the need for a co-
cationic lipid. Accordingly, a small group of succinic-based cationic lipids was constructed, of
which some members were successfully formulated into lipid-DNA complexes (lipoplexes) and
evaluated for their potential to deliver genetic material into cells in culture.
Herein we describe the construction of four cationic lipids, 1-4, based on either an acyclic
(non-rigid) or macrocyclic (rigid) hydrophobic domain, each featuring an embedded succinic
unit and either a dimethylamine (DMA) or trimethylamine (TMA) headgroup (Fig. 1).
Furthermore, an investigation was conducted to evaluate the impact that structural and
formulation differences between the lipids employed in the lipoplexes has towards transfection
efficiency and cytotoxicity. Three of the synthetic lipids, 2-4, each combined with a co-lipid of
neutral charge, namely the non-rigid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or
the rigid cholesterol (Chol), in a 3:2 cationic lipid/co-lipid molar ratio were hydrated giving a
total of six liposome combinations. The six corresponding lipoplexes, formed when the
liposomes were mixed with pDNA, were evaluated for binding and protection from DNase I
degradation via gel electrophoresis. The packing morphology of selected lipoplexes was
determined by small angle X-ray diffraction (SAXD). Finally, in vitro assays were performed in
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cell culture using Chinese hamster ovarian (CHO-K1) cells to evaluate the relative transfection
efficiency and cell viability of all six lipoplex formulations, using the well-known commercial
formulation Lipofectamine 2000TM as a positive control. The lipoplex formulations were
organized into two groups according to a matched or mismatched relationship between the
hydrophobic domain structures associated with both the synthetic cationic lipid and co-lipid
employed. The structures of the cyclic lipids 2 and 4, and co-lipid cholesterol, lack
conformational freedom and were therefore considered rigid relative to the structures of the
acyclic lipid 3, and the co-lipid DOPE, which exhibit conformational freedom and were
considered non-rigid (Fig. 1). Herein, a matched formulation refers to one in which the synthetic
lipid and co-lipid were either both rigid (e.g. 2/Chol) or both non-rigid (e.g. 3/DOPE).
Conversely, a mismatched formulation refers to a combination of a rigid and a non-rigid lipid
(e.g. 2/DOPE or 3/Chol). From this investigation our main finding was a significant correlation
between the relative transfection efficiencies and the structural rigidity differences between the
cationic lipid and co-lipid employed within each formulation. Among the six formulations, those
based on a rigid lipid “mismatched” with a flexible (or non-rigid) lipid partner exhibited the
highest transfection performance. This applied whether the rigid lipid was cationic and the non-
rigid lipid was of neutral charge, and vice versa. Conversely, “matched” lipid formulations
exhibited the lowest transfection performance.
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Figure 1. The structures of (non-rigid) succinic-based acyclic DMA and TMA cationic lipids 1
and 3, and the corresponding (rigid) macrocyclic analogues 2 and 4; as well as the commercial
(non-rigid) zwitterionic co-lipid DOPE and (rigid) neutral co-lipid cholesterol.
2. Results
2.1. Synthesis of the succinic-based cationic lipid series
Our planned synthesis of lipids that possess succinic units embedded in the hydrophobic
domain, designed to improve lipid hydration, was based on a route we previously developed for
the construction of an acyclic and macrocyclic cationic lipid library [22]. The synthetic route,
which employed solketal (8) as the glycerol backbone and starting material, involved;
installation of the headgroup linker unit, deprotection and installation of the two hydrophobic
acyl groups, macrocyclization, and finally installation of the DMA or TMA headgroup. Our
synthetic studies began with the preparation of a succinic acid mono-ester for use in the
construction of the target lipids. Reduction of 10-undecenoic acid (5) using lithium aluminum
hydride in tetrahydrofuran (THF) gave the alcohol 6 in 65% yield (Scheme 1A). Succinic
anhydride was then treated with 4–(dimethylamino)-pyridine (DMAP) and the alcohol 6 in
CH2Cl2 to give the mono-ester 7 in 89% yield [23,24]. Next, we pursued the construction of a
small library of unsaturated (U), acyclic (A) or cyclic (C) cationic lipids 1-4 that possess a
succinic (Succ) unit in the hydrophobic domain, derived from the mono-ester 7, and either a
DMA or TMA headgroup. For example, construction of the unsaturated, acyclic cationic lipid 1
(UA11Succ DMA) started from the diol 10, which was obtained in two steps from solketal (8)
via the ester 9 [22a]. Accordingly, the reaction between the diol 10 and two equivalents of the
mono-ester 7, using N,N’-dicyclohexylcarbodiimide (DCC) and DMAP in CH2Cl2, gave the tri-
ester 11 in 89% yield (Scheme 1B). Treatment of the alkyl bromide 11 with an excess of
dimethylamine in THF, followed by treatment of the resulting amine with a 2 M solution of HCl
in diethyl ether gave the cationic lipid 1 (UA11Succ DMA) in 97% yield. Toward the TMA-
based lipid, the bromide 11 was treated with a 45% aqueous solution of trimethylamine in
chloroform (CHCl3), isopropanol (iPrOH) and dimethylformamide (DMF) to give the quaternary
ammonium cationic lipid 3 (UA11Succ TMA) in quantitative yield. Macrocyclization of the
diene 11 using 10 mol% of Grubbs’ 2nd generation pre-catalyst in CH2Cl2 at 45 °C gave an
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inseparable 1.6:1 mixture of the (E)- and (Z)-isomers 12 in 64% combined yield. Treatment of
the isomeric mixture of alkenes 12 with either dimethylamine, or separately trimethylamine,
under the conditions described previously, led to the cationic lipids 2 (UC11Succ DMA) or 4
(UC11Succ TMA), in 41% and 97% yield, respectively.
Scheme 1. Synthesis of the succinic-based (A) mono-ester 7, and (B) acyclic and macrocyclic,
DMA and TMA cationic lipids 1-4. Reagents and conditions: (i) LAH, THF, 0 °C, 65%; (ii)
succinic anhydride, DMAP, CH2Cl2, 45 °C, 89%; (iii) 7 (2 equiv.), DCC, DMAP, CH2Cl2, RT,
89%; (iv) 10 mol% Grubbs’ 2nd generation pre-catalyst, CH2Cl2, 45 °C, 64% (E/Z, 1.6:1); (v) a. 2
M Me2NH in THF, RT; then b. 2 M HCl in diethyl ether, RT, 97% (1: UA11Succ DMA) or 41%
(2: UC11Succ DMA) (2 steps); (vi) 45% (v/v) Me3N in water, CHCl3, iPrOH and DMF, RT,
quantitative (3: UA11Succ TMA) or 97% (4: UC11Succ TMA).
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Construction of the four succinic-based cationic lipids, 1-4, which possess an acyclic or
macrocyclic hydrophobic domain structure, and either the DMA or TMA headgroup, set the
stage for their evaluation as gene delivery vehicles. This work involved formulation, biophysical
characterization, including SAXD studies, and in vitro assays performed using CHO-K1 cells to
assess the relative transfection efficiency and cytotoxicity of the synthetic lipid-DNA
formulations.
2.2. Lipoplex formulation and particle sizing
Liposomes based on a synthetic cationic lipid, 2-4, were formulated with either DOPE or
cholesterol as a co-lipid in a 3:2 cationic to neutral molar ratio. The liposomes were prepared by
sonication of a hydrated film, which was formed by concentration of an ethanolic solution of the
lipid mixture. Unfortunately, in our hands we were unable to hydrate a thin film composed of the
synthetic cationic lipid 1 combined with either DOPE or cholesterol as co-lipid. Lipoplexes were
prepared from the liposomes and pDNA at various N:P (+/-) molar charge ratios, ranging from
0.5:1 to 10:1.
Liposome particle size data recorded for the cationic lipid/co-lipid formulations was
obtained by dynamic light scattering (DLS). The data revealed a liposome diameter range of
approximately dH = 170 to 1300 nm, together with polydispersity index (PDI) values ranging
from 0.3 to 1.0 (see SI Table S1). Furthermore, a trend of increasing liposome size, on the order
of 2 < 4 < 3, regardless of the co-lipid employed, was observed. With respect to the co-lipid,
liposomes formulated with cholesterol were generally larger than those with DOPE. A particle
size diameter range of the synthetic lipid/co-lipid/pDNA lipoplexes, at various N:P (+/-) molar
charge ratios, was recorded between dH = 180 to 6600 nm (see SI Table S2). In general, among
the formulations based on the cyclic lipid 2, the size of the lipoplex increased with the N:P (+/-)
molar charge ratio. However, the lipoplex particle size of formulations based on the lipids 3 or 4
followed a Gaussian distribution that reached a maximum at N:P (+/-) molar charge ratio 3.
2.3. Small angle X-ray diffraction analyses of selected lipoplex formulations
In order to elucidate structural information, such as packing morphology and interlayer
distances, SAXD experiments were carried out at the BioSAXS beamline, BM29, at the
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European Synchrotron Radiation Facility (ESRF), Grenoble, France. The highly automated setup
offered by this beamline provides a monochromatic X-ray beam of energy 12.5 keV
(corresponding to a wavelength of 0.992 Å). Further details of the data acquisition and the
subsequent analysis are given in the supplementary material. The main results are summarized in
Figure 2 and Table 1. The lipoplex formulations exhibited lamellar ordering with interlayer
distances in the range of 63-68 Å.
Table 1. Packing morphology and interlayer distance δ (Å) of succinic-based lipid 3 and
4/DOPE and Chol/DNA lipoplex formulationsa based on the small angle X-ray diffraction
analysesb
Formulation
Type
δ (Å) packing
morphology
Lα *
Lα
Lα
3/Chol/DNA
4/Chol/DNA
3/DOPE/DNA
4/DOPE/DNA
Mismatched
Matched
63±1
66±1
67±2
68±2
Matched
Lα
Mismatched
a
Formulations at N:P (+/-) molar charge ratio of 3:1.
b
δ = interlayer distance, Lα = lamellar. *There are, for this formulation, indications of another superimposed
simultaneously occurring lamellar packing regime having a repetition distance of approximately 69 Å.
Figure 2. SAXD curves recorded for synthetic lipid 3 or 4/DOPE or cholesterol/DNA lipoplexes
at a N:P (+/-) molar charge ratio = 3:1. The ordinate axis: intensity in arbitrary units (curves
shifted for clarity). Abscissa axis: the modulus of the scattering vector (denoted as s).
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2.4. Gel retardation assay of lipoplex (liposome-DNA) formulations
The binding interaction between each of the synthetic cationic lipids, 2-4, and pDNA was
assessed using a gel retardation assay. In general, the DNA binding improved with increasing
N:P (+/-) molar charge ratio (see SI Fig. S1). Near complete retention was achieved at N:P (+/-)
= 10 for the formulations based on the cationic lipid 2, and at N:P (+/-) = 5 for the formulations
based on the cationic lipids 3 or 4.
2.5. DNase I degradation assay of the lipoplex formulations
A DNase I degradation assay was employed to evaluate the protection of the lipid-
associated pDNA lipoplex formulations from nucleases. The lipoplexes, in general, offered some
degree of pDNA protection from the nuclease. The formulations that employed cholesterol as co-
lipid offered improved pDNA protection, with increasing N:P (+/-) molar charge ratio, which
was better in general than the protection offered by the DOPE-based formulations that reached a
maximum at N:P (+/-) = 3 for all cationic lipids (see SI Fig. S2). Surprisingly, at high molar
charge ratios, the formulations that employed DOPE as co-lipid were associated with weaker
DNA protection despite complete DNA binding evidenced by the gel retardation assay. Overall,
the formulation that offered the highest degree of pDNA protection was based on the cationic
lipid 3 with cholesterol as co-lipid.
2.6. Relative transfection efficiencies
In our previous work we analyzed the relative transfection efficiency of formulations
based on structural differences in the hydrophobic domain and headgroup structure of the
synthetic cationic lipid [22]. In the present work, analysis along these lines involved comparisons
between synthetic lipids that possessed acyclic or non-rigid (lipid 3) and cyclic or rigid (lipids 2
and 4) hydrophobic domain structures, and DMA (lipid 2) and TMA (lipid 4) headgroup
structures. The transfection efficiency data reported herein, for each lipoplex formulation,
represents the average of six duplicate assays, performed over two sets of experiments, each in
triplicate. Initial inspection of the transfection efficiency data recorded for the formulations that
employed the synthetic lipids 2-4 and DOPE as co-lipid revealed the formulations based on the
cyclic lipids outperformed the formulation based on the acyclic lipid (Fig. 3A). In contrast, when
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using formulations based on the synthetic lipids 2-4 and cholesterol as the co-lipid the opposite
trend was observed (Fig. 3B). That is, when cholesterol was employed as co-lipid the
formulation based on the acyclic lipid outperformed the formulations based on the cyclic lipids.
(A)
(B)
Figure 3. Transfection efficiency (bars) and cytotoxicity ( ) of succinic-based lipids 2-4 (48 h
after incubation with CHO-K1 cells) using 3:2 synthetic lipid:co-lipid / DNA lipoplexes (A: co-
lipid = DOPE; and B: co-lipid = cholesterol) at various N:P (+/-) molar charge ratios, ranging
from 0.5:1 to 10:1, compared to Lipofectamine 2000TM/DNA (L2k) as a positive control, and
plasmid DNA alone (D) and CHO-K1 cells alone (C) as negative controls (n=6; mean ± SE). The
transfection efficiency of all formulations within each set appears as a relative percentage of the
Lipofectamine 2000TM/DNA control.
A closer inspection of the cyclic versus acyclic hydrophobic domain structure analysis
revealed a significant, general trend governed by the matched (rigid paired with rigid, or non-
rigid paired with non-rigid) or mismatched (rigid paired with non-rigid) relationship between the
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synthetic cationic lipid and co-lipid employed in each formulation. Statistical analysis of the data
illustrated in Figure 3 revealed the mismatched formulations significantly outperformed the
matched formulations when using either DOPE (p = 0.013) or cholesterol (p = 0.003) as co-lipid
(Fig. 4A). Furthermore, across all formulations, and irrespective of the co-lipid employed, a
general transfection efficiency trend emerged in which the mismatched formulations
significantly outperformed the matched formulations (p <0.001).
A statistical analysis of the transfection efficiency data based on structural differences in
the DMA (lipid 2) or TMA (lipid 4) headgroup, in which all other cationic lipid structural
features were identical, revealed the DMA-based lipid formulations significantly outperformed
the TMA-based lipid formulations when cholesterol was employed as co-lipid (p = 0.031) (Fig.
4B). However, the higher relative performance of the DMA-based lipid formulations was not
statistically significant when using the co-lipid DOPE (p = 0.19). Taken together across all
formulations, a general transfection efficiency trend emerged in which the DMA-based lipid
formulations significantly outperformed the TMA-based formulations (p = 0.030). The
comparison between formulations, based on the headgroup structure and co-lipid employed,
revealed trends that were consistent with those observed in our previous study of lipoplex
formulations based on a cationic lipid library characterized by macrocyclic and acyclic, saturated
and unsaturated hydrophobic domain structures derived from either 7-carbon or 11-carbon acyl
units, a glycerol backbone and a DMA- or TMA-based headgroup [22a].
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(A)
(B)
Figure 4. Calculated means for transfection efficiency data, as a relative percentage of the
Lipofectamine 2000TM control based on formulation sub-groups according to (A) mismatched
(black bars; 2 or 4/DOPE and 3/Chol) or matched (white bars; 2 or 4/Chol and 3/DOPE)
formulation, and (B) DMA (black bars) or TMA (white bars) headgroup structure. Significant
differences in paired Student's t-test p-values were expressed as follows: *p < 0.05; ** p < 0.01
and *** p < 0.001.
2.7. Relative cytotoxicity
The cytotoxicity of the lipoplex formulations generally increased with increasing lipid
content (i.e. N:P (+/-) molar charge ratio). The cytotoxicity data reported herein, for each
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lipoplex formulation, represents the average of six duplicate assays, performed over two sets of
experiments, each in triplicate. Statistical analyses were performed to evaluate the relative
cytotoxicity of the formulations based on structural differences between the synthetic cationic
lipids, such as cyclic versus acyclic hydrophobic domain structure and DMA versus TMA
headgroup structure, together with the matched versus mismatched relationships between the
lipids employed in the formulations. A statistical analysis of the cell viability data based on
structural differences between the cyclic versus acyclic lipid structure (p = 0.43), or DMA versus
TMA headgroup structure (p = 0.33) did not reveal a significant trend across all formulations. In
contrast, statistical analysis of the formulations based on the co-lipid employed revealed the cell
viability of the mismatched formulations was significantly higher than that of the matched
formulations when using cholesterol as co-lipid (p = 0.016). Across all formulations a general
trend emerged in which the cell viability of mismatched formulations was significantly higher
than that of the matched formulations (p = 0.008).
3. Discussion
The present study aimed to compare the relative transfection efficiency and cytotoxicity
of the succinic-based cationic lipids, 1-4, in formulation, based on differences in the hydrophobic
domain structure, such as cyclic versus acyclic, and in the headgroup, such as DMA versus
TMA. Our previous studies indicated these, and other lipid structural features, can impart a
significant impact on the performance of the lipoplex formulation [22]. The successful
construction of the cationic lipids, 1-4, was based on a synthesis route previously developed in
our laboratory, and required the assembly of a 16-atom succinic-based acyl unit for installation
onto the glycerol backbone. Once installed, the embedded succinic unit did not present any
challenges or compatibility issues with the chemistry employed to form the macrocyclic
hydrophobic domain or install the headgroups used in this study. The succinic-based lipids,
which were designed to achieve greater hydration, were readily formulated into liposomes, with
the exception of lipid 1, and subsequently lipoplexes with a co-lipid, DOPE or cholesterol.
Finally, analysis of the transfection efficiency and cytotoxicity data was based on comparisons
between the matched and mismatched lipoplex formulations, defined by the structure rigidity
relationship between the two lipids employed.
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The liposomes, composed of a cationic lipid and a co-lipid, were prepared from thin films
using standard hydration protocols. In our hands, it was not possible to hydrate the cationic lipid
1 in the presence of either co-lipid employed in this study, and therefore it was not employed in
the subsequent physicochemical studies and assays. In general, the liposomes increased in size
based on the lipid employed, e.g. 2 ꢀ 4 ꢀ 3, and the liposomes formulated with cholesterol
were generally larger than those with DOPE. The lipoplexes were prepared from the liposomes
by addition of pDNA at different N:P (+/-) molar charge ratios. We observed particle size trends
which were specific to each cationic lipid employed, however the trends did not correlate with
the transfection or cytotoxicity data recorded for the formulations. The gel retardation and DNase
I degradation assays revealed, in general, improved pDNA binding and protection at higher N:P
(+/-) molar charge ratios irrespective of the lipids employed in the formulation. These results are
consistent with the trends observed with most other cationic lipids reported in the literature,
including our previous results [22], and therefore indicate that the succinic unit does not impact
upon either pDNA binding or its protection in the lipoplex formulations. Finally, the SAXD
results reveal that each lipoplex formulation exhibited lamellar ordering, commonly found in
lipoplex packing, which offers greater particle stability when compared with hexagonally packed
systems. We note that the lipoplexes which employed DOPE as co-lipid gave rise to less ordered
structures than the corresponding lipoplexes that employed cholesterol. The cholesterol-based
formulations exhibited a denser packing. The SAXD-signature of the mismatched lipoplex
3/Chol/DNA appears to be consistent with the presence of a minor fraction of (lamellar) particles
having a repetition distance of approximately 69 Å, as compared to 63 Å for the majority of
particles. However, further investigations are required to determine if such mixed particle
morphologies play any role in governing the higher relative transfection performance we
observed with the mismatched lipoplex formulations over the matched formulations.
The current lipid design, based on an embedded succinic unit, led to improved lipid
hydration, which is in contrast to that shown by some of the cationic lipids we previously
reported [22]. However, the relative transfection efficiency was not enhanced. For example, the
transfection efficiency of each lipoplex formulation studied in this work was lower than that of
the positive control Lipofectamine 2000TM. In fact, the formulation that showed the best
performance was the acyclic lipid 3/Chol / pDNA lipoplex at N:P (+/-) = 3, which exhibited 60%
relative Lipofectamine 2000TM transfection. This formulation is associated with the highest
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degree of pDNA protection, which may explain, in part, its strong transfection performance
relative to the other formulations. In a study of related surfactant structures, Sherrington
suggested dipolar interactions between the succinic carbonyl groups and the cationic headgroup
could lead to charge delocalization [24]. It is unclear whether such an interaction occurred within
the cationic lipids at some stage during transfection, and thus whether it could account for the
lower relative transfection. However, the gel retardation assay indicated a lipid-DNA binding
interaction was achieved (see SI Fig. S1). Alternatively, a previous report suggested that the
orientation of one or both of the ester bonds in a succinic unit could have a profound impact on
transfection efficiency, which may account for the lower relative transfection of the formulations
based on the cationic lipids 2-4 [25].
Among the set of six lipoplex formulations composed of a synthetic lipid 2-4, co-lipid
and pDNA a number of interesting trends emerged from a statistical analysis of the transfection
and cytotoxicity assay results, in spite of the lower relative performance of the lipoplex
formulations. The most striking trend revealed the transfection efficiency of formulations based
on mismatched lipid structures, such as the formulation composed of the rigid cationic lipid 2
and the non-rigid co-lipid DOPE, significantly outperformed the matched formulations,
composed of either two rigid or two non-rigid lipids. Furthermore, we found the relative
transfection performance of lipoplex formulations, based on a cyclic versus acyclic hydrophobic
domain structure analysis, was co-lipid dependent. However, the analysis that led to the matched
versus mismatched trend in the data proved to be a more elegant and conclusive explanation for
these results. In addition to these analyses, we observed the DMA-based lipid 2 outperformed the
TMA-based lipid 4, which was consistent with our previous findings [22a]. Finally, statistical
analysis revealed the mismatched formulations exhibited higher cell viability than the matched
formulations, which is in line with the ideal transfection conditions; highest levels of DNA
expression with minimal toxicity.
The transfection efficiency trend observed between the matched and mismatched
formulations can be attributed to the relative structural rigidity difference between the lipids
employed in the formulation. In our previous research, a transfection efficiency study of lipoplex
formulations based on structural differences in the hydrophobic domain revealed a similar trend
[22]. For example, formulations based on synthetic cationic lipids that possess two short 7-
carbon hydrophobic groups outperformed those with longer 11-carbon groups, when formulated
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with the co-cationic lipid EPC, which possesses two 14-carbon groups. Accordingly, EPC is
mismatched with respect to the 7-carbon chain lipids. However, as in the present case, this trend
was not based on the structural differences between the synthetic cationic lipids alone, i.e. 7-
versus 11-carbon chains, but the mismatched or matched structural relationship between the
synthetic cationic lipid and the co-cationic lipid employed in the formulation. The correlation
between the higher relative performance of mismatched lipid formulations, characterised by
significant hydrophobic domain structure differences between the lipids employed in the
formulation, was elegantly demonstrated and described by MacDonald and co-workers [26]. In
their transfection studies, formulations composed of two cationic lipids that differed in the
properties of the hydrophobic domain structure were no longer in a single phase, and thus
exhibited poor lipid packing, which constituted a defect that facilitated DNA release from the
lipoplexes within the endosome [27,28]. In the present work, a defect may arise in selected cases
due to rigidity differences between the hydrophobic domain structures of the synthetic cationic
lipid and the co-lipid employed in the lipoplex formulation, resulting in higher transfection
efficiency of the mismatched formulations relative to the matched formulations.
The results of this study revealed the introduction of a succinic unit into the hydrophobic
domain of the cationic lipids improved, in most cases, their hydration into liposomes.
Furthermore, the lipoplex formulations based on a combination of rigid and non-rigid, or
mismatched lipids exhibited the highest levels of transfection efficiency, when in comparison
with matched lipid formulations. Further research is required to determine whether this trend is
limited to the succinic-based cationic lipids, or could be generally applied to a wide range of
lipid structure types. Although the purpose of this work was to study structure-transfection
efficiency relationships, further research toward the discovery of lipoplex formulations that
exhibit transfection levels beyond that of the commercial formulation lipofectamine 2000TM are
required. Nonetheless, the trend that emerged from this work will aid in the future development
of safe and efficient nonviral gene delivery systems.
4. Conclusions
An effective synthesis route was developed for the construction of four succinic-based
cationic lipids, 1-4, and with the exception of lipid 1 all were readily hydrated into liposomes,
which employed either DOPE or cholesterol as co-lipid. All six lipoplex formulations screened
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exhibited effective pDNA binding and protection from DNase I degradation. Furthermore, most
formulations exhibited concentration dependent cytotoxicity against the CHO-K1 cells employed
in this work. Of note, a higher relative transfection performance of mismatched formulations,
based on a combination of rigid and non-rigid lipids, over that of matched formulations was
found. Accordingly, the impact on transfection efficiency due to the structure-rigidity difference
between the two lipids employed in the formulation was more significant than the differences
between the macrocyclic or acyclic hydrophobic domain structure, or the co-lipids employed in
the formulation. In comparison with the lipids we previously reported, which required the co-
cationic lipid EPC to facilitate hydration [22], the inclusion of the succinic unit within the
hydrophobic domain generally led to improved hydration. The succinic-unit did not impact
pDNA binding or protection, however it could be the source of the lower relative transfection
efficiency. These results emphasize the importance of lipid and formulation design in the pursuit
of safe and effective gene transfer reagents, and thus form the basis for further investigations.
Acknowledgements
This research was funded in part by the British Council (PMI2 Gulf States Cooperation
Grant No. RCGS206) and the Biomedical Research Program intramural funding at Weill Cornell
Medicine in Qatar. Synchrotron beam time on the BioSAXS-beamline (BM29) was provided by
the European Synchrotron Radiation Facility in Grenoble, France. The authors wish to thank M.
Bowler for technical assistance with setting up the instrumentation.
Supplementary data
Experimental procedures, data and NMR spectra for the cationic lipids 1-4, together with
all synthetic intermediates leading to these compounds are provided. In addition, procedures and
protocols for liposome and lipoplex preparation, β-Galactosidase, total protein and cytotoxicity
assays, small angle X-ray diffraction, statistical analysis, particle size and polydispersity index
data for liposome and lipoplex formulations based on the cationic lipids 2-4, and gel retardation
and DNase degradation assays of lipoplex formulations based on the cationic lipids 2-4 are also
found within the supplementary data. Supplementary data related to this article can be found at
[insert article doi].
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List of Figure, Scheme and Table Captions
Figure 1. The structures of (non-rigid) succinic-based acyclic DMA and TMA cationic lipids 1
and 3, and the corresponding (rigid) macrocyclic analogues 2 and 4; as well as the commercial
(non-rigid) zwitterionic co-lipid DOPE and (rigid) neutral co-lipid cholesterol.
Scheme 1. Synthesis of the succinic-based (A) mono-ester 7, and (B) acyclic and macrocyclic,
DMA and TMA cationic lipids 1-4.
Table 1. Packing morphology and interlayer distance δ (Å) of succinic-based lipid 3 and
4/DOPE and Chol/DNA lipoplex formulationsa based on the small angle X-ray diffraction
analysesb
Figure 2. SAXD curves recorded for synthetic lipid 3 or 4/DOPE or cholesterol/DNA lipoplexes
at a N:P (+/-) molar charge ratio = 3:1. The ordinate axis: intensity in arbitrary units (curves
shifted for clarity). Abscissa axis: the modulus of the scattering vector (denoted as s).
Figure 3. Transfection efficiency (bars) and cytotoxicity ( ) of succinic-based lipids 2-4 (48 h
after incubation with CHO-K1 cells) using 3:2 synthetic lipid:co-lipid / DNA lipoplexes (A: co-
lipid = DOPE; and B: co-lipid = cholesterol) at various N:P (+/-) molar charge ratios, ranging
from 0.5:1 to 10:1, compared to Lipofectamine 2000TM/DNA (L2k) as a positive control, and
plasmid DNA alone (D) and CHO-K1 cells alone (C) as negative controls (n=6; mean ± SE). The
transfection efficiency of all formulations within each set appears as a relative percentage of the
Lipofectamine 2000TM/DNA control.
Figure 4. Calculated means for transfection efficiency data, as a relative percentage of the
Lipofectamine 2000TM control based on formulation sub-groups according to (A) mismatched
(black bars; 2 or 4/DOPE and 3/Chol) or matched (white bars; 2 or 4/Chol and 3/DOPE)
formulation, and (B) DMA (black bars) or TMA (white bars) headgroup structure. Significant
differences in paired Student's t-test p-values were expressed as follows: *p < 0.05; ** p < 0.01
and *** p < 0.001.
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Highlights
Cationic lipids bearing succinic-based, acyclic and macrocyclic
hydrophobic domains: synthetic studies and in vitro gene transfer
Emile Jubelia, Amanda B. Magintyb, Nada Abdul Khaliquec, Liji Rajuc, David G.
Nicholsond, Helge Larsene, Michael D. Pungentef,* and William P. D. Goldringb,1,
*
Highlights:
•
Succinic-based acyclic (non-rigid) and macrocyclic (rigid) cationic lipids were
synthesized.
•
•
The synthetic lipids were formulated with plasmid DNA and a neutral lipid.
The in vitro transfection and cytotoxicity of formulations was evaluated using CHO-K1
cells.
•
•
Introduction of the succinic unit within the hydrophobic domain improved lipid
hydration.
Mismatched (rigid lipid blended with a non-rigid lipid) outperformed matched
formulations.
1Present address: Department of Science and Environment, Roskilde University, PO Box 260,
Universitetsvej 1, DK-4000 Roskilde, Denmark
1