The discovery and enhanced properties of trichain lipids in lipopolyplex gene delivery systems

Article abstract of DOI:10.1039/c8ob02374c

The formation of a novel trichain (TC) lipid was discovered when a cationic lipid possessing a terminal hydroxyl group and the helper lipid dioleoyl l-α-phosphatidylethanolamine (DOPE) were formulated as vesicles and stored. Importantly, the transfection efficacies of lipopolyplexes comprised of the TC lipid, a targeting peptide and DNA (LPDs) were found to be higher than when the corresponding dichain (DC) lipid was used. To explore this interesting discovery and determine if this concept can be more generally applied to improve gene delivery efficiencies, the design and synthesis of a series of novel TC cationic lipids and the corresponding DC lipids was undertaken. Transfection efficacies of the LPDs were found to be higher when using the TC lipids compared to the DC analogues, so experiments were carried out to investigate the reasons for this enhancement. Sizing experiments and transmission electron microscopy indicated that there were no major differences in the size and shape of the LPDs prepared using the TC and DC lipids, while circular dichroism spectroscopy showed that the presence of the third acyl chain did not influence the conformation of the DNA within the LPD. In contrast, small angle neutron scattering studies showed a considerable re-arrangement of lipid conformation upon formulation as LPDs, particularly of the TC lipids, while gel electrophoresis studies revealed that the use of a TC lipid in the LPD formulation resulted in enhanced DNA protection properties. Thus, the major enhancement in transfection performance of these novel TC lipids can be attributed to their ability to protect and subsequently release DNA. Importantly, the TC lipids described here highlight a valuable structural template for the generation of gene delivery vectors, based on the use of lipids with three hydrophobic chains.

Full text of DOI:10.1039/c8ob02374c

Organic &
Biomolecular Chemistry
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The discovery and enhanced properties of trichain
lipids in lipopolyplex gene delivery systems†
Cite this: DOI: 10.1039/c8ob02374c
Atefeh Mohammadi,‡a Laila Kudsiova,‡§b M. Firouz Mohd Mustapa,a
Frederick Campbell,a Danielle Vlaho,a Katharina Welser,a Harriet Story,a
a
Aristides D. Tagalakis,c Stephen L. Hart,c David J. Barlow, b Alethea B. Tabor,
a
M. Jayne Lawrence
*
b,d and Helen C. Hailes
*
The formation of a novel trichain (TC) lipid was discovered when a cationic lipid possessing a terminal
hydroxyl group and the helper lipid dioleoyl L-α-phosphatidylethanolamine (DOPE) were formulated as
vesicles and stored. Importantly, the transfection efficacies of lipopolyplexes comprised of the TC lipid, a
targeting peptide and DNA (LPDs) were found to be higher than when the corresponding dichain (DC)
lipid was used. To explore this interesting discovery and determine if this concept can be more generally
applied to improve gene delivery efficiencies, the design and synthesis of a series of novel TC cationic
lipids and the corresponding DC lipids was undertaken. Transfection efficacies of the LPDs were found to
be higher when using the TC lipids compared to the DC analogues, so experiments were carried out to
investigate the reasons for this enhancement. Sizing experiments and transmission electron microscopy
indicated that there were no major differences in the size and shape of the LPDs prepared using the TC
and DC lipids, while circular dichroism spectroscopy showed that the presence of the third acyl chain did
not influence the conformation of the DNA within the LPD. In contrast, small angle neutron scattering
studies showed a considerable re-arrangement of lipid conformation upon formulation as LPDs, particu-
larly of the TC lipids, while gel electrophoresis studies revealed that the use of a TC lipid in the LPD formu-
lation resulted in enhanced DNA protection properties. Thus, the major enhancement in transfection per-
formance of these novel TC lipids can be attributed to their ability to protect and subsequently release
DNA. Importantly, the TC lipids described here highlight a valuable structural template for the generation
of gene delivery vectors, based on the use of lipids with three hydrophobic chains.
Received 24th September 2018,
Accepted 19th December 2018
DOI: 10.1039/c8ob02374c
rsc.li/obc
formulations, and since the report of the cytofectin DOTMA2
which is often used with the neutral lipid dioleoyl
Introduction
Non-viral vectors continue to have significant potential to L-α-phosphatidylethanolamine (DOPE) (Fig. 1), a range of
facilitate DNA or RNA delivery into target cells.1 Synthetic cat- related lipids have been described.1e,3 The aim in developing
ionic lipids (cytofectins) have been used in many lipid-DNA new cytofectins has been to optimise the delivery properties of
the resulting vector for a wide variety of cell types, and for
in vivo applications.2,3 Cytofectins are typically used in lipoplex
(lipid-DNA; LD) binary formulations or lipopolyplex (lipid-poly-
aDepartment of Chemistry, University College London, Christopher Ingold
Laboratories, 20 Gordon Street, London WC1H 0AJ, UK. E-mail: h.c.hailes@ucl.ac.uk
bFaculty of Life Sciences and Medicine, King’s College London,
Franklin-Wilkins Building, Stamford Street, London SE1 9NH, UK
cWolfson Centre for Gene Therapy of Childhood Disease, Institute of Child Health,
University College London, 30 Guilford Street, London WC1N 1EH, UK
dDivision of Pharmacy and Optometry, School of Health Sciences, Faculty of Biology,
Medicine and Health, Stopford Building, Manchester University, Oxford Road,
Manchester, M13 9PT, UK. E-mail: jayne.lawrence@manchester.ac.uk
†Electronic supplementary information (ESI) available: This includes detailed
experimental procedures, characterisation data, and supplementary Fig. S1–S6.
See DOI: 10.1039/c8ob02374c
cation/peptide-DNA; LPD) ternary formulations where the poly-
cation/peptide has DNA-binding functionality and peptides
can also have cell-targeting capability.1e,4–6
Cationic lipids are made up of a positively charged head
group with a linker to a hydrophobic component. Since
DOTMA was first described, analogues with ether or ester link-
ages to the glycerol skeleton, alternative alkyl chain groups
and functionalized head groups have been reported. Examples
include the diester DOTAP,7 diester DORI and corresponding
diether DORIE, and DMRIE (Fig. 1).3,8 Such hydroxyalkyl cyto-
fectins were reported to be more active than DOTMA.8 It was
postulated that they were particularly effective, either because
‡Joint first authors.
§Current address: School of Pharmacy and Biomolecular Sciences, Centre for
Stress and Age-Related Disease, University of Brighton, Lewes Road, Brighton
BN2 4GJ, UK.
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Fig. 1 Structures of a selection of cationic lipids: DOTMA, DOTAP,
DORI, DORIE, DMRIE, βAE-DMRIE and the helper lipid DOPE.
Scheme 1 Acyl transfer from DOPE to give lyso-PE (1 and 2) and ami-
dation products of GL-67 such as 3.23
the hydroxyl group was able to increase the liposome inter-
action with DNA or cellular membranes, or because it was able
to stabilize the cationic lipid : DOPE bilayer structure, through
charge neutralization and/or hydrogen bonding.8 Others have
also incorporated hydroxyalkyl chains onto quaternary amine DOPE revealed the formation of lyso-PE (1 and 2) and ami-
species with varying results.9,10
dated GL-67 such as 3. Analysis of the reaction revealed a 6%
Alterations to the alkyl chain, including factors such as conversion after 5 h in hydrated mixtures, but very low levels
chain saturation, double bond geometry, and chain length when stored as dried lipid films.22 Lyso-PE (1 and 2) and 3
have been shown to be important in lipoplex and lipopolyplex were tested in transfection experiments which confirmed they
formulations.11–17 In addition, other head groups such as were ineffective transfection reagents, and when lyso-PE was
aminopropyl groups, and short polyethylene glycol (PEG) added to GL-67/DOPE formulations, lower levels of transfec-
shielded glycerol-based lipids with non-cleavable and cleavable tion were also observed.22 The acyl chain transfer effect was
functionalities have been reported.14,16–20 Studies established also highlighted with an analogue of GL-67.22 A similar acyl
that shielding nanoparticles with long PEG-lipid conjugates, chain transfer reaction has been noted with diester lipids pos-
while enhancing circulation times and reducing vesicle aggre- sessing a primary amine head group, where some fluorinated
gation, resulted in lower transfections efficiencies.16 However, analogues were less prone to acyl transfer.9 It is unclear
the use of short n-ethylene glycol (n-EG) moieties attached to whether the acyl transfer reaction from DOPE to cationic lipids
lipid headgroups still gave improved LPD particle stabilities possessing a terminal primary amine group is a general
but enabled nanoparticle targeting and good in vitro and phenomenon, but reduced levels of transfection were observed
in vivo transfection efficiencies.16–20
in the compounds studied, presumably due to reduced
Addition of the neutral co-lipid DOPE is often found to capacity of the lipids to be protonated and induce endosomal
enhance transfection levels, and is attributed to improved breakdown as well as changes to the molecular architecture of
endosomal membrane fusion, due to the presence of DOPE the nanoparticles formed.
changing the bilayer Lα phase into the inverted hexagonal HII
Here we report that in the course of our studies on gene
phase, promoting endosomal escape.3,8,9 An enhanced trans- delivery using cytofectins possessing hydroxyl terminated
fection however is not always observed, for example in formu- short n-EG head groups, it was noted that when lipid DODEG4
lations of DOPE with βAE-DMRIE.21
was co-formulated as vesicles with DOPE and stored for
Interestingly, some groups have reported that on storage, periods of up to several months before peptide was added to
an acyl chain transfer reaction can occur from an acyl chain on prepare an LPD, a trichain (TC) lipid TC-DODEG4 was formed
the DOPE lipid to the terminal primary amine of the co-formu- that gave improved levels of LPD transfection (Scheme 2A).
lated lipid.22,23 It was noted in experiments with the chole- This transacylation of an acyl chain from DOPE to the terminal
sterol lipid GL-series, such as GL-67, and DOPE (Scheme 1) co- hydroxyl group occurred in an analogous fashion to that
formulated in aqueous media, that transfection levels dimin- observed with GL-67 (Scheme 1).22 Therefore, several hydroxyl
ished by 10–20 fold.22 When the GL-lipids were formulated terminated short n-EG lipids were prepared and the corres-
with other co-lipids this was not the case and transfections ponding TC-lipids, then LPD transfection experiments and
levels were enhanced. HPLC analysis of mixtures of GL-67 and biophysical characterisations performed to understand this
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Scheme 2 (A) Formation of TC-DODEG4 from DODEG4 and DOPE. (B) Structures of lipids DOesDEG4 and Me-DOesDEG3, Peptide A, sc and K16.
(C) Synthesis of TC-lipids. Reagents and conditions: (i) oleic acid, DMAP, DIC, CH2Cl2, 54%; (ii) for TC-DODEG4 acetone, 90 °C, 36%; for
TC-DOesDEG4 acetone, 80 °C, 11%; (iii) 5, DMAP, py, Ac2O, for 10 36%; 5, H15C7COCl, DMAP, Et3N, for 11 52%; 5, H23C11COCl, DMAP, Et3N, for 12
54%; (iv) acetone, 90 °C, AC-DODEG4 30%, AC-DOesDEG4 55%, OC-DODEG4 60%, DO-DODEG4 19%, DO-DOesDEG4 52%.
change in efficacy. The transacylation reaction is of importance of the lipid, DODEG4 (in the presence or absence of DOPE)
because there are many hydroxyl, n-EG and PEG terminated was investigated, over a period of one month, using TLC ana-
lipids in use that when formulated with DOPE may be affected lysis and LC-MS. As expected, when the lipids were stored in
by this process. In addition, this may lead to the identification an organic solvent (CHCl3) no changes in lipid structure were
of new improved compounds for delivery applications.
observed by LC-MS. When DODEG4 was formulated, in the
absence of DOPE, as liposomes dispersed in water at pH 5 and
pH 7.5, no new compounds were again formed. For liposomal
formulations made using
DODEG4 : DOPE dispersed in water at pH 7 however, the slow
formation of TC-DODEG4 (m/z 1061) together with lyso-PE
a
1 : 1 molar ratio of
Results and discussion
Synthesis and lipid transacylation
We have previously reported the use of the lipid, DODEG4, a (1 and/or 2) (m/z 479) was observed (Fig. S1 ESI†) as a conse-
glycerol-based diether lipid possessing a short n-EG headgroup quence of transfer of an oleoyl chain from DOPE. When the
for applications in LPD targeted delivery.17 The lipid has been liposomes containing DODEG4 : DOPE (1 : 1) were dispersed in
used with the co-lipid DOPE, to provide a short n-EG layer to water at pH 5, the transacylation to give TC-DODEG4 and lyso-
shield nanoparticles for enhanced stabilities, while still PE (1 and 2) was more pronounced (Fig. 2).
enabling peptide targeting to be achieved.17,20 Interestingly
To probe the behaviour of these dichain (DC) and trichain
when liposomal formulations of DODEG4 : DOPE were stored (TC) lipids in detail, several structurally related terminal n-EG
at 4 °C for several months, then used in the preparation of DC and the corresponding TC lipids were prepared for investi-
LPDs containing peptide A (Scheme 2B) and transfections gation and application in LPD gene transfections (Scheme 2
examined in a range of cell types including HAE and NIH3T3 and Table 1, referred to as Group
1 lipids). DODEG4
cells, improved transfection efficiencies were noted when com- (Scheme 2A) was synthesized as previously reported, from the
pared to LPDs prepared from freshly made vesicles. MS-ana- diether 4 and 4-EG bromide 5. A diester analogue DOesDEG4
lysis revealed that a transacylated trichain lipid, TC-DODEG4, (structure in Scheme 2B) was similarly prepared via the quater-
had formed (Scheme 2A). Consequently, the chemical stability nisation of 6 8 with 5. The corresponding TC lipids
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hydrolyse when undergoing purification on silica. A methoxy-
capped analogue of DOesDEG4, Me-DOesDEG3 (structure in
Scheme 2B), was also synthesized from amine 6 and 1-(2-bro-
moethoxy)-2-(2-methoxyethoxy)ethane24 as this is a lipid that
cannot undergo transacylation with DOPE (Scheme 2).
A further set of lipids were prepared based upon a pre-
viously reported short n-EG terminated lipid DOSEG3, effective
in targeted LPD transfections (Table 1; Group 1 lipids).19 This
lipid possesses an endosomal esterase cleavable functionality
to release the n-EG group to enhance endosomal breakdown,
and was prepared as previously described via the quaternisa-
tion of 4 and the n-EG ester-bromide 8.19 In addition to
DOSEG3, the novel dichain diester analogue DOesSEG3 was
prepared, which may have greater biodegradability, and the
corresponding methoxy capped analogue Me-DOesSEG3 (struc-
tures in Scheme 3A). They were synthesized by the reaction of
bromides 8 19 and 9 19 with the tertiary amine 6, in 78% yield
(4, 6, 8, 9 are in Scheme 3B).
Two trichain lipids were also synthesized based on DOSEG3
and DOesSEG3, TC-DOSEG3 and TC-DOesSEG3. The n-EG
bromide 8 was coupled to oleic acid in 75% yield, and the
product reacted with the corresponding tertiary amines 4 and
6 to give TC-DOSEG3 and TC-DOesSEG3 (Scheme 3C). Again,
purification of the final TC-lipids was not straightforward, and
compounds were purified at least twice by silica column
chromatography.
Fig. 2 Formation of TC-DODEG4 and lyso-PE (1 and 2) from DODEG4
and DOPE, at pH 5 monitored by LC-MS.
Table 1 Summary of the dichain and trichain lipid prepared (Group 1
lipids) and dichain lipids capped with different ester chain lengths
(Group 2 lipids)
DOTMA-based
ether series
DOTAP-based
ester series
Group 1 lipids
DODEG4
DOesDEG4
TC-DODEG4
TC-DOesDEG4
Me-DOesDEG3
DOesSEG3
DOSEG3
TC-DOSEG3
TC-DOesSEG3
Me-DOesSEG3
Group 2 lipids
AC-DODEG4
AC-DOesDEG4
OC-DODEG4
DO-DODEG4
TC-DODEG4
DO-DOesDEG4
TC-DOesDEG4
, Ether lipid; , ester lipid;
, C18-chain; , acetyl group; , octa-
noyl group;
3-PEG;
, dodecanoyl group;
4-PEG; , directly linked; , longer cleavable linker.
, 3-PEG
, Me capped
TC-DODEG4 and TC-DOesDEG4 were synthesized by the qua-
ternisation of 4 and 6 with the 4-EG oleoyl ester 7, prepared
from oleic acid and bromide 5 (Scheme 2C).
Compounds TC-DODEG4 and TC-DOesDEG4 were formed
in 36% and 11% yield respectively, reflecting the general
difficulties in purifying the lipids. In addition, the di- and tri-
Scheme 3 (A) Structures of lipids DOSEG3, DOesSEG3, Me-DOesSEG3.
(B) Structures of substrates 4, 6, 8, 9. (C) Synthesis of TC-DOSEG3 and
TC-DOesSEG3. Reagents and conditions: (i) oleic acid, DMAP, DIC,
CH2Cl2, 75%; (ii) for TC-DOSEG3, 4, acetone, 90 °C, 11%; TC-DOesSEG3,
esters DOesDEG4 and TC-DOesDEG4 showed a tendency to 6, acetone, 90 °C, 45%.
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In addition to this first group of cytofectins (Group 1 lipids)
comprised of traditional dialkenyl chains and the new TC
series, a second group of cytofectins (Group 2 lipids) based on
DODEG4, but capped with acyl chains of different lengths,
were prepared to probe the effect of the length of the third
chain. This type of the TC lipid was denoted by the length of
the third acyl chain, namely either an acetyl (AC–), octanoyl
(OC–) or a dodecanoyl (DO–) chain.
Analogues of DODEG4 and DODesDEG4 with acetyl groups
were synthesised using analogous routes to those described
above. The 4-EG bromide 5 was reacted with acetic anhydride
to give 10 (Scheme 2C), which was reacted with amines 4 and 6
to give AC-DODEG4 and AC-DOesDEG4 in 30% and 55% yield,
respectively. Compound OC-DODEG4, with an intermediate
terminal octanoyl chain length, was prepared similarly from 5
and octanoyl chloride to give bromide 11, which was then
reacted with 4 to give the lipid in 60% yield. Dodecanoyl chlor-
ide was also used to make bromide 12, which was reacted with
4 and 6 to give DO-DODEG4 and DO-DOesDEG4 (Scheme 2C
and Table 1).
Fig. 3 In vitro transfection efficiency (expressed as RLU mg−1 protein)
obtained in B104 cells after exposure to LPDs prepared at
a
lipid : peptide : DNA charge ratio of 0.25 : 6.5 : 1 in either Opti-MEM (grey
bars) or in 25% v/v water then diluted with Opti-MEM (black bars).
Cationic lipid used to prepare the LPDs shown on the abscissa were for-
mulated with DOPE or *DOPC. PDs prepared at a peptide : DNA charge
ratio of 6.5 : 1, and LDs of Lipofectamine and L2K prepared at weight
ratios of 5 : 1 and 4 : 1, respectively. Peptide and DNA used in all cases
were peptide A and gWiz plasmid DNA (pDNA), respectively. Data is the
mean of three measurements standard deviation.
Lipid : Peptide : DNA transfections
Transfections were performed in rat neuroblastoma B104 cells,
using LPDs prepared from vesicles containing the lipids, in
the presence of a 1 : 1 molar ratio of DOPE (or 1,2-dioleoyl-sn- while differing absolute levels of transfection were obtained on
glycero-3-phosphocholine (DOPC) in the case of DO-DODEG4 different occasions the order of transfection efficiencies were
and DO-DOesDEG4 since it was impossible to formulate these consistent between experiments.
lipids into vesicles in the presence of DOPE) and peptide A.
As can be seen in Fig. 3, regardless of whether the LPDs
The cyclic sequence -CRGDC-LG of peptide A was chosen were initially prepared in Opti-MEM or water, the LPDs made
because it binds to αv integrin receptors and we have pre- at a L : P : D charge ratio of 0.25 : 6.5 : 1 using the TC lipids of
viously demonstrated that LPDs formulated using peptide A Group 1 exhibited superior transfection abilities to those pre-
mediate recognition and cell-specific uptake in neuroblastoma pared using DC lipids. Increases in transfection ranged from a
cells in vitro and in vivo.19,20 Control LPD formulations were 5-fold in the case of LPDs containing DOSEG3 and DODEG4 to
also
K16RVRRGA-CGRDC-LG (sc) which should not be recognised DOesSEG3 and DOesDEG4, respectively, suggesting that the
by the αv integrin receptors.
presence of a short ethylene glycol chain had a detrimental
prepared
using
a
scrambled
sequence a 200 and 300-fold increase in the case of LPDs prepared using
The LPDs were prepared either in Opti-MEM or in water effect on transfection in vitro. Although the acetyl and, to a
and diluted by 1 in 4 with Opti-MEM, and the results are lesser extent, the methoxy-capped analogues exhibited a slight
shown in Fig. 3. The preparation of LPDs in Opti-MEM was to improvement in transfection efficiency, this increase was not
allow a comparison with other researchers who have used this as high as that obtained when using the corresponding oleoyl
protocol.
All
LPDs
tested
were
formulated
at containing TC analogues. In contrast the OC- and DO-deriva-
lipid : peptide : DNA (L : P : D) charge ratios of 0.25 : 6.5 : 1 tives of DODEG4 (Group 2 lipids), gave comparable transfec-
(equivalent to a weight ratio in the range 0.7–1 : 4 : 1, the exact tion levels to the LPDs prepared using TC-lipids. Note that the
weight ratio depending upon the particular cationic lipid DO-derivative of DOesDEG4 exhibited a low level of transfec-
used) since this was the charge ratio found to produce the tion, less than that achieved with AC-DOesDEG4. LPDs pre-
highest transfection results during a preliminary screen using pared using DOTMA and DOTAP exhibited similar transfection
B104 cells (Fig. S2, ESI†). The preliminary screen (B104 cells) efficiencies to the TC- and OC-lipids, suggesting that the pres-
with LPDs were made from cationic vesicles containing DOPE ence of a third acyl chain negates the detrimental effect of the
and either DODEG4, TC-DODEG4, DOSEG3 and TC-DOSEG3 short ethylene glycol chain on in vitro transfection.
together with peptide A at final L : P : D weight ratios of
For the purposes of comparison, polyplexes (PD) of peptide
0.5 : 4 : 1, 1 : 4 : 1, 2 : 4 : 1 and 3 : 4 : 1. The level of transfection A: DNA (prepared at a charge ratio of 6.5 : 1) and lipoplexes of
achieved using LPDs with the higher lipid weight ratio of Lipofectamine and L2K (prepared at lipid : DNA weight ratios
3 : 4 : 1 was ∼20% of that obtained for the lower lipid ratios of of 5 : 1 and 4 : 1, respectively) were also examined for their
1 : 4 : 1 or 0.5 : 4 : 1. LPDs prepared using these lower weight transfection ability (Fig. 3). The polyplex demonstrated an
ratios produced similarly high levels of transfection. extremely poor transfection efficiency, a result in line with pre-
Transfections were repeated on more than one occasion and vious studies15 while the use of complexes prepared using
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Lipofectamine and L2K generally resulted in similar or lower efficiency. A screen of LPDs prepared using an equimolar
transfection levels and higher toxicities than those achieved amount of TC-lipid and lyso-PE in place of DOPE to mimic the
with LPDs prepared using the TC lipids, some DC lipids, fact that lyso-PE would be formed during the transacylation
DOTMA or DOTAP. Transfection experiments were performed process (Fig. S4, ESI†) showed that LPDs containing both TC-
on at least three separate occasions and the trends observed lipid and lyso-PE tended to be similar to or slightly more
(Fig. 3) were reproducible. Scrambling of peptide A resulted in effective at transfecting B104 cells, with no significant detri-
a 70–75% decrease in transfection confirming selective target- mental effect on toxicity as assessed by determination of the
ing of the B104 cells by peptide A. Complexes prepared in the percentage protein in each well. This observation supports the
absence of a targeting peptide i.e. TC lipid : DNA (LDs) have hypothesis that it is the TC-lipid and not the lyso-PE formed
recently been published in a separate study.25 The LDs pre- that is the beneficial transfection agent in these studies.
pared from TC lipids also showed superior transfection
A series of biophysical experiments were then performed to
efficiencies compared to their DC equivalents, however the understand the reasons for the enhancement in transfection
absolute transfection efficiency of the LDs was lower by around observed with the Group 1 and Group 2 TC lipids. Of particular
an order of magnitude.25
interest was whether the use of a TC lipid resulted in a
LPDs prepared fully in Opti-MEM generally showed better different LPD structure than has been previously observed
transfection efficiency than those prepared in 25% v/v water, for LPDs formed by DC lipids,5 where biophysical studies
followed by dilution with Opti-MEM before applying onto the showed that the lipopolyplexes consisted of a core of DNA
cells. The reasons are not currently understood but could be complexed with peptide surrounded by a bilayer of lipid.
due to differences in the size of the complexes when prepared Studies using LDs prepared from TC lipids showed a major
in Opti-MEM as opposed to water (particle size of the com- structural re-organisation of the lipid bilayer, suggesting
plexes prepared fully in Opti-MEM were larger than those pre- that the third chain may fold over and create inter-digitated
pared in water), and/or the looser association of DNA with the structures.25 It is not known, however how the trichain lipids
peptide and the lipid when formulated in Opti-MEM due to behave in ternary systems in the presence of a targeting
the presence of electrolyte that partially neutralises the cat- peptide.
ionic charge on the peptides and lipid. However, similar
trends in transfection were observed regardless of the Opti-
MEM or water content, the only exception being DO-DODEG4
Size and zeta potential
which exhibited a far lower transfection efficiency when pre- The apparent hydrodynamic size (assuming spherical par-
pared in water rather than Opti-MEM.
ticles) and zeta potential of the cationic lipids formulated in
An assay to determine the percentage protein in each well water were determined, either as vesicles with DOPE at
compared to blank (untreated) cells was simultaneously per- 1 : 1 molar ratio or as LPDs at lipid : peptide A : gWIZ plasmid
formed with every transfection experiment to give an indi- (pDNA) charge ratios of 0.25 : 6.5 : 1, measured at cationic lipid
cation of any toxicity caused by the various formulations concentrations of 5 μg mL−1 and 1 μg mL−1, respectively
(Fig. S3, ESI†). Significantly, the cells exposed to the LPD for- (Fig. 4). With the exception of the vesicles formed using the
mulations generally exhibited greater than 80% protein Group 2 lipid, DO-DOesDEG4, which were ∼110 nm, all vesi-
content, suggesting that the formulations caused little or no cles prepared using Group 1 and Group 2 lipids exhibited sizes
toxicity. The only cells exhibiting a lower protein content of in the range 45–80 nm, and polydispersities of between 0.1–0.3
∼60% were those exposed to LPDs prepared using the (Fig. 4a and b). The size of the DC-containing vesicles, includ-
TC-DOesSEG3 and then only when the LPDs were formulated ing those prepared using methyl capped lipids, tended to be at
in water and diluted with Opti-MEM, i.e. not fully prepared in the lower end of the size range suggested that these vesicles
Opti-MEM. Notably, both Lipofectamine and L2K exhibited are predominately unilamellar in nature, although the pres-
considerable toxicity towards the cells as evidenced by the ence of a small population of multilamellar structures cannot
∼65–70% and 35–55% protein contents obtained when in the be dismissed.
cells exposed to LDs prepared either fully in Opti-MEM or in
By way of comparison, vesicles prepared using the longer
water and diluted with Opti-MEM, respectively.
acyl chain TC lipids (i.e. a third acyl chain of C8 and above)
In addition, some transfection studies were performed to tended to be slightly larger than their DC counterparts. The
investigate the influence of the neutral co-lipid lyso-PE on the observation that the longer acyl chain TC lipids, when in com-
effectiveness of DNA delivery. In these studies, lyso-PE was bination with DOPE, formed vesicles was unexpected. Indeed,
used in the place of DOPE during the preparation of LPDs. because of the relatively large hydrophobic regions of both the
Previous reports have suggested that replacing DOPE with lyso- TC lipids containing an acyl chain of C8 and above and DOPE,
PE in cationic liposome formulations reduces the effective mixtures of such lipids with DOPE had been expected to
volume of the lipid hydrophobe, thus lowering the curvature promote the formation of reverse structures such as reverse
stress of the liposomes which in turn, results in liposome for- hexagonal phases.
mulations that cannot effectively induce cell–cell fusion.26 It
was possible therefore that the presence of lyso-PE in an LPD exhibited a zeta potential of at least ∼30 mV (DOSEG3-contain-
All vesicles (prepared from Group 1 and Group 2 lipids)
formulation might lead to
a
reduction in transfection ing vesicles), although the typical value was in the range
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dominately spherical in nature, irrespective of whether the cat-
ionic lipid used was a DC or TC, supporting the presence of
vesicular structures (Fig. 5a–d). Although vesicles are not visu-
alised well using TEM, since the sample is dehydrated upon
staining, TEM was performed in order to rule out the for-
mation of other aggregate types, since to our knowledge this is
the first time that LPDs have been prepared from lipids con-
taining three long (C18:1) acyl chains.
In contrast to the spherical nature of the vesicles, the LPD
formulations (Fig. 5e–h) consisted of a mixture of spherical
and rod-shaped particles, with no apparent visible internal
structure: an observation in agreement with LPD formulations
reported previously.15,27 This observation is probably a conse-
quence of the fact that the ratio of lipid : peptide : DNA was not
fully optimised in the present study. Furthermore, there were
some observable differences between the LPDs prepared using
the DC and TC lipids in that the LPDs formulated from the DC
lipids were relatively monodisperse with individual particles of
between 50–100 nm in diameter, while the LPDs prepared
using the TC lipids, although similar in shape were more poly-
disperse and displayed some particle aggregation (Fig. 5f
and h). It is not known at present whether these aggregates are
due to drying and/or surface effects, although it should be
noted that the size of the LPDs measured by dynamic light
scattering suggested that when in solution the LPDs are not
(significantly) aggregated. Furthermore, the LPDs were
prepared freshly for the TEM study.
Fig. 4 The apparent hydrodynamic size (grey bars) and zeta potential
(black squares) of (a) cationic lipid vesicles prepared using at 1 : 1 molar
ratio of DOPE or *DOPC and (b) LPD complexes prepared using cationic
lipid vesicles, peptide
A and pDNA at L : P : D charge ratios of
Small angle neutron scattering
0.25 : 6.5 : 1. Measurements were performed at a cationic lipid concen-
tration of 5 μg mL−1 for vesicles and a DNA concentration of 1 μg mL−1
for LPDs. Data is the mean of three independent measurements stan-
dard deviation.
Table 2 shows the structural parameters obtained through
model fitting of small angle neutron scattering (SANS) data
obtained for cationic vesicles containing 50 mol% DOPE (at a
cationic lipid concentration of 1 mg mL−1) and LPDs prepared
at a L : P : D charge ratio of 0.25 : 6.5 : 1 (containing 0.175
mg mL−1 of K16 peptide and 0.0625 mg mL−1 of ctDNA) as
single, flat sheets (or bilayers) with or without the presence of
stacks thereof. The data for the corresponding vesicles and
LPDs formed from DOTMA and DOTAP are also included in
Table 2. A representative selection of the SANS profiles
recorded for the cationic lipid : DOPE vesicles (those made
from DOesDEG4, TC-DOesDEG4 and Me-DOesDEG4) together
with the LPDs (prepared using ctDNA and K16 at L : P : D
charge ratios of 0.25 : 6.5 : 1) are shown in Fig. 6.
40–50 mV, indicating that the vesicles should exhibit a high
stability (Fig. 4a and b).
The apparent hydrodynamic sizes recorded for the LPDs
were similar, although slightly larger than those obtained for
the vesicles at between 55–115 nm, with most being less than
80 nm, with polydispersities in the range 0.1–0.4. Significantly,
the zeta potentials measured for the LPDs were still positive,
albeit lower than the vesicles at 15–45 mV, suggesting that the
LPDs may be less stable. Interestingly, no correlation between
the size and zeta potential of the LPDs and the structure of the
cationic lipids used to prepare the complexes was evident.
Regardless of the presence and length of a third chain, all
the cationic vesicles examined in the present study were well
modelled assuming the presence of single, flat sheets (i.e. uni-
lamellar vesicles) and, where necessary, with the addition of
Transmission electron microscopy
Transmission electron microscopy (TEM) in combination with stacks (i.e. multilamellar vesicles). The size of all the cationic
negative staining using a 4% w/v solution of uranyl acetate was vesicles in the present study are too large to be analysed by the
used to examine the structure of the vesicles (co-formulated at SANS instrument used here as spherical structures. Indeed,
a 1 : 1 molar ratio with DOPE) and LPDs (prepared from cat- the assumption of flat sheets is in line with the apparent
ionic vesicles, peptide A and gWIZ plasmid pDNA at a hydrodynamic size of the vesicles made in earlier studies on
0.25 : 6.5 : 1 charge ratio) made using a selection of Group 1 related, DC cationic lipid-containing systems.13 As with the
DC and TC lipids, namely DODEG4, TC-DODEG4, DOSEG3 vesicles, it was possible to analyse the LPDs formed by all the
and TC-DOSEG3 (Fig. 5). Significantly, although some polydis- Group 1 and Group 2 DC and TC lipids assuming the presence
persity in size was apparent, the aggregates observed were pre- of sheets or bilayers of lipid. Significantly, in the absence of
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Fig. 5 Transmission electron micrographs of lipid vesicles composed of a molar ratio of DOPE and cationic lipid (a) DODEG4, (b) TC-DODEG4, (c)
DOSEG3 and (d) TC-DOSEG3; and LPDs formulated with peptide A and pDNA at charge ratios of 0.25 : 6.5 : 1 using (e) DODEG4, (f) TC-DODEG4, (g)
DOSEG3 and (h) TC-DOSEG3; and (i) PD prepared from peptide A : pDNA complexes at 6.5 : 1 charge ratio. All samples were stained with a 4% w/v
uranyl acetate solution.
any data suggesting the presence of non-vesicular structures, it period. When the LPDs were prepared from freshly made vesi-
was reasonable to assume that the LPDs prepared using the cles there was little evidence of the presence of any multilamel-
TC lipids were, as with the LPDs prepared using DC lipids, lar structures, regardless of the nature of the lipid (DC or TC)
composed of a lipid bilayer (or bilayers). This observation is used. However, if the LPDs were prepared from vesicles in
supported by the results of the transmission electron which a significant amount of multilamellar structure was
microscopy study.
present, the LPDs exhibited a greater content of multilamellar
The SANS profiles of the vesicles and LPDs were measured structure. The data shown in Table 2 was acquired using
on more than one occasion. This was important as vesicles freshly prepared vesicles and LPDs so the level of multilamel-
prepared from the TC cationic lipids were expected to be less lar content was very low.
stable than their DC counterparts. Indeed, preliminary studies
Inspection of the values of the bilayer thickness, L,
suggested that the amount of multilamellar structure present obtained for vesicles prepared from Group 1 and Group 2 DC
in the vesicles containing the DC lipids was consistently very and TC lipids in the presence of DOPE (Table 2) showed,
low, while the amount in vesicular preparations containing a perhaps somewhat surprisingly, that there was no change in
TC lipid tended to be higher depending on the age of the vesi- the bilayer thickness of the lipids as a function of bond type
cles, particularly if the vesicles were over 1 month old. (ester or ether), length of the n-EG linker and the absence/pres-
Significantly, however, the thickness of bilayer present in the ence or length of a third chain. Furthermore, regardless of
TC vesicles did not change over the corresponding time whether the cationic lipid was a DC or TC, the thickness of the
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0.15 mg mL−1 of ctDNA and either 0.42 mg mL−1 of K16
peptide or 0.6 mg mL−1 of peptide A to see the effect of con-
centration on the structure of the LPDs (Fig. S5, ESI† shows
the vesicles and LPDs prepared from TC-DODEG). The thick-
ness of the bilayer and lamellar d-spacing present in the LPDs
prepared using the higher level of peptides K16 and A were L =
9.3 0.1 and D = 13.4 0.1 (K16) and L = 8.8 0.2 and D =
12.5 0.1 (peptide A). These compared favourably to values of
L = 9.5 0.3 and D = 11.8 0.1 nm when the LPDs were pre-
pared at a ctDNA concentration of 0.0625 mg mL−1 and con-
tained 0.175 mg mL−1 of K16 peptide, suggesting that the
concentration of LPDs had no effect under the present experi-
mental conditions. Similar results were obtained for LPDs pre-
pared using all of the cationic lipids tested.
Table 2 Structural parameters obtained through model fitting of small
angle neutron scattering data of freshly prepared cationic vesicles con-
taining 50 mol% DOPE at a cationic lipid concentration of 1 mg mL−1
,
and LPDs prepared at a L : P : D charge ratio of 0.25 : 6.5 : 1 and contain-
ing K16 peptide at a DNA concentration of 0.0625 mg mL−1
Cationic lipid
DOTMA
L (nm)
D (nm)
V 4.2 0.1
LPD 4.2 0.3
V 4.1 0.1
V 6.2 0.1
LPD 6.5 0.1
ND
DODEG4
LPD 6.3 0.2
V 4.2 0.1
LPD 9.5 0.3
V 4.1 0.1
ND
TC-DODEG4
DOSEG3
V 5.9 0.1a
LPD 11.8 0.1
ND
LPD 6.4 0.2
V 4.0 0.1
LPD 6.8 0.2
V 4.2 0.1
LPD 4.2 0.3
V 3.9 0.1
LPD 6.9 0.3
V 4.1 0.1
LPD 7.8 0.4
V 4.0 0.1
LPD 7.6 0.5
V 4.1 0.1
LPD 7.3 0.3
V 4.1 0.1
LPD 9.1 0.4
V 4.0 0.1
ND
TC-DOSEG3
DOTAP
V 5.9 0.1a
LPD 11.6 0.1
V 6.2 0.1
LPD 6.5 0.1
ND
Circular dichroism
DOesDEG4
TC-DOesDEG4
Me-DOesDEG4
DOesSEG3
Circular dichroism (CD) spectra of LPDs prepared using repre-
sentative Group 1 DC and TC lipids, DOSEG3 and TC-DOSEG3
respectively, either peptide A or K16 peptide and calf thymus
DNA (ctDNA) at a 0.25 : 6.5 : 1 charge ratio was measured and
compared to a solution containing the same concentration of
ctDNA (0.05 mg mL−1) (Fig. 7). All samples were prepared in
D2O in order to increase the optical transparency of the solu-
tion at low wavelengths. Dynamic light scattering measure-
ments of vesicles and LPDs made in D2O showed no difference
in size suggesting that the replacement of H2O with D2O does
not significantly alter the structure of the complexes. The CD
spectrum obtained for ctDNA in solution (Fig. 7) showed that
ctDNA adopts a B type conformation (namely a positive peak
at ∼275 nm and a negative region between 260–230 nm)
typical of DNA. A similar CD spectrum was obtained for pDNA
(data not shown).
ND
V 5.8 0.3
ND
ND
ND
V 6.0 0.1
ND
ND
LPD 13.0 0.1
ND
TC-DOesDEG3
Me-DOesDEG3
LPD 6.6 0.3
V 3.9 0.1
V 3.8 0.1
ND
ND
V 5.9 0.1
ND
AC-DODEG4
OC-DODEG4
DO-DODEG4
V 3.9 0.1
L is the vesicle bilayer thickness (in nm), D the lamellar d-spacing (in
nm). Standard errors on the fitted parameters are shown. V is data
obtained for vesicles and LPD is data obtained for LPDs. ND = no
stacks (multilamellar structure) determined. a Here M (the number of
stacks in a multilayer structure) = 5.
The CD spectra obtained for the DC and TC LPDs prepared
using either peptide exhibited a red shift in the CD spectrum
water layer between the bilayers, when present, varied between above ∼240 nm. Since there is no absorbance either from the
1.7 and 1.9 nm, suggesting a water layer between each bilayer peptide or the lipid above 240 nm (Fig. S6, ESI† shows the
of ∼2 nm, in line with previous results for other DC cationic spectra of free peptide and lipid at the same concentration as
vesicles.13
used in the LPDs) this suggests that the conformation of
Interestingly the thickness of the bilayer was much greater ctDNA is altered when in the form of an LPD, most likely
when the cationic lipids were part of an LPD, with the thick- adopting a more condensed structure. Similar results have
ness of the bilayers containing the DC lipids typically increas- been obtained in previous studies using LPD formulations.27
ing by between ∼2.2 and 3.2 nm, while the increase in the Interestingly, while no difference in the extent of the red shift
bilayer containing the TC lipids was even larger in the range was observed with the type of lipid used, a greater red shift
2.8–5.3 nm. The methoxy capped lipids behaved more like was seen in the LPDs containing peptide A. These results
the DC lipids exhibiting an increase in bilayer thickness of suggest that the ctDNA in the LPD complex is predominantly
2.6–3.6 nm. These results suggest a significant re-arrangement condensed by the peptide with relatively little contribution
of the lipids in the presence of DNA, to accommodate the from the lipid present. This result also agrees with the LPD
interaction of the cation with the negative charges on DNA, structure model described previously5 wherein the LPDs were
with the re-arrangement being greatest for the TC lipids. In proposed as having
a core composed of predominately
contrast, DOTMA and DOPE and indeed previous SANS studies DNA : peptide surrounded by a bilayer of cationic lipid.
on vesicles and their corresponding LPDs prepared from cat-
Gel electrophoresis
ionic lipids without a short ethylene glycol chain did not
exhibit any difference in bilayer thickness between the vesicles Gel electrophoresis was performed on LPDs prepared using
and LPDs.15
the Group 1 and Group 2 DC and TC lipids, peptide A and
A limited SANS study was performed using LPDs prepared pDNA at charge ratios of 0.25 : 6.5 : 1 and the corresponding
at a L : P : D charge ratio of 0.25 : 6.5 : 1 but containing polyplex (PD) to determine the extent of complexation of,
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Fig. 6 Small angle neutron scattering data (dots) recorded at 298 K and the best fit to the data (solid line) obtained using the mixed sheet and stack
model for freshly prepared vesicles prepared from DOPE and 1 : 1 molar ratio of cationic lipid: (a) the dichain lipid, DOesDEG4, (c) the corresponding
trichain lipid TC-DOesDEG4, and (e) their methoxy capped dichain counterpart Me-DOesDEG4, at a cationic lipid concentration of 1 mg mL−1 and
LPDs prepared from these vesicles containing (b) DOesDEG4, (d) TC-DOesDEG4, and (f) Me-DOesDEG3, peptide K16 at a ctDNA concentration of
0.0625 mg mL−1 and a L : P : D charge ratio of 0.5 : 6 : 1.
enzyme protection afforded to, and release of DNA (Fig. 8, using the Group 1 TC lipids, DOTMA or DOTAP protected
Lanes A, B and C, respectively). The extent of protection of pDNA to a greater extent than LPDs formulated with the DC
pDNA in the LPDs against DNAse I, was measured using two lipids and, to a lesser extent, the Group 2 lipids. In the case of
amounts of DNAse I, namely single strength (1 IU per µg of the DC lipids, a greater amount of low molar weight pDNA
pDNA) and double strength (2 IU per µg of pDNA) DNAse I fragments were particularly evident. These differences were
(Fig. 8 Lane B). Double strength DNAse I was used to help elu- particularly noticeable when double strength DNAse I was
cidate trends.
used (Fig. 8b). Taken together these results suggest that the
It was evident that all the LPDs as well as the PD were able pDNA is well complexed by the peptide and that the lipids add
to condense pDNA completely as no migration of pDNA down little benefit in this respect. The different lipids however
any Lane A was observed (Fig. 8). Significant differences protect the pDNA to various extents supporting the hypothesis
between the protection from DNAse I afforded by the various that the lipid forms a barrier between the lipid and DNA. This
lipids was, however, observed when the complexes were treated suggestion is further supported by the fact that no uncomplexed
with DNAse I (Fig. 8, Lanes B). Generally, LPDs formulated DNA was observed.
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Fig. 7 CD spectra in (x-axis nm) of ctDNA and LPDs prepared using
DOSEG3 or TC-DOSEG3 in combination with either peptide A (green
and pale blue) or K16 (black and purple) at charge ratios of 0.25 : 6.5 : 1.
Measurements were performed using a 0.5 cm path length at a final
DNA concentration of 0.05 mg mL−1
.
All complexes were able to release pDNA on the addition of
excess pAsp (Lanes C), although the extent of release obtained
varied with the nature of the lipid present. LPDs prepared
using Group 1 TC lipids appeared to release more pDNA due
to the presence of brighter pDNA bands and a lower residual
fluorescence remaining in the wells. pDNA release from the
LPDs prepared using the Group 2 TC lipids was intermediate
with relatively low levels of fluorescence remaining in the
wells. In an attempt to quantify the protection and release of
pDNA from the complexes formed using the Group 1 lipids as
well as those formed using DOTMA or DOTAP, the intensities
of the DNA bands in Lane B were analysed and compared
either to the amount of pDNA in their corresponding Lane C
(i.e. the pDNA release in the absence of DNAse I) or the corre-
sponding (untreated) pDNA control band using the data
obtained in Fig. 8b (first sample on every row). The results of
this intensity analysis are shown in Table 3. With the exception
of TC-DOesDEG4, the complexes prepared with the Group 1
TC lipids, DOTMA and DOTAP exhibited much higher DNA
band intensity (52% and higher) compared to their DC equiva-
lents (28% and lower), suggesting that more DNA was pre- Fig. 8 Gel condensation, protection and release assay of LPDs or PDs
prepared at 0.25 : 6.5 : 1 or 6.5 : 1 charge ratios, respectively with peptide
A, and gWIZ plasmid DNA (pDNA) at a concentration of 0.25 μg per
10 μL per well. LPDs were prepared using the DC and TC lipids and
treated in either one of three ways: Lane A = untreated LPDs (showing
served. This was also mirrored by observing brighter low mole-
cular weight degradation bands for LPDs containing DC lipids
compared to Group 1 TC lipids, DOTMA or DOTAP, providing
further evidence that the DNA was digested to lower molecular
DNA condensation); Lane B = LPDs treated with DNAse and pAsp
weight fragments to a greater extent. These observations (showing pDNA protection against enzymatic degradation); Lane C =
LPDs treated with pAsp only (showing efficiency of DNA release from
complex). Free (untreated) pDNA and pDNA treated with DNAse I were
used as controls in each row. (a) Single strength (0.25 IU) DNAse I was
used per well and (b) double strength (0.5 IU) DNAse I was used.
suggest that the bilayer formed by the TC lipids provides a
more effective barrier to the degradation of DNA.
Since transfection efficiency was highest in LPDs prepared
using DOTMA, DOTAP or the Group 1 and Group 2 TC lipids
as opposed to their DC counterparts, and a similar trend was
observed in the pDNA protection capacity, it is possible that
the ability of the LPD complex to protect the pDNA and release tion and the extent of protection and subsequent release of
it from the LPD particle plays a major role in enhancing its the DNA payload a complex carried has been observed in our
transfection efficiency. A similar relationship between transfec- previous studies15 as well as those reported by others.28–30
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Table 3 Density analysis of gel electrophoresis data from Fig. 8ba
chain into the bilayer or partial inter-digitation of the third
alkyl chain into the other bilayer component. In summary, a
new design concept for use in effective gene delivery lipids has
been identified. Further studies are now underway to probe
this effect.
Intensity of lane B
DNA band as % of
untreated DNA
Intensity of lane B DNA
band (as % of
Group 1 lipid
present in the
LPD
corresponding lane C
band)
control (the first
sample in each row)
DODEG4
TC-DODEG4
DOSEG3
11
81
20
57
28
5
4
7
52
20
81
76
35
7
95
18
46
22
4
4
5
52
19
86
81
41
Experimental
TC-DOSEG3
DOesDEG4
TC-DOesDEG4
Me-DOesDEG3
DOesSEG3
TC-DOesSEG3
Me-DOesSEG3
DOTMA
Detailed experimental procedures, characterisation data, and
ESI Fig. S1–S6 are in the ESI.†
Conflicts of interest
DOTAP
PD
There are no conflicts to declare.
a Data analysed using GelAnalyzer.
Acknowledgements
The EPSRC is thanked for the award of a Nanotechnology
Grand Challenge grant (EP/G061521/1) to K. W., F. C., L. K.,
A. T. and for DTG studentships to A. M and H. S. The BBSRC is
thanked for the award of a grant to M. F. M. M (BBS/B/03645).
STFC is thanked for the award of neutron beam time on the
instrument LoQ at the ISIS Facility, The Rutherford Appleton
Laboratories.
The expert help of Dr Alex Drake and Dr Tam Bui
(Biomolecular Spectroscopy Centre, King’s College London) is
gratefully acknowledged for the circular dichroism/UV
measurements. We thank the UCL Mass Spectrometry and
NMR Facilities in the Department of Chemistry. The EPSRC
National Mass Spectrometry Service Centre, Swansea
University, is thanked for the provision of selected MS data.
We are also grateful to Liliane Jimenez Van Hoorn for perform-
ing the spectroscopic measurements.
Conclusions
The identification of a novel TC lipid, formed from the helper
lipid DOPE and a cationic lipid possessing a terminal hydroxyl
group, led us to design and synthesise several trichain lipids
and the corresponding DC cytofectins. The transfection
efficacies (in Opti-MEM and Opti-MEM/water) of the TC cyto-
fectins in LPD ternary vectors was significantly enhanced com-
pared to the DC analogues and therefore biophysical studies
were performed to enhance understanding of the behaviour of
lipids with three alkyl chains attached. In general, the size of
the TC lipid vesicles and LPD nanoparticles were slightly
larger than the corresponding DC-containing systems. TEM
revealed that the TC lipids formed spherical vesicles, whereas
for LPDs both spherical and rod-shaped particles were formed,
with monodisperse particles for the DCs and more polydisper-
sity with the TCs. SANS studies indicated that for vesicles with
DC and TC lipids, multilamellar structures were present: when
DC lipids were used this was low, rising to 25–50% for TC lipids.
For LPD formulations, however, there was no evidence for the
formation of multilamellar structures, only unilamellar struc-
tures of very large layer thickness, suggesting a re-arrangement
of the lipids in the presence of DNA to accommodate the inter-
action between the TCs and the negatively charged DNA. This
is the first time a re-arrangement of the lipid bilayer, on for-
mation of lipopolyplexes, has been reported. CD spectra of the
LPDs indicated that the peptide interacted with DNA preferen-
tially to the lipid, which suggests that the structure of the LPD
consists of a DNA-peptide core surrounded by a lipid bilayer as
previously shown for other DC lipids.15 Finally gel electrophor-
esis studies revealed that the TC lipids in LPD formulations
had enhanced pDNA protection properties, compared to the
corresponding DC analogues. Thus, the major enhancement
in transfection performance of the TC lipids is probably due to
their ability to protect and release DNA. At a molecular level
this may be achieved by folding back of the third hydrophobic
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