Efficient cell transfection with melamine-based gemini surfactants

Article abstract of DOI:10.1021/bc3004292

Gemini surfactants consisting of two melamine scaffolds connected by a n-hexyl linker and functionalized with a 1-propylammonium polar head and a lipophilic chain having variable carbon length (from C8 to C16) were synthesized. These were then used successfully for the transfection of A549, U87 MG, and Bristol 8 cell lines with maxGFP expressing plasmid. The transfection protocol was optimized appropriately (confluence, reagent/pcDNA ratio, compaction time, and transfection time) for each cell line. Under optimized conditions, the C12 and C14 melamine gemini surfactants showed little toxicity and remarkable transfection efficiency, superior to the gold-standard Lipofectamine 2000. These reagents were also able to efficiently transfect primary DRG neurons, which are notoriously difficult to transfect. The presence of serum completely inhibited the transfection capacity of these reagents. Owing to their ready availability, straightforward synthesis, high chemical stability (even in solution), ease of use (no formulation is required), improved transfection ability, and low toxicity, melamine-based gemini surfactants are very promising reagents for cellular DNA transfection.

Full text of DOI:10.1021/bc3004292

Article
pubs.acs.org/bc
Efficient Cell Transfection with Melamine-Based Gemini Surfactants
Serena Perrone,† Michele Usai,§ Paolo Lazzari,§,‡ Steven J. Tucker,†,* Heather M. Wallace,*,†
and Matteo Zanda*,†,∥
†Kosterlitz Centre for Therapeutics, Institute of Medical Sciences, School of Medical Sciences, University of Aberdeen, Foresterhill,
Aberdeen AB25 2ZD, Scotland, United Kingdom
§KemoTech s.r.l., Parco Scientifico della Sardegna, Edificio 3, Loc. Piscinamanna, 09010 Pula (CA), Italy
‡Department of Chemistry and Pharmacy, University of Sassari (SS), Via F.Muroni 23/A, 07100 Sassari, Italy
∥C.N.R.-I.C.R.M., via Mancinelli 7, 20131 Milano, Italy
S
* Supporting Information
ABSTRACT: Gemini surfactants consisting of two melamine
scaffolds connected by a n-hexyl linker and functionalized with
a 1-propylammonium polar head and a lipophilic chain having
variable carbon length (from C8 to C16) were synthesized.
These were then used successfully for the transfection of A549,
U87 MG, and Bristol 8 cell lines with maxGFP expressing
plasmid. The transfection protocol was optimized appropri-
ately (confluence, reagent/pcDNA ratio, compaction time, and transfection time) for each cell line. Under optimized conditions,
the C12 and C14 melamine gemini surfactants showed little toxicity and remarkable transfection efficiency, superior to the gold-
standard Lipofectamine 2000. These reagents were also able to efficiently transfect primary DRG neurons, which are notoriously
difficult to transfect. The presence of serum completely inhibited the transfection capacity of these reagents. Owing to their ready
availability, straightforward synthesis, high chemical stability (even in solution), ease of use (no formulation is required),
improved transfection ability, and low toxicity, melamine-based gemini surfactants are very promising reagents for cellular DNA
transfection.
delivery are usually divided into two major groups: viral and
nonviral.
INTRODUCTION
■
Gene transfection is a powerful tool to study protein expression
and gene function in cells. The development of efficient and
reliable gene delivery systems remains a very active area of
research. The currently available systems are affected by a
number of drawbacks, most notably the prohibitive costs of
most transfectants and the need to rely on complex
formulations, which affect the long-term reagents’ stability
and require careful storage.1
As a result of size and charge, and several cellular barriers, the
spontaneous entry of naked DNA and its subsequent
expression by the replicative machinery of the cell is a very
inefficient process. For this reason, the success of gene
transfection is largely dependent on the development of a
suitable carrier molecule (“vector” or “vehicle”) that can deliver
DNA selectively and efficiently to target cells, leading to the
expression of the therapeutic gene.
Over the years, a range of techniques and vehicles for the
transfer of nucleic acids into cultured eukaryotic cells
(transfection) have been developed. Early transfection methods
include calcium phosphate coprecipitation2 and physical
methods, such as direct microinjection of nucleic acids into
cells or nuclei3 and electroporation,4 where cell membranes
become permeable to DNA by exposure to electrical fields.
More recent approaches involve the use of a carrier molecule
to deliver genetic material to the cells. Vehicles for gene
Although viral vectors (mainly recombinant retrovirus and
adenovirus) have been found to be effective in gene transfer,
they have several intrinsic drawbacks. For adenoviral vectors,
packaging capacity is low, and production is labor-intensive.5
With retroviral vectors, there is the potential for activation of
latent disease and, if there are replication-competent viruses
present, activation of endogenous retrovirus and limited
transgene expression.6 Furthermore, viral capsids have the
potential to generate a severe immune response in vivo, as has
been demonstrated in a number of animal experiment and
clinical trials.7−10 As a result, nonviral gene delivery is currently
the subject of increasing attention because of its relative safety
and ease of use.
Among nonviral-based approaches, cationic lipid-mediated
gene transfer or lipofection represents the most extensively
investigated method.11−15 Since first being used as novel
transfection vectors in 1987 by Felgner et al.,16 numerous
cationic lipids (also called cytofectins or lipofection reagents)
have been synthesized and used for delivery to cell culture,
animals, and patients enrolled in phase I, II, and III of clinical
trials (http://www.wiley.co.uk/genmed/clinical/).
Received: August 3, 2012
Revised: January 8, 2013
Published: January 8, 2013
© 2013 American Chemical Society
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These reagents contain amphiphilic molecules with a
hydrophobic hydrocarbon domain connected to a cationic
polar headgroup via a linker structure. The positive charge on
the headgroup facilitates spontaneous electrostatic attraction to
the phosphate backbone of DNA, leading to compaction of
DNA and the formation of cationic lipid/DNA complexes
(lipoplexes). In addition, the positive charge facilitates binding
of the resulting lipoplexes to the negatively charged
components of the cell membrane prior to cellular uptake.17−19
Cationic lipids possess many features of an “ideal” gene
delivery vector. Although the gene expression levels obtained
using these systems are lower than with viral vectors, they
present several advantages including safety, large-scale
production, and capacity to deliver large gene fragments.
Moreover, since they are easy to produce and to modulate
chemically for improved transfection efficiency, research efforts
in this particular area have increased dramatically in recent
years.18
Since the initial description, many cationic lipids, generally
bearing either a single tertiary or quaternary ammonium
headgroup or containing protonatable polyamines linked to
dialkyl or cholesterol anchors, have been designed for
transfection of a variety of cell types in culture.18−22
These lipids are usually formulated as an aqueous liposome
suspension containing two lipid species, a cationic amphiphile
and a neutral phospholipid, typically dioleoyl phosphatidyl
ethanolamine (DOPE). DOPE is often used in conjunction
with cationic lipids to achieve high transfection efficiency
because of its membrane destabilizing effects which promote
endolysosomal escape.23
Cationic liposomes with the polycationic lipid 2,3-dioleyloxy-
N-[2-spermine carboxamide] ethyl-N,N-dimethyl-1-propanam-
monium trifluoroacetate (DOSPA) and DOPE at 3:1 w/w are
available commercially as Lipofectamine Reagent; Lipofectin
Reagent is a 1:1 w/w liposome formulation of the cationic lipid
N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium
(DOTMA) and DOPE. Other important commercially
available reagents for lipofection include 1,2-bis(oleoyloxy)-3-
(trimethylammonio)propane (DOTAP), N-[1-(2,3-
dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl) am-
monium bromide (DMRIE), 3β-[N-(N′,N′-dimethylamino-
ethane) carbamoyl]cholesterol (DC-Chol), and dioctadecyl
amidoglyceryl spermine (DOGS).
In the panorama of new positively charged amphiphiles,
gemini surfactants (GS),24,25 featuring two hydrophobic tails
and two polar head groups connected by a linker, seem
promising compounds. They typically show greatly enhanced
physical−chemical properties compared to their corresponding
monomeric (single chain, single headgroup) counterparts.24−26
This makes them of special interest for biomedical applications,
such as gene delivery, where they are generally much more
efficient transfection reagents than the corresponding mono-
meric structures.26,27
In this study, we report new nonviral gene transfer vectors
belonging to the GS class. Five novel triaminotriazine
(melamine)-based GS, that differ only in the hydrophobic tail
length, have been synthesized; and their ability to transfect
plasmid DNA containing maxGFP reporter gene into adherent
and suspension cell lines, as well as primary cells, has been
examined.
In vitro transfection experiments demonstrated a remarkable
correlation between the length of the lipophilic tail and the
transfection efficiency, and a structure−activity relationship was
identified.
Unlike many cationic lipids used in gene transfection studies,
these new melamine-based GS are synthesized from relatively
inexpensive starting materials, are easy to handle, and are
readily soluble at room temperature in aqueous medium.
Moreover, some of these compounds are very effective in cell
transfection using a simple experimental protocol, in which no
formulation or liposome preparation is required. Last but not
least, these reagents are stable indefinitely in aqueous solution
at room temperature (they can be stored for months on the
bench, without any apparent deterioration or loss of perform-
ance). This makes these molecules efficient, cost-effective, and
therefore very attractive and competitive reagents for gene
delivery applications.
EXPERIMENTAL SECTION
■
Materials. pMaxGFP vector (3.4 kb), encoding the green
fluorescent protein from Potellina sp., was purchased from
Lonza. Lipofectamine 2000 was obtained from Invitrogen Life
Technologies. All chemicals were of reagent-grade or higher
quality and were purchased from Sigma unless otherwise
specified.
Methods. Chemistry. Experimental procedures for the
synthesis of gemini surfactants GS C8−C16 and spectro-
scopic/analytical characterization of all the compounds
described herein are provided in the Supporting Information
section.
Cell Culture. Transfection studies were performed using two
adherent cell lines, A549 (human lung adenocarcinoma
epithelial) and U-87 MG (human glioblastoma-astrocytoma,
epithelial-like) cell lines, and a suspension cell line, Bristol 8
(normal human HLA-A2 positive B lymphoblastoid) cell line.
These cell lines were maintained in culture according to
ECACC guidelines.
Primary dorsal root ganglion (DRG) neurons were also used
for in vitro transfection experiments and were prepared as
previously described (Murray et al., 2009).
In preparation for transfection experiments of adherent cell
lines, cells were seeded at a density of 7.5 × 104 cells/cm2 in 35
mm culture dishes the day prior to transfection. The cells were
incubated for 24 h at 37 °C to reach ∼70% confluence on the
day of transfection.
For the Bristol 8 suspension cell line, on the day of
transfection the cell pellet was suspended in serum-free
medium and 500 μL of cell suspension was seeded at a density
of 7.5 × 104 cells/cm2 in 35 mm culture dishes.
For the primary DRG neurons, cells were removed from the
animal and dissociated into culture dishes coated with
polylysine (1 μg/mL) and laminin (10 μg/mL) to promote
adherence to the culture surface.
Although a large number of cationic lipids have been
synthesized, most of the present liposomal formulations are not
useful due to their poor performance, including cytotoxicity and
low transfection efficiency,28 especially compared to viral
vectors. Furthermore, the prices associated with most
commercially available transfection tools are very high. For
these reasons, there is still a need to develop cationic lipid-
based gene transfer systems with improved transfection
properties and low cytotoxicity that are readily accessible,
easy to use, cheap, and cost-effective.
In Vitro Transfection. Transfection Protocol for Adherent
Cells. The A549 and U-87 MG transfection protocol using GS
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a
Table 1. A549 Cells and GS C8-C16
parameters optimized
seeding density
GS C8
GS C10
GS C12
GS C14
GS C16
7.5 × 104 cells/cm2 7.5 × 104 cells/cm2 7.5 × 104 cells/cm2 7.5 × 104 cells/cm2 7.5 × 104 cells/cm2
concentration of GS used in lipoplex preparation
---
---
6 μM
6 μM
6 μM
6 μM
GS-pcDNA complex formation time (compaction
time)
60 min
60 min
60 min
30 min
exposure time
---
4 h
4 h
4 h
4 h
a
Optimal transfection conditions. Transfection efficiency for each GS was evaluated by visualization of maxGFP positive cells under fluorescent
microscope and confirmed quantitatively using flow cytometry.
a
Table 2. U-87 MG Cells and GS C8-C16
parameters optimized
GS C8
GS C10
GS C12
GS C14
GS C16
seeding density
7.5 × 104 cells/cm2 7.5 × 104 cells/cm2 7.5 × 104 cells/cm2 7.5 × 104 cells/cm2 7.5 × 104 cells/cm2
concentration of GS used in lipoplex preparation
---
---
6 μM
6 μM
3 μM
6 μM
GS-pcDNA complex formation time (compaction
time)
60 min
30 min
45 min
30 min
exposure time
---
4 h
4 h
4 h
4 h
a
Optimal transfection conditions. Transfection efficiency for each GS was evaluated by visualization of maxGFP positive cells under fluorescent
microscope and confirmed quantitatively using flow cytometry.
C8-C16 was developed and optimized throughout the passage
of this work.
GFP Detection. Cellular expression of maxGFP in A549, U-
87 MG, Bristol 8, and DRG cells was evaluated 24 h post-
transfection using fluorescence microscopy. The cells were
imaged and photographed using Nikon Eclipse E400 micro-
scope equipped with Q-Imaging QICAM Fast 1394 Color 12
bit digital camera. Images were captured using ImageJ software.
FACS Analysis. A549 and U-87 MG cells were transfected
using GS C8-C16 surfactants, according to the optimized
protocols, and using optimized Lipofectamine 2000 (positive
control), according to the manufacturer’s protocols. Non-
transfected samples (negative control) were cultured at the
same time and had the same period of time in serum-free
medium as transfected samples.
One day post-transfection, the cells were dissociated from
their culture dishes with trypsin-EDTA solution, and trans-
fection efficiency, evaluating the percentage of cells expressing
maxGFP in each sample, was measured by means of
fluorescence-activated cell sorting (FACS) analysis.
Gates and instrument settings were set according to forward
and scatter characteristics, and a total of 10 000 events were
collected for each sample. Data, based on triplicate measure-
ments, were expressed as means as mean SEM.
Briefly, GS 20× stock solutions were prepared by dissolving
cationic lipids in distilled water. Preparations of GS molecules
in distilled water are stable for at least 6 months at 4 °C.
GS-pmaxGFP lipoplexes were obtained by simple mixing 1
μg of pmaxGFP vector (diluted in 500 μL of serum free
medium) with an equal volume of the cationic lipid solution in
serum free medium. At this stage, pcDNA concentration was 1
μg/mL and GS lipid was at the desired concentration.
The GS-pcDNA mixture was incubated at room temperature
for the indicated compaction time to allow the transfection
complexes to form and these were added to dishes containing
cells. Before adding lipoplexes, the cells were rinsed in serum
free medium to remove traces of serum.
Following the indicated exposure time at 37 °C (typically 4
h), the transfection medium was replaced by fresh serum-
containing culture medium. A similar protocol was used to
assess the capacity of GS C12 and C14 to transfect primary
DRG neurons. Control transfections were carried out using
optimized Lipofectamine 2000, according to the manufacturer’s
protocols.
Transfection Protocol for Suspension Cells. Bristol 8 cells
were transfected with GS C12 or C14 surfactants using the
optimal lipofection conditions found for both A549 (Table 1)
and U-87 MG (Table 2) cells. Briefly, GS C12 or GS C14 20×
stock solutions were prepared by dissolving cationic lipids in
distilled water.
GS-pmaxGFP lipoplexes were prepared by simple mixing 1
μg of pmaxGFP vector (diluted in 250 μL of serum free
medium) with an equal volume of the cationic lipid solution in
serum free medium. At this stage, pcDNA concentration was 2
μg/mL and GS lipid was at 2× concentration.
RESULTS AND DISCUSSION
■
The novel synthetic transfection reagents GS C8, C10, C12,
C14, and C16 are melamine-based gemini surfactants with an
overall net positive charge determined by the presence of 8
equiv of trifluoroacetic acid. Their structures are shown in
Figure 1.
These molecules are composed of two units, each consisting
of a triaminotriazine (melamine) scaffold supporting three
different side chains: (1) a linear lipophilic tail with increasing
The GS-pcDNA mixture was incubated at room temperature
for the indicated compaction time to allow the transfection
complex to form, and these were added to dishes containing
500 μL of cells. At this stage, plasmid DNA concentration was 1
μg/mL and GS lipid was at the desired concentration.
Following the indicated exposure time at 37 °C (typically 4
h), 2 mL of serum-containing culture medium was added to the
dish.
Figure 1. Chemical structure of the five cationic amphiphiles used in
this study.
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a
Scheme 1. Synthesis Scheme for GS C8-C16
a
Reagents and conditions: Step 1: CH3(CH2)nNH2, DIPEA, acetonitrile, room temperature (RT), 2 h, 45−60%; Step 2: NH2(CH2)6NH2, DIPEA,
acetonitrile, RT, 3 h, 32−35%; Step 3: NH2(CH2)3NHBoc, benzene (anhydrous), 125 °C, sealed tubes, 24 h, 52−83%; Step 4: TFA, DCM
(anhydrous), RT, 3 h, 90−95%. Key: DIPEA = N,N-diisopropylethylamine, TFA = trifluoroacetic acid, DCM = dichloromethane.
alkyl chain length of 8 (GS C8), 10 (GS C10), 12 (GS C12),
14 (GS C14), and 16 (GS C16) carbon atoms, (2) a 3-
propylammonium group which acts as cationic polar head, and
(3) a n-hexyl spacer connected to the melamine headgroup of
the other unit (Figure 1).
melamine (C3H6N6) has been shown to crystallize with two
molecules of trifluoroacetic acid, each protonating a ring
nitrogen.33b It is therefore likely that the samples of GS 8−16
used in this study contained 2 to 4 equiv of TFA in excess, as a
result of the specific workup, isolation, and purification
procedures used herein (see Supporting Information for
details). However, no significant cell toxicity that could be
ascribed to TFA was ever observed at the concentrations of GS
8−16 used in the transfection experiments. It is worth noting
that the serum free medium used in the transfection
experiments contained a bicarbonate buffering system that
was supplemented by storing cells in an incubator supplying 5%
CO2. Additionally, the medium contained the pH indicator
phenol red. Throughout all of the experiments described, no
change in the color of the indicator was observed, and
therefore, no significant changes in the pH of the cell cultures
were evident.
Optimization of the Transfection Protocol for Each GS
Using A549 and U87mg Cells. The cationic compounds
used in this study maintain common features in the head
groups and spacer, but vary the length of the lipophilic tails
(Figure 1). It is well-known that the hydrophobic moiety of
amphiphiles is a key determinant of lipoplex structure and
lipofection activity. Therefore, we looked for a structure−
activity relationship to determine whether differences in the
alkyl side chain length resulted in altered biological activity.
The gene transfer properties of the surfactants GS C8-C16
were studied using a maxGFP expressing plasmid in two
different adherent cell lines, A549 (human lung adenocarcino-
ma epithelial) and U-87 MG (human glioblastoma-astrocyto-
ma, epithelial-like) cells. Since GS C8-C16 are novel
compounds, before making a comparison across all GS, the
optimal conditions for transfection of both cell lines were
determined. A successful transfection requires several variables
to be optimized. In fact, a number of parameters can greatly
influence transfection efficiency, such as: (a) degree of cell
confluence, (b) ratio of transfection reagent to DNA, (c) length
of incubation time between DNA and cationic lipid
These amphiphilic GS molecules are highly stable, with a
considerably improved chemical stability compared to the
triazine-based redox-sensitive agents previously described in the
literature.29−32 The presence of a saturated alkyl spacer makes
GS C8-C18 compounds resistant to air oxidation and to the
intracellular reducing environment with respect to the cationic
lipids based on the thiol/disulfide (-SH/-SS-) equilibrium.29−32
The preparation of the new cationic lipids is relatively quick,
produces gram amounts of the target GS molecules, and uses
relatively inexpensive materials, which certainly increases the
value of these melamine-based GS as transfection reagents. The
synthesis of GS 8−16 proceeded via a simple multistep strategy
making use of cyanuric chloride 1 as starting material, as shown
in Scheme 1. The activated triazine ring of 1 allows sequential
dechloroamination substitution reactions with nitrogen nucle-
ophiles. The hydrophobic hydrocarbon moiety was introduced
first on the triazine core (Scheme 1). The single tail
dichlorotriazine 2 was then reacted with 1,6-diaminohexane
to provide the dimeric compounds 3a−e featuring two
symmetric single tail-monochlorotriazine units connected via
the n-hexyl spacer (step 2, Scheme 1). The subsequent
dechloroamination reaction (step 3), followed by cleavage of
the N-protecting group (step 4) produced the amphiphilic GS
8−16 as trifluoroacetic salts, which were purified by flash
chromatography on silica gel (Scheme 1).
The number of equivalents of CF3CO2H (TFA) incorpo-
rated by each GS was assessed by 19F NMR titration
experiments, using trifluoromethylbenzene as an internal
probe. Each GS was found to incorporate 8 equiv of TFA;
therefore, the basic primary amine functions and some nitrogen
atoms of each melamine unit are actually protonated in these
molecules. The pKb of melamine is 9.00 vs a pKb = 3.29 of a
basic dialkylamine such as dimethylamine.33a Recently,
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(compaction time), (d) length of the time cells are exposed to
DNA-cationic lipid complex (exposure time), and (e) presence
or absence of serum. The optimization results of each GS and
A549 and U-87 MG cells are summarized in Table 1 and 2,
respectively, and discussed below.
Seeding Density (Confluence). Usually it is necessary to
transfect cells at 60−80% confluence. Too few cells will cause
the culture to grow poorly as a result of lack of cell-to-cell
contact. Too many cells results in contact inhibition, making
cells resistant to uptake of foreign DNA. Ideally, cells should be
in an exponential phase of growth. For the in vitro experiments,
A549 and U-87 MG cells were seeded on 35 mm dishes the day
prior to transfection at 7.5 × 104 cells/cm2 density and allowed
to adhere overnight (Tables 1−2). For both cell lines, this
seeding density was found to produce ∼70% confluent cells on
the day of transfection.
maximum GFP expression in both cell lines (Tables 1−2).
Despite the use of this optimal lipid concentration, the
transfection efficiency of GS C10 or C16 based complexes
remained modest (approximately 10%). A higher transfer
activity was observed with GS C12 and C14. For GS C12
surfactant, 6 μM worked most effectively with U-87 MG cells
(Figure 2) and with A549 cells (Tables 1−2). For the GS C14
Concentration of Cationic Transfection Reagent and
Compaction Time. The key step in the lipofection process is
the formation of the complex between cationic lipids and DNA
(lipoplex) and the consequent compaction of the extended,
high molecular-mass, negatively charged DNA into a dense,
positively charged particle small enough to be taken up by the
cell.26 Electrostatic interactions between the positively charged
cationic lipid head-groups and the negative phosphate DNA
backbones are the main driving forces for lipoplex formation
and DNA compaction.19 Hydrophobic interactions between the
apolar hydrocarbon tails enhance compaction further.34 This
step determines the characteristics and thus the transfection
potential of lipoplexes. Consequently, both the cationic
transfection reagent-to-DNA ratio and the compaction time
are factors that should be considered carefully in any protocol
of lipoplex preparation.
Concentration of GS Reagent (Ratio of Cationic Trans-
fection Reagent to pcDNA). In the lipoplex, the amount of
positive charge contributed by cationic lipids should equal or
exceed the amount of negative charge contributed by the
phosphates on the DNA backbone, resulting in neutral or
positively charged cationic lipid/nucleic acid complex. It is
widely accepted that preparation of lipoplexes with an overall
net positive charge confers a higher transfer efficiency,
presumably because this allows a more efficient interaction of
the complexes with the negatively charged cell surface.16,35,36
For each GS molecule, by varying the concentration of the
gemini surfactant (3 μM, 6 μM, 12 μM, 25 μM) and keeping
constant the concentration of pmaxGFP (1 μg/mL), different
GS-pcDNA lipoplexes, at increasing charge ratio (CR ), were
prepared. Transfection ability of each GS-pcDNA preparation
was then evaluated qualitatively, by visualization of the cells
under fluorescence microscope. No formulation or liposome
preparation was required for our transfection protocol and all
the GS were readily dissolved in distilled water. GS-pcDNA
complexes were obtained by simple mixing of the GS solution
(at the desired concentration) and the pcDNA solution in
serum free medium. The GS-pcDNA mixture was incubated for
a compaction time of 15 min at room temperature and then the
cells were exposed to GS-pcDNA complexes for 4 h at 37 °C
(see Experimental Section). By observation of maxGFP positive
cells, it was found that GS C8 based lipoplexes were not able to
transfer DNA into A549 or U-87 MG cells, whereas
transfection was observed with all the other gemini
amphiphile-based lipoplexes (GS C10−C16).
Figure 2. U-87 MG cells transfected with different concentrations of
GS C12 amphiphile. Images collected 24 h post-transfection by
fluorescence microscopy (10× magnification); images are representa-
tive of at least three other independent experimental repeats. In vitro
transfection experiments were performed using lipoplexes prepared
with increasing GS C12 concentration (3 μM, 6 μM, or 25 μM) and a
pmaxGFP concentration of 1 μg/mL. The GS C12-pcDNA mixture
was incubated for 15 min (compaction time) at room temperature.
The cells were then exposed to GS C12-pcDNA lipoplexes for 4 h.
(A) GS C12 concentration of 3 μM led to a nonoptimal GFP
expression in U-87 MG cells. (B) A very effective U-87 MG
transfection and a low toxicity was obtained using GS C12
concentration of 6 μM. (C) GS C12 concentration of 25 μM resulted
in a decrease in the number of fluorescent cells and an increase in cell
death.
molecule, a concentration of 6 μM (CR = 16) and 3 μM (CR
= 8) led to an effective transfection with A549 and U-87 MG
cell line, respectively (Tables 1−2). A visible decrease in the
number of U-87 MG fluorescent cells was observed using GS
C14 concentrations lower than 3 μM, confirming 3 μM as the
optimal concentration for this cell line. As shown in Figure 2
for GS C12 and U-87 MG cells, an increase in lipid
concentration improved transfection. However, high levels of
GS C12 (above the optimal concentration) resulted in
decreased fluorescence (Figure 2C). The decline in transfection
activity at high concentrations of GS C12 correlated with a
A GS C10 and C16 concentration of 6 μM (lipoplexes at CR
= 16), among the different concentrations tested, led to the
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visible reduction in the number of viable cells per dish (Figure
2C). This increase in cell death may be due to the
incorporation of the cationic chemical reagents into cellular
membranes. At high concentrations, the cationic lipid content
of cellular membrane may be sufficient to significantly alter the
net charge of the membranes and adversely affect the activity of
a number of enzymes, membrane receptors, and ion channels.
Alternatively, the cationic lipids could interfere with the
attachment of the cells to the extracellular matrix, leading to
the detachment of cells from the dish.37,38 It should be noted
though that, throughout, toxicity was only observed at high
surfactant concentrations (>12 μM), and the effective trans-
fection concentrations were well tolerated as determined by
MTT assay. At the concentrations of GS C12 and GS C14 used
in optimized transfections, the cell populations remained at an
equivalent (or higher) level of viability than those cell
populations transfected with Lipofectamine 2000 (data not
shown).
GS-pcDNA Complex Formation Time (Compaction Time).
A key aspect of the transfection protocol optimization is the
time required for complex formation between the pcDNA and
GS lipids (compaction time). The compaction of DNA by
cationic surfactants presumably involves an initial interaction of
a small cluster of surfactant molecules with DNA. Cationic
amphiphiles can compact and stabilize polyanionic nucleic acids
by a combination of attractive electrostatic interactions and
hydrophobic interactions between the apolar hydrocarbon
tails.34 In order to evaluate the best pcDNA-GS incubation
time, the appropriate amount of pmaxGFP vector (diluted in
serum free medium) was mixed with diluted GS used at optimal
concentration established by the previous set of transfection
experiments (GS C14 concentration was 3 μM for U-87 MG
cells; all the other GS concentrations were 6 μM for both cell
lines). The GS-pcDNA mixture was incubated for different
times (0 min, 15 min, 30 min, 45 min, 60 min, 90 min) at room
temperature and then added to dishes containing cells.
The results of the in vitro transfection experiments, analyzed
by fluorescence microscopy, show for each GS molecule an
increase of maxGFP expression levels with compaction time,
until an optimum is reached (Figure 3A,B). Exceeding the
optimal time decreased maxGFP positive cell numbers (Figure
3C). This decrease in fluorescence may be related to a loss of
energy or degradation of the complexes with time. Therefore, if
left too long the complexes became progressively less effective
at transfecting cells.
For GS C10 and C16, the optimal GS-pcDNA compaction
time was observed to be cell-type independent. In particular, for
GS C10, fluorescence microscopy revealed a pcDNA-GS
incubation time of 60 min as optimal for successful transfection
of both cell lines (Tables 1−2). For GS C16-pcDNA
lipoplexes, an optimal transfection of both cell lines was
obtained after a 30 min compaction time (Tables 1−2). For GS
C12 and C14, the optimal time of compaction changed
depending on the cell line being studied. A GS C12-pcDNA
compaction time of 60 and 30 min corresponded to the most
effective transfection of A549 and U-87 MG cells, respectively
(Tables 1−2). By comparing the images of the A549
transfection experiments (Figure 3), it is clear that 60 min is
the best GS C14-pcDNA compaction time. When U-87 MG
cells were used, an incubation time of 45 min led to a very
effective transfection (Table 2).
Figure 3. A549 cells and GS C14-pcDNA compaction time. Images
collected 24 h post-transfection by fluorescence microscopy (10×
magnification); images are representative of at least three other
independent experimental repeats. The GS C14-pcDNA mixture,
prepared using a surfactant concentration of 6 μM and pmaxGFP
concentration of 1 μg/mL, was incubated at room temperature for
increasing compaction times (0, 60, or 90 min). The cells were then
exposed to GS C14-pcDNA lipoplexes for 4 h. (A) A nonoptimal
maxGFP expression in A549 cells was achieved with a GS C14-pcDNA
compaction time of 0 min. (B) A very effective transfection was
obtained using a GS C14-pcDNA compaction time of 60 min. (C) A
GS C14-pcDNA compaction time of 90 min led to a decrease in A549
fluorescent cells.
Different cell lines, having different membrane properties,
composition, and charge respond differently to GS-pcDNA
complexes. The compaction time will determine the nature and
″maturity″ of these complexes, and their most optimal form is
slightly different between the two cell types.
Exposure Time (or Transfection Time). In the previous
transfection experiments, both A549 and U-87 MG cells were
incubated with GS-pcDNA mixture for 4 h. The growth
medium containing lipoplexes was then replaced with fresh
medium. To help minimize toxic effects of the GS-pcDNA
complexes and find the best exposure time, the initial
incubation time was varied between 30 min and 24 h (30
min, 1 h, 2 h, 4 h, 6 h, 24 h). As shown in Figure 4 for GS C12
based lipoplexes and U-87 MG cells, replacement of the
medium after 30 min of cell incubation with GS-pcDNA
mixture led to few fluorescent cells per dish (Figure 4A). As the
treatment time with GS C12-pcDNA complexes increased,
higher transfection efficiency was observed. A 4 h transfection
time was confirmed to be optimal for U-87 MG cells, achieving
the best compromise between the number of cells transfected
and alive at the end of the process (Figure 4B). Exposure times
The results obtained with GS C12 or GS C14 amphiphiles
show a clear cell-type dependence for compaction time.
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transfect, even over extended periods of exposure (data not
shown).
FACS Analysis. Once the optimal transfection conditions
were found, a quantitative comparison of transfection proper-
ties between all the GS molecules was carried out. Furthermore,
the capacities of compounds GS C8-C16 to mediate the
transfer of maxGFP gene reporter across A549 and U-87 MG
cell membranes were compared to that of Lipofectamine 2000
(Life Technologies), regarded as the gold standard of
commercially available transfection tools. Fluorescence-acti-
vated cell sorting (FACS) analysis was performed in order to
get some quantitation for each of the optimized surfactant
protocols, and consistently these confirmed the qualitative
judgements made using the fluorescence microscope. Figures 5
and 6 show FACS analysis for selected experiments, specifically
maxGFP expression levels, presented as the fluorescence
intensity per cell number (fluorescent intensity, x-axis; cell
number, y-axis) in A549 and U-87 MG cells using Lipofect-
amine 2000, GS C10, C14, and Lipofectamine 2000, GS C12,
C16 vectors, respectively. The results indicate that A549
transfection with GS C14 (Figure 5D) yielded a higher
fluorescence intensity per population than Lipofectamine 2000
(Figure 5B) (i.e., maxGFP expression in population D is shifted
further to the right than population B). On the contrary, the
level of maxGFP expression obtained in GS C10 transfected
cells (Figure 5C) was lower than the Lipofectamine 2000
transfected sample.
Compared to the positive control (Figure 6B), U-87 MG
cells transfected with GS C12 vector exhibited a significantly
higher fluorescence intensity (Figure 6C), while lower levels of
maxGFP expression were obtained with GS C16 surfactant
(Figure 6D). FACS analysis also showed that cell populations
were consistent, healthy, and comparable across all experiments
(data not shown).
Figure 4. U-87 MG cells and different exposure times to GS C12-
pcDNA lipoplexes. Images collected 24 h day post-transfection by
fluorescence microscopy (10× magnification); images are representa-
tive of at least three other independent experimental repeats. The cells
were incubated with GS C12 based lipoplexes (GS C12 concentration
= 6 μM, pmaxGFP concentration = 1 μg/mL, compaction time GS
C12-pmaxGFP = 30 min) for 30 min, 4 h, or 24 h. (A) A low U-87
MG transfection efficiency was obtained with 30 min transfection time.
(B) U-87 MG cells incubated with GS C12-based lipoplexes for 4 h
showed high levels of maxGFP expression and the morphology of the
nontransfected cells. (C) Exposure time of 24 h resulted in an
inhibition of cell growth.
FACS results are summarized and population fluorescence is
quantified in Figures 7 and 8, where the percentage of the
sample population that has shifted outside the M1 regions
indicated in Figures 5 and 6 is plotted. This gives a measure of
the comparative distribution and level of fluorescence within
the population. In both cell types, the transfection efficiency of
surfactants GS C8-C16 exhibited a Gaussian bell-shaped
distribution with the highest transfection performance corre-
sponding to the GS C12 amphiphile. Particularly remarkable is
that its transfection properties are significantly better (3−4
times higher) than that of Lipofectamine 2000. As shown in
Figures 7 and 8, 44% and 74% of cells were fluorescent using
GS C12, whereas only 11% and 23% were fluorescent following
Lipofectamine 2000 transfection in A549 and U-87 MG cells,
respectively. The percentage of A549 reporter gene-expressing
cells transfected with the compound GS C14 was also higher
than for the Lipofectamine 2000-transfected cells (Figure 7).
GS C14 was at least as effective as Lipofectamine 2000 in U-87
MG cells (Figure 8). The compounds with a lipophilic tail
shorter than C12 (GS C10) or longer than C14 (GS C16)
demonstrated a low transfection capacity. The GS C8 molecule
was completely ineffective.
longer than 4 h resulted in mild inhibition of cell growth
(Figure 4C).
Removal of the medium containing the GS C12-pcDNA
complexes after 4 h also resulted in the highest transfection
efficiency for A549 cells (Table 1). Similar results were
obtained from the in vitro transfection experiments with all the
other GS based lipoplexes showing an optimal exposure time of
4 h for both cell lines (Tables 1−2). In each case, light
microscopy analysis of transfected cells showed that the
morphology of the nontransfected cells was preserved (data
not shown).
Effect of Serum. Transfection protocols often require
serum-free conditions for optimal performance because serum
can interfere with many commercially available transfection
reagents. It is thought that serum components, such as
negatively charged proteins, may destabilize the positively
charged lipid/DNA complex, leading to lipid vector disintegra-
tion, with DNA release and degradation.39,40 The experiments
performed with GS molecules, in both A549 and U-87 MG cell
lines, showed that these cationic lipids also need serum free
conditions for successful transfection. In fact, the presence of
serum completely inhibited the ability of these compounds to
These results allowed us to identify a consistent structure−
activity relationship. In particular, the activity of the cationic
surfactants is affected significantly by changes in the
composition of the aliphatic tail. As the aliphatic chain length
was increased from 12, 14, and 16 carbon atoms, the
transfection capacity of the resulting compounds GS C12,
C14, and C16 progressively declined (Figures 7 and 8). Our
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Figure 5. Expression of maxGFP in A549 cells evaluated by FACS analysis 24 h post-transfection. Optimized Lipofectamine 2000 was used as
positive control according to the manufacturer’s procedures (B). Transfections of A549 cells with GS C10 (C) and C14 (D) were performed in
serum free conditions according to the optimized parameters reported in Table 1. (A) A549 cells in the absence of transfectants (negative control).
(B) A549 cells transfected with Lipofectamine 2000 (positive control). (C) A549 cells transfected with GS C10. (D) A549 cells transfected with GS
C14.
Figure 6. Expression of maxGFP in U-87 MG cells evaluated by FACS analysis 24 h post-transfection. Optimized Lipofectamine 2000 was used as
positive control according to the manufacturer’s guidelines (B). Transfections of U-87 MG cells with GS C12 (C) and C16 (D) were performed in
serum free conditions according to the optimized parameters reported in Table 2. (A) U-87 MG cells in the absence of transfectants (negative
control). (B) U-87 MG cells transfected with Lipofectamine 2000 (positive control). (C) U-87 MG cells transfected with GS C12. (D) U-87 MG
cells transfected with GS C16.
results were consistent with previous studies.41−43 Surfactant-
mediated gene delivery requires a membrane destabilization of
endosomal compartments, which allows translocation of the
gene into the cytosol via an as yet poorly defined
mechanism.19,44 An exchange between cationic lipid molecules
in the lipoplex and endosomal membrane has been suggested to
weaken the integrity of the endosome leading to pcDNA
cytoplasmic delivery.19,44 The shorter alkyl tails, favoring a
higher intermembrane transfer rate and faster rate of lipid
mixing,45,46 may enhance the endosome escape and thus the
transfection capacity of GS molecules. The low transfection
efficiency or the complete ineffectiveness of the compounds
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for both surfactants, A549 optimal transfection conditions
worked very effectively with Bristol 8 cells.
Figure 9. Bristol 8 cells transfected with GS C12.
Figure 7. Transfection efficiency of compounds GS C8-C16 in A549
cells 24 h post-transfection. In vitro transfection experiments, for each
GS, were performed according to the optimal conditions reported in
Table 1. Transfection capacity of GS C8-C16 was compared with that
of optimized Lipofectamine 2000 (used according to the manufac-
turer’s guidelines). Fluorescent values are derived from Figure 5 and
quantify the % of each cell population that elicits fluorescent signal
beyond the M1 threshold indicated in Figure 5. Data are expressed as
Figure 10. Bristol 8 cells transfected with GS C14.
mean
SEM, and are compared using one-way ANOVA with a
Bonferroni post hoc test. *** = p < 0.005.
Images collected 24 h post-transfection by fluorescence
microscopy (10× magnification); images are representative of
at least three other independent experimental repeats. Trans-
fection experiments were performed in serum free conditions
according to A549 optimized parameters reported in Table 1.
Images collected 24 h post-transfection by fluorescence
microscopy (10× magnification); images are representative of
at least three other independent experimental repeats. Trans-
fection experiments were performed in serum free conditions
according to A549 optimized parameters reported in Table 1.
Transfection of Primary DRG Neurons Using GS C12
and GS C14. The surfactants GS C12 and C14 were also
tested for their ability to transfect primary cell cultures, such as
primary DRG (dorsal root ganglion) neurons, which are
notoriously difficult to transfect. These DRG neurons, obtained
from a rat one day prior to transfection, were plated in 35 mm
culture dishes with the growth area coated with polylysine (1
μg/mL) and laminin (10 μg/mL) to aid adherence of the cells
to the substratum as previously described.47,48 DRG neurons
were transfected in the absence of serum with increasing
concentrations of GS C12 or C14 (3 μM, 6 μM, 12 μM).
Lipofectamine 2000 was used as positive control in the
transfection experiments, although it lacks efficacy with cells of
this type. Images in Figure 11 clearly show 6 μM of GS C14
not only transfected these cells, but also did so with a greater
potency than Lipofectamine 2000. Interestingly, the neurons
visible in the bright field channel of Figure 11B show clear
evidence of neurite outgrowth, and this is more pronounced in
GS-transfected than Lipofectamine-transfected cells (compare
Figure 11A,B). This provides evidence that these cells have
tolerated this procedure particularly well, as such outgrowth
only occurs if cells are physiologically functional and “settled”.
Indeed, there is a further suggestion here that a greater level of
outgrowth is occurring post transfection with GS C14 and this
transfection method is therefore at least as well tolerated as
Lipofectamine. For the GS C12 surfactant, a 6 μM
concentration of surfactant also led to transfection substantially
more effective than with Lipofectamine 2000 (data not shown).
Figure 8. Transfection efficiency of compounds GS C8-C16 in U-87
MG cells 24 h post-transfection. In vitro transfection experiments, for
each GS, were performed according to the optimal conditions reported
in Table 2. Transfection capacity of GS C8-C16 was compared with
that of optimized Lipofectamine 2000 (used according to the
manufacturer’s guidelines). Fluorescent values are derived from Figure
6 and quantify the % of each cell population that elicits fluorescent
signal beyond the M1 threshold indicated in Figure 6. Data are
expressed as mean SEM, and are compared using one-way ANOVA
with a Bonferroni post hoc test. *** = p < 0.005.
with a hydrophobic moiety shorter than 12 carbon atoms, GS
C10 and GS C8, could be explained by a significant decrease or
loss of surfactant properties of the compounds, respectively.
Transfection of Suspension Cells Using GS C12 and
GS C14. In the previous experiments, the gene transfer
properties of the lipids GS C8-C16 were studied in two
adherent cell types, A549 and U-87 MG cells. The GS C12 and
C14 surfactants, showing the best transfection efficiency in
A549 and U-87 MG cells, were also assessed for their capacity
to transfer pcDNA into suspension cell lines such as Bristol 8
(normal human HLA-A2 positive B lymphoblastoid) cells.
Bristol 8 cells were transfected with GS C12 or C14 using the
optimal lipofection conditions found for both A549 (Table 1)
and U-87 MG (Table 2) cells. As shown in Figures 9 and 10,
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surfactants GS a very attractive and effective alternative to
conventional lipofecting reagents.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures and spectroscopic characterization of
compounds 1−4 and GS C8−C16. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Fax: (+44) 01224 437465, e-mail: m.zanda@abdn.ac.uk.
■
Notes
The authors declare the following competing financial
interest(s): M.U. is current employee of KemoTech s.r.l.,
whereas P.L. and M.Z. are cofounders of KemoTech s.r.l.
(www.kemotech.it).
ACKNOWLEDGMENTS
■
Figure 11. Comparative transfection efficiency of Lipofectamine 2000
and GS C14 in DRG neurons. Expression of maxGFP evaluated by
fluorescence microscopy. Images collected 24 h post-transfection (10×
magnification). Images are representative of at least three other
independent experimental repeats. (A) DRG neurons transfected with
Lipofectamine 2000 (according to the manufacturer’s procedures). (B)
DRG neurons transfected with GS C14. GS C14-based lipoplexes
were prepared using 6 μM of GS C14 (pmaxGFP concentration = 1
μg/mL, compaction time GS C14-pmaxGFP = 1 h, transfection time =
4 h, serum-free conditions).
We thank Professor L. Troisi and the University of Lecce
(Italy) for a visiting Ph.D. studentship to S.P. Dr. Sergio
Dall’Angelo (University of Aberdeen) is gratefully acknowl-
edged for technical and logistic support. This study was
supported in part by a Sardinia Regional Council grant
(project: ”Cation Lipid Surfactants″; Pacchetti Integrati di
Agevolazione PIA, Industria, Artigianato e Servizi; Bando 2007;
Project #229).
REFERENCES
■
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CONCLUSIONS
■
We have described the synthesis of novel gemini surfactants GS
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