Histidine-rich amphipathic peptide antibiotics
promote efficient delivery of DNA into
Antoine Kichler*†, Christian Leborgne*, Josefine Ma ¨rz‡, Olivier Danos*, and Burkhard Bechinger‡§
*Ge ´ne ´thon III–Centre National de la Recherche Scientifique Unite ´ de Recherche Associe ´e 1923, 1 Rue de l’Internationale, F-91002 Evry, France; and
‡Max-Planck-Institut fu ¨r Biochemie, am Klopferspitz 18A, D-82152 Martinsried, Germany
Communicated by Jean-Marie P. Lehn, Universite ´ Louis Pasteur, Strasbourg, France, December 17, 2002 (received for review August 23, 2002)
Gene delivery has shown potential in a wide variety of applica-
tions, including basic research, therapies for genetic and acquired
diseases, and vaccination. Most available nonviral systems have
serious drawbacks such as the inability to control and scale the
production process in a reproducible manner. Here, we demon-
strate a biotechnologically feasible approach for gene delivery,
number of histidine residues. Gene transfer to different cell lines
in vitro was achieved with an efficiency comparable to commer-
efficiency depends on the number and positioning of histidine
residues in the peptide as well as on the pH at which the in-plane
to transmembrane transition takes place. Endosomal acidification
is also required. Interestingly, even when complexed to DNA these
possibility of treating the genetic defect and the bacterial infec-
tions associated with cystic fibrosis with a single compound. Thus,
this family of peptides represents a new class of agents that may
have broad utility for gene transfer and gene therapy applications.
peptides. Although these compounds offer in addition the
possibility of a reproducible and scaleable production process,
synthetic peptide-based gene delivery systems are among the
Until recently, peptides were mostly used as auxiliary agents
in combination with other systems (1), providing either a cell
targeting moiety (2), a membrane destabilization activity (3), or
a nuclear localization function (4). A strategy that is beginning
to emerge is the use of multifunctional peptides. Peptides such
as KALA (5), ppTG20 (6), and Vpr52–96 (7) at the same time
bind DNA and destabilize membranes, two properties that are
indispensable for efficient gene transfer in dividing cells. Of
note, the two former peptides combine a positively charged
lysine or arginine stretch required for DNA binding and an
amphipathic membrane-destabilizing domain deriving from the
fusogenic peptides GALA (5) and JTS-1 (8). These transfecting
peptides have a strong propensity for an ?-helical conformation
that positions the lysines or arginines on one face of the helix.
Another strategy to achieve efficient release of the delivered
material from endosomes consists in using compounds such as
polyethyleneimines (PEIs) that are able to capture protons
entering the endosome. This induces swelling of the endosomes
that leads to membrane disruption (9, 10).
Here we present peptides that exhibit efficient gene transfer
activity by mimicking partially the proton sponge activity of
PEIs. Amphipathic peptides that are rich in alanine and leucine
residues were used as a template. Various numbers of lysine and
histidine residues were included in the peptide sequence (Tables
1 and 2). Whereas the lysines at both ends of the peptides might
serve for DNA condensation, the histidine residues might favor
endosomal escape of the DNA (11).
n contrast to most polymers, key parameters such as product
identification and quality control are possible for synthetic
Our results demonstrate that the presence of histidine and
lysine residues is not sufficient to obtain an efficient transfection
agent. Of crucial importance are the number of histidine resi-
dues, their position in the sequence, and the pH at which the
peptides change from an in-plane to a transmembrane align-
ment. Because some of the peptides possess antimicrobial ac-
tivities (12), we propose that antibacterial peptides with high
transfection efficiency may have potential for the treatment of
cystic fibrosis (CF) as the airways of CF patients are chronically
infected with a variety of bacteria.
Materials and Methods
Plasmids. SMD2-Luc?ITR (7.6 kb) and eGFP-C1 (4.7 kb) are
expression plasmids encoding the firefly luciferase gene and the
green fluorescent protein gene (GFP), respectively, under the
control of the human cytomegalovirus (CMV) immediate-early
Peptides. Histatin-5 (purity ?99%) was purchased from Bachem.
H4-LAK4 (purity ?98%) was from Synt:em (Nimes, France).
LAH4 was synthesized by the authors and by Neosystem [Stras-
bourg, France; theoretical molecular weight ? 2,779.5, molec-
ular weight determined by ES?(electrospray) ? 2,780, purity ?
98%]. The different batches of LAH4 had indistinguishable
transfection properties. All other peptides were prepared by
automated solid-phase synthesis on Millipore 9030 and Applied
Biosystems 431 synthesizers, using fluorenylmethoxycarbonyl
(Fmoc) chemistry. In crude peptide preparations a predominant
peak was observed when analyzed by HPLC with acetonitrile?
water gradients. During HPLC purification the main peak was
collected and the identity of the product confirmed by matrix-
assisted laser desorption ionization (MALDI) MS.
Cell Culture. DMEM (GIBCO?BRL) was supplemented with 2
mM L-glutamine, 100 units?ml penicillin, 100 ?g?ml streptomy-
cin, and 10% FCS. Human hepatocarcinoma cells (HepG2 cells;
Abbreviations: CF, cystic fibrosis; PEI, polyethyleneimine.
†To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
§Present address: Faculte ´ de Chimie, Institut le Bel, 4 Rue Blaise Pascal, F-67000 Strasbourg,
Table 1. Sequence of histidine-containing cationic peptides
?g of peptide*
*Amount of peptide (?g) needed for complete retardation of 1 ?g of DNA.
February 18, 2003 ?
vol. 100 ?
American Type Culture Collection), rabbit aorta smooth muscle
cells (Rb1 cells; kindly given by M. Nachtigal, University of
South Carolina, Columbia), transformed HEK 293 cells (Amer-
ican Type Culture Collection), and mouse fibroblasts (NIH 3T3,
American Type Culture Collection) were used.
Preparation of Polycation?DNA Complexes. Four micrograms of
DNA and the desired amount of 25-kDa PEI, poly L-lysine with
a degree of polymerization of 180 (pLys180; Sigma), 1,2-
dioleoyl-3-trimethylammonium propane (DOTAP, Avanti Polar
Lipids), or peptide were each diluted in 100 ?l of 150 mM NaCl.
Transfection reagent and DNA solution were mixed and incu-
bated for 20 min at room temperature. Before adding the
complexes to the cells, the mixtures were diluted with serum-free
medium to a final volume of 1 ml. For some peptides, the
complexes were prepared in citrate buffer (pH 5.3).
DNA Retardation Assay. DNA binding was studied by agarose gel
retardation assays. DNA (1 ?g) and increasing amounts of
peptide were each diluted in 25 ?l of 150 mM NaCl or citrate
buffer and mixed. After 20 min, samples were electrophoresed
through a 1% agarose gel by using TBE buffer (89 mM Tris?89
mM boric acid?2 mM EDTA, pH 8.3). DNA was visualized after
ethidium bromide staining.
Transfection Experiments. Cells were plated in 24-well plates and
transfected at 50–80% confluency. For transfection, 0.5 ml of
serum-free medium containing the DNA complexes was trans-
ferred into each well. After incubation for 2.5–4 h at 37°C, the
medium was replaced with DMEM containing 10% FCS. Lu-
ciferase activity was measured 24–48 h posttransfection. For
transfection experiments with bafilomycin A1 (Sigma, 175 nM)
or chloroquine (100 ?M), the drugs were added to the DNA
complexes after dilution with medium. The luciferase assay was
performed as described (13). Luciferase background was sub-
tracted from each value and the transfection efficiency was
expressed as total light units per 10 s per well (1 light unit ? 10
counts) and are the means of duplicates. The protein content of
transfected cells was measured by using the Bio-Rad protein
assay. Cytotoxicity was evaluated by performing the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT,
Sigma) assay. Briefly, 1 day after transfection, the cell culture
medium was removed and replaced by serum-free DMEM
containing 0.5 mg?ml MTT. After incubation at 37°C for 4 h, the
medium was removed and 200 ?l of DMSO was added to each
well to dissolve the formazan crystals produced from the reduc-
tion of MTT by viable cells. Absorbance was then measured at
570 nm. Untreated cells were used as control.
Antibacterial Activity. Escherichia coli (DH5?) was grown in LB
until the mid-logarithmic phase. Cells were diluted with LB to
OD600 ? 0.15, and 200-?l aliquots were added to a 96-well
microtiter plate containing 50 ?l of a serial dilution of LAH4
with or without DNA. The pH of the culture was adjusted either
to 7.4 or 5.5. The OD600was measured after an incubation of 6 h
at 37°C with constant shaking. One hundred percent of relative
growth was determined with bacteria cultured in LB medium.
Southern Blot Analysis. HepG2 cells were transfected with 3 ?g of
plasmid SMD2-Luc?ITR in six-well plates, washed once with
PBS, trypsinized, centrifuged, and washed again with PBS. After
selective low-molecular-weight DNA extraction (14), the South-
ern blot analysis was performed as described (7).
Gel-Mobility Shift Assay. The peptides encompassing the series
LAH1–LAH5 were first tested for their capacity to interact with
a constant amount of plasmid and the complexes were electro-
phoresed through an agarose gel. As shown in Table 1, with 2.5
amounts of peptide, the DNA was no more visible, showing that
its condensation was sufficiently strong to prevent ethidium
Determination of the Transfection Activity.Thetransfectionactivity
of the LAH series was evaluated on HEK 293 and HepG2 cells.
The results presented in Fig. 1A show that the most efficient
peptides were those containing four and five histidine residues.
LAH1–LAH3 are at least 10 times less efficient than LAH4 or
LAH5. In light of these results, we chose LAH4 to continue our
By using GFP as a reporter gene, we determined that ?50%
of the HEK 293 cells were transfected with LAH4. Toxicity of
LAH4?DNA complexes was evaluated after transfection by
comparing the total amount of protein per well and the cell
number of transfected and nontransfected cells. Thirty hours
after transfection the protein content was 88% when compared
with nontreated cells. The number of cells remained unchanged
when transfection experiments were compared with controls.
Thus, the treatment with this peptide was well tolerated by the
Next, the transfection efficiency of LAH4 was compared with
that of pLys180 and 25-kDa PEI. The optimal transfection
conditions were determined by using increasing concentrations
of reagents. The results show that LAH4 is significantly more
efficient than polylysine on four different cell lines and that it
Table 2. LAH4 derivatives and histatin-5
?g of peptide*
Optimal ratio, ?g of
peptide??g of DNA‡
Italics in first row indicate the peptide that was used as reference.
*The indicated amount of peptide is the minimal amount required to completely retard 1 ?g of plasmid DNA.
†We gave the value 1,000 to the luciferase activity obtained with LAH4. Transfection experiments were performed on HepG2 cells.
‡Amount of peptide per ?g of DNA that gives the highest reporter gene expression.
§Some of the DNA was retarded, but not all.
Kichler et al.
February 18, 2003 ?
vol. 100 ?
no. 4 ?
exhibits an efficiency comparable to PEI (Fig. 1B). In compar-
ison to the cationic peptide KALA, LAH4 mediates 200- and
40-times-higher luciferase expression in HepG2 and HEK 293
cells, respectively (not shown).
Inhibition of the Acidification of Endosomes. It has recently been
shown that inhibition of endosome acidification by H?-ATPase
inhibitors such as bafilomycin A1 diminishes significantly the
transfection efficiency of PEI. Because this polymer is proton-
ated under physiological conditions, it is assumed that buffering
of endosomes by PEI leads to their destabilization (13). We
asked whether LAH4, which contains imidazole groups, exhibits
similar properties. In the presence of bafilomycin A1, the
transfection efficiency of PEI and LAH4 was diminished by 17-
and 8-fold, respectively (Fig. 1C). In contrast, the efficiency of
the monocationic lipid DOTAP, which is already completely
protonated at pH 7, was not altered.
The neutralizing effect of chloroquine, a weak base that
accumulates in acidic endocytic vesicles (15), did not influence
luciferase expression (data not shown).
Evaluation of the Transfection Activity of LAH4 Variants and Hista-
tin-5. To obtain better insight into the mechanism of action of
LAH4, different modifications were introduced into the peptide
allow the visualization of the distribution of hydrophobic and polar residues
with respect to the helical axis. The histidine residues are in italics.
Edmundson helical wheel diagrams. The helical wheel projections
amounts of transfection reagent were mixed with a constant amount of
reporter plasmid [2 ?g of cytomegalovirus (CMV)-Luc per duplicate]. The
complexes were incubated for 3 h in serum-free medium. Luciferase activity
was measured 24–43 h posttransfection. The transfection efficiency is ex-
pressed as total light units per 10 s per well, and the average of duplicates is
(poly L-lysine with a degree of polymerization of 180) and 25-kDa PEI on four
different cell lines. Only the best condition for each reagent is shown (open
bars, PEI; gray bars, pLys; black bars, LAH4). (C) HepG2 cells were transfected
with PEI [N?P ? 13; N?P is the molar ratio of amino nitrogen (present in the
transfection agent) to DNA phosphate], DOTAP (N?P ? 4), or LAH4 in the
presence (black bars) or absence (gray bars) of 175 nM bafilomycin A1.
Evaluation of the transfection efficiency of LAH peptides. Increasing
www.pnas.org?cgi?doi?10.1073?pnas.0337677100Kichler et al.
sequence. First, we tested whether the presence of histidine
residues is required for efficient reporter gene expression. A
peptide having only leucine, alanine, and lysine residues was
synthesized and its ability to complex DNA and to transfect cells
was determined. As shown in Table 2, although LAK4 retarded
well the migration of plasmid DNA, it exhibited only poor gene
Second, we analyzed whether the presence of a well defined
Therefore, we included the salivary antimicrobial peptide hista-
tin-5, which presents an equal distribution of hydrophilic resi-
dues along its surface (Fig. 2), in our assays (Table 2). Surpris-
ingly, histatin-5 did not fully retard DNA migration, even though
high amounts of peptide were used. This was still the case when
protonation of the imidazole groups. The transfection experi-
ments confirmed that histatin-5 is an inefficient DNA carrier.
Third, we studied whether the position of the four histidine
residues in the hydrophilic face is of importance. The peptides
LAH4, LAH4-L3, and LAH4-L4 (Table 2, Fig. 2) were used to
transfect HepG2 cells. Although all three peptides mediated high
gives reproducibly slightly lower luciferase levels than LAH4 and
LAH4-L3, suggesting that optimal transfection efficiency requires
diagram leave a high hydrophobic angle (Fig. 2).
Fourth, it was shown that LAH4 completely reorients from
parallel to the surface into a transmembrane configuration
when the pH is increased from 5 to 7 (16). The midpoint of
this transition is pH50 ? 6.1. To test whether this behavior
is important for the mechanism of transfection, derivatives
of LAH4 differing in the midpoint of their in-plane-to-
transmembrane transition were tested: pH50? 4.8 for LAH4-L6,
6.0 for LAH4-A6, and ?8.5 for LAH4-G6 (17). Table 2 shows
that LAH4-L6 and -A6 were able to complex DNA efficiently
and to retard its electrophoretic migration, whereas LAH4-G6,
which is considerably more hydrophilic, is unable to fully retard
In transfection experiments, the three mutants are significantly
less efficient than LAH4 (Table 2). Interestingly, LAH4-A6, the
peptide having the closest transition midpoint to LAH4, was also
the most efficient when compared with LAH4-L6 or -G6.
Finally, we tested whether inversion between lysine and his-
tidine residues (H4-LAK4) affects the transfection efficiency.
The results presented in Table 2 show that H4-LAK4 is signif-
icantly less active in gene transfer than LAH4.
The toxicity of the peptide?DNA complexes on HepG2 cells
after transfection was monitored by measuring the total amount of
protein per well and by estimating cell proliferation by the MTT
colorimetric assay. The results indicate that the lower transfection
efficiency of certain LAH4 mutants when compared with LAH4 is
not due to an increased cytotoxicity (data not shown).
To determine whether the observed differences in reporter
gene expression are due to varying efficiencies of DNA delivery,
we transfected cells by using LAH4, LAH4-A6, or LAH4-L6.
The cells were harvested 7 and 25 h posttransfection and the
DNA of low molecular weight was isolated and quantified by
Southern blot analysis. At the same time, expression of the
reporter gene was monitored. The results demonstrate that
LAH4-L6 delivers almost as much DNA as LAH4, although this
peptide is significantly less efficient in mediating reporter gene
expression (Fig. 3). Surprisingly, LAH4-A6 delivers 80 times less
DNA into cells than LAH4, although 1 day posttransfection the
difference in luciferase activity is only one log unit.
These results show that minor modifications of the sequence
of LAH4 can result in significant changes in the capacity to
mediate gene transfer. Moreover, our data show that there is not
a simple correlation between the amount of delivered DNA and
the level of transgene expression.
We have evaluated the transfection efficiency of a series of
peptides including LAH4 and its derivatives. Our results show
that although DNA retardation is observed for peptides encom-
passing four lysines and one to five histidines (Table 1), only
those with four to five histidine residues in the central region of
the sequence exhibit high transfection efficiencies (Table 2). In
fact, LAH4 allows for gene transfer levels on several cell lines
that are comparable to those observed with 25-kDa PEI, which
is one of the most efficient transfection reagents (9, 18).
cells were harvested and lysed and the low-molecular-weight DNA was recovered. At the same time, the luciferase activity was measured. The blot at Left serves
as a standard curve and corresponds to the results obtained after hybridization of 25, 50, 100, and 150 ng of CMV-Luc. Graphs below the blots show the
corresponding luciferase activity.
Efficiency of plasmid DNA delivery. HepG2 cells were transfected with peptide?cytomegalovirus (CMV)-Luc complexes. At 7 and 25 h after transfection,
Kichler et al.
February 18, 2003 ?
vol. 100 ?
no. 4 ?
The pK values of the four histidine imidazole groups of LAH4,
determined in the presence of dodecylphosphocholine micelles,
are 5.4, 5.8, 5.9, and 6.0 (16). The importance of protonation of
the imidazole groups was shown with transfections performed in
the presence of the proton pump inhibitor bafilomycin A1.
Luciferase expression was significantly diminished, but not abol-
ished, in the presence of the drug. Interestingly, the mere
presence of four or more histidine residues is not sufficient for
efficient gene transfer. This indicates that the mechanism of
action of these peptides is different from that of histidylated
From the experiments performed with LAH4 mutants, the
following conclusions can be drawn: (i) the ability of the peptides
to complex DNA seems not to be solely linked to the global
charge of the peptide, suggesting that structural requirements
need to be fulfilled (LAH4-G6 and histatin-5 for example do not
retard DNA migration well); (ii) comparison of results obtained
with LAH4 and H4-LAK4 shows that the histidine residues have
to be positioned in the core of the peptide; and (iii) the peptides
LAH4-L3, LAH4-L4, and LAH4 all show pronounced transfec-
tion efficiencies, indicating that the position of the histidine
residues within the polar face is important, even though minor
modifications are tolerated.
Structural investigations using circular dichroism (CD)-
fourier transform infrared (FTIR) and attenuated total reflec-
tion (ATR)-FTIR spectroscopy indicate that in the presence of
membranes the LAH4 family exhibits a high propensity for
?-helical conformations with all histidine residues located on
are reconstituted into bilayers aligned with the normal parallel
to the magnetic direction,15N solid-state NMR spectroscopy
indicates that the interactions of the LAH4 peptides with
membranes are strongly modulated by the pH-dependent chem-
ical potential of the histidine side chains. As a result, the LAH4
helix acts as a pH-dependent molecular switch, having a trans-
membrane orientation at neutral pH (15N chemical shift close to
200 ppm) and an in-plane orientation at pH ?6 (15N chemical
shift ?90 ppm) (16). The transition midpoint around pH 6.2 was
confirmed by following the dichroic ratio measured in oriented
ATR-FTIR spectra. Experiments with this latter technique also
indicated that the in-plane–transmembrane transition is revers-
ible (19). Interestingly, in this study all peptides exhibiting high
transfection efficiencies also show pH-dependent membrane
alignments with transition midpoints around pH 6 (16, 17). This
suggests that pH-dependent membrane insertion of the peptides
constitutes an important part of the DNA import mechanism.
In patients with CF the airways are colonized and chronically
infected with a variety of bacteria (20), despite the presence of
many antibacterial proteins and peptides. It has been proposed
that in CF an increase in the airway surface liquid salt concen-
tration inhibits the activity of antibacterial factors (21). How-
ever, increased amounts of antibiotics can partially compensate
have therapeutic value (22). LAH4 was shown to possess signif-
icant antibiotic activity, although it does not lyse human eryth-
rocytes at the same concentrations (12). We tested whether
LAH4 still possesses its antibiotic activity once complexed to
DNA. Our results, obtained with E. coli, show that the antibac-
As previously reported, this activity was found to be pH depen-
dent (Fig. 4; ref. 12). Interestingly, a similar pH-dependent
antibacterial activity was recently characterized for calcitermin,
a histidine-rich peptide found in human airways (23).
Although more experiments have to be performed, including on
bacterial strains commonly found in CF patients (e.g., Pseudomo-
nas aeruginosa and Staphylococcus aureus), administration of pep-
tide antibiotic?DNA complexes has potential in the treatment of
the genetic defect and bacterial infections associated with CF.
This work was performed with the financial support of the Association
Franc ¸aise contre les Myopathies and Vaincre la Mucoviscidose.
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pH 7.4 (open symbols) and 5.5 (filled symbols) as a function of peptide
concentration for LAH4 in the absence (circles) or presence (squares) of DNA
(LAH4?DNA with a wt/wt ratio of 6?1).
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www.pnas.org?cgi?doi?10.1073?pnas.0337677100Kichler et al.