Advanced Drug Delivery Reviews 56 (2004) 967 – 985

www.elsevier.com/locate/addr

GALA: a designed synthetic pH-responsive amphipathic peptide

with applications in drug and gene delivery
Weijun Li a, Francßois Nicol a,b, Francis C. Szoka Jr. a,*
a
Department of Biopharmaceutical Sciences and Pharmaceutical Chemistry, School of Pharmacy,
University of California at San Francisco, San Francisco, CA 94143-0446, USA
b
ALZA Corporation, 1900 Charleston Road, Mountain View, CA 94039-7210, USA
Received 16 July 2003; accepted 31 October 2003

Abstract

GALA is a 30 amino acid synthetic peptide with a glutamic acid-alanine-leucine-alanine (EALA) repeat that also
contains a histidine and tryptophan residue as spectroscopic probes. It was designed to explore how viral fusion protein
sequences interact with membranes. The sequence selected was long enough to span a bilayer in the a-helix, the
glutamic acids (Glu) were selected to provide a pH-dependent negatively charged side-chain and the EALA repeat was
adjusted so that the peptide would have a hydrophobic face of sufficient hydrophobicity to interact with the bilayer when
the peptide was in an a-helix. GALA converts from a random coil to an amphipathic a-helix when the pH is reduced
from 7.0 to 5.0. At neutral pH, GALA is water soluble while at acid pH, GALA binds to bilayer membranes. The
nature of the association and the type of peptide – peptide interactions in the membrane depend upon the physico-
chemical properties of the bilayer such as the acyl chain composition of the phospholipids and the presence of
cholesterol. Neutral and negatively charged bilayers composed of saturated phospholipids of 14 – 16 acyl chain length are
solubilized into peptide – lipid discs by GALA. GALA can induce fusion between small unilamellar vesicles (SUV)
composed of unsaturated phospholipids. Most importantly GALA forms a transmembrane peptide pore comprised of
approximately 10 GALA a-helical monomers that are arrayed in an a-helix perpendicular to the plane of the membrane.
Membrane leakage from neutral or negatively charged vesicles at pH 5.0 can be adequately explained by a mathematical
model assuming that GALA becomes incorporated into the vesicle bilayer and aggregates to form a transbilayer pore
consisting of 10 ( F 2) peptides. The lipid compositions of model bilayer have important effects on the GALA
transbilayer insertion mechanism and peptide orientation. Insertion of the pore into the membrane dramatically accelerates
transmembrane phospholipid flip-flop. Cationic peptides designed based upon GALA but containing a lysine-alanine-
leucine-alanine (KALA) motif can interact with nucleic acids and perturb biomembranes. The pH-controlled membrane

Abbreviations: ANTS, 1-aminonaphthalene-3,6,8-trisulfonic acid; ATR, infrared attenuated total-reflection spectroscopy; BODIPY,
dipyrrometheneboron difluoride; CD, circular dichroism; DE, 1,2-dielaidoyl; DM, 1,2-dimyristoyl; DO, 1,2-dioleoyl; DP, 1,2-dipalmitoyl; DPe,
1,2-dipetroselinoyl; DPX, p-xylene-bis-pyridinium bromide; FITC, fluorescein isothiocyanate; FTIR, fourier transform infrared spectroscopy;
HA, hemagglutinin; LUV, large unilamellar vesicle; NBD, N-4-nitrobenzo-2-oxa-1,3-diazole; ODN, oligonucleotide; PC, phosphatidylcholine;
PE, phosphatidylethanolamine; PEG, poly(ethylene glycol); PEI, polyethylenimine; PG, phosphatidylglycerol; PO, 1-palmitoyl-2-oleoyl;
Poly(DMAEMA-NVP), poly(2-(dimethylamino)ethyl methacrylate-co-N-vinyl-2-pyrrolidone); Rh, rhodamine; SUV, small unilamellar vesicle.
* Corresponding author. Tel.: +1-415-476-3895; fax: +1-415-476-0688.
E-mail address: szoka@cgl.ucsf.edu (F.C. Szoka).

0169-409X/$ - see front matter D 2004 Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2003.10.041
968 W. Li et al. / Advanced Drug Delivery Reviews 56 (2004) 967–985

permealization induced by GALA and related peptides serve as a paradigm for the design of environmentally responsive
peptidic delivery vehicles for drugs and genes.
D 2004 Elsevier B.V. All rights reserved.

Keywords: GALA; KALA; Amphipathic peptide; Pore formation; Bilayer; Gene delivery; pH-sensitivity

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 968
2. Peptide mimic of the natural membrane active elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 969
2.1. Natural amphipathic a-helical peptide, ion channel motif and viral fusion protein . . . . . . . . . . . . . . . . . . 969
2.2. GALA and related peptides: simplified synthetic amphipathic a-helices. . . . . . . . . . . . . . . . . . . . . . . 970
3. Pore formation mechanism of GALA and related peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 971
3.1. GALA properties in aqueous solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 971
3.2. GALA interactions with model lipid membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 973
3.2.1. pH-dependent GALA binding to vesicles made of unsaturated PC lipids . . . . . . . . . . . . . . . . . . 973
3.2.2. GALA induces the fusion of SUV made of unsaturated PC lipids at low pH . . . . . . . . . . . . . . . . 974
3.2.3. GALA induces the fragmentation of LUV made of saturated PC lipids at neutral pH . . . . . . . . . . . . 975
3.2.4. pH-dependent pore formation by GALA in LUV . . . . . . . . . . . . . . . . . . . . . . . . . . . . 975
3.2.5. Peptide orientation on membrane . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 978
4. Applications for the delivery of plasmid DNA and oligonucleotide (ODN) . . . . . . . . . . . . . . . . . . . . . . . . . 980
5. What are the advantages of GALA-like peptides as components of a drug and gene delivery system? . . . . . . . . . . . . 982
6. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 982
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 983
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 983

1. Introduction 16 years exploring the details concerning how mem-

Biological membranes are the barriers that separate
the machinery of life from the chaotic environment. In
mammalian cells membranes are predominantly com-
posed of proteins and amphipathic lipids: sphingoli-
pids, phospholipids and cholesterol. The lipids
spontaneously assemble into a bilayer which is a
two dimensional solvent for the membrane proteins.
It has been postulated that the self-organizing ability
of lipids into bilayer vesicles was one of the earliest
steps in the development of life. Indeed, an intact
membrane is critical for proper cell function but it is
also one of the major impediments for the delivery of
therapeutic agents into cells. This is particularly true
for macromolecular drugs such as oligonucleotide
(ODN) and DNA. Viruses have evolved proteins to
breach membranes, so we originally designed and
synthesized GALA to serve as a simple model of a
viral fusion peptide sequence. We believed GALA
would aid in the understanding of how an amphipathic
sequence can perturb bilayers and induce fusion
between membranes [1,2]. We have spent the past
brane composition influences the strength of the
GALA –lipid interaction as well as the nature of the
resulting peptide structure in the bilayer [1– 10]. This
information is the basis of more recent attempts to
design peptides that can be applied for the delivery of
genes and other nucleic acid drugs [11– 13]. Such
bioresponsive synthetic peptides have the potential to
be the basis of a highly efficient nucleic acid delivery
system.
Rules for the design of peptide-based gene delivery
systems must take into account DNA condensation,
active targeting to the biological membrane, efficient
transmembrane transport and nucleus access [14].
GALA and GALA-like peptides are concerned with
the regulated permeabilization of membranes. Natural
pore-forming peptide, ion channel motif and viral
fusion proteins provide classic examples on the natural
process of peptide-induced membrane permeabiliza-
tion [15,16]. With the knowledge on the amphipathic
peptide– membrane interaction, we designed a series
of amphipathic peptide vectors for the delivery of
gene-coded plasmid DNA or ODN [11,12,17,18]. In
 W. Li et al. / Advanced Drug Delivery Reviews 56 (2004) 967–985
this review, we will discuss the factors that influence
pore-forming phenomena as revealed in the mecha-
nisms of GALA and related amphipathic peptides, and
suggest the ways they might be applied for gene
delivery.
2. Peptide mimic of the natural membrane active
elements
2.1. Natural amphipathic a-helical peptide, ion
channel motif and viral fusion protein
The amphipathic helix is a peptide motif in which
the amino acids are arrayed in an a-helix with one
face, composed of hydrophilic residues (mainly com-
posed of polar/charged amino acids) while the oppo-
site face is hydrophobic (composed primarily of
apolar amino acids) [19]. The amphipathic helix is a
widespread feature in many biologically active pep-
tides and proteins of diverse origin and function [20 –
23]. The amphipathicity enables the a-helix to form
an interface between the apolar region of macromo-
lecules and the polar environment. In particular,
amphipathic a-helices can self-associate in water by
hydrophobic interactions between apolar side chains
and hence can control protein folding pathways or
anchor tertiary and quaternary protein structures. They
are also able to associate with the hydrophobic regions
of a lipid bilayer.
This ability to interact with interfaces is a double
edge sword and numerous organisms secrete short
cytotoxic peptides that adopt an amphipathic a-
helical conformation when they are bound to lipid
membranes. In turn there are a spectrum of cells that
are sensitive to the cytotoxic action of these pep-
tides, from bacteria, viruses and fungi to tumor and
mammalian cells [24,25]. The cytotoxic peptides kill
the target cells by inducing permeabilization or
disruption of the plasma membrane [21]. It is now
generally accepted that many of these peptides exert
their cytotoxic activities directly on the lipid matrix
of the cell membrane. These natural amphipathic a-
helical toxins induce drastic changes in the structure,
dynamics and properties of the lipid bilayers they
interact with. In particular, they permeabilize model
lipid membranes, i.e. they generate ion conduction
across planar lipid bilayers in a voltage-independent
969
fashion and induce the leakage of molecules pre-
encapsulated in liposomes. In the most common
model proposed to explain the amphipathic a-helices
permeabilizing lipid bilayer, an aqueous pore is
generated in the membrane as a result of the
oligomerization of membrane-bound peptides. These
ion pores are generally described as bundles of
aligned transbilayer helices, with the hydrophilic
side of the helices forming the water-exposed inner
surface of the pore and the hydrophobic side
interacting with the fatty acyl chains of the lipids
[26 – 28].
Based on their potential to form aqueous pores in
model lipid membranes, amphipathic a-helical pep-
tides are relevant models of ion channel proteins
[29,30]. Ionic channels generally are large, complex
multimeric transmembrane glycoproteins that facili-
tate the selective translocation of ions across lipid
bilayer. For example, a basic structural motif of the
nicotinic acetylcholine receptor superfamily is con-
stituted by five parallel transbilayer amphipathic a-
helices in the central pore of the ion channel [31,32].
Bundles of transbilayer helices are thought to occur
also in a wide range of transport proteins such as
bacteriorhodopsin [33], the sugar transporter lactose
permease [34] and ‘‘ABC’’-transport proteins [35].
Amphipathic a-helical toxins that are able to asso-
ciate in membranes to form ion channels are valu-
able models for studying the properties of the pore
formed by these proteins [27]. This is particularly the
case because the pore comprises only one type of
helix, which simplifies its structure. The study of
these peptides, therefore, increases our understanding
of certain biological processes, such as nerve con-
duction, mass and information transport, energy
conversion and cellular signaling [36].
Membrane fusion proteins play important roles in
the process of viral entry into living cells. Most
viruses have a single integral fusion protein which
can bind to the cell membrane upon a conformation-
al change by a physiological trigger such as the pH-
drop in endosome, binding to a receptor, or proteol-
ysis which facilitate the viral membrane fusion with
the target cell membrane [37]. The fusion peptide
usually presents on the N-terminal hydrophobic re-
gion of transmembrane proteins of various viruses,
such as retroviruses, orthomyxoviruses and para-
myxoviruses [38]. The viral fusion process can be
970 W. Li et al. / Advanced Drug Delivery Reviews 56 (2004) 967–985

cataloged into two classes, low pH-dependent or pH-
independent.
The influenza virus hemagglutinin (HA) has been
extensively investigated as a model of membrane
fusion protein. To infect the host cell, the influenza
virus binds to the cellular membrane first, and then
internalized into the endosome. The low pH in the
endosome induces a loop – helix conformational
change in HA, leading to the insertion of a hydro-
phobic fusion peptide domain (HA-2) into both the
viral and host membranes. This induces the two
adjacent membranes to form a ‘‘stalk’’ intermediate
structure, followed by a complete fusion of the two
membranes [37]. The viral genome is subsequently
released into the cytosol. Contrasted to this, the HIV
fusion protein (gp41) is pH-independent, inducing
membrane fusion at neutral pH. The ability of
synthetic gp41 fusion peptides to perturb and disrupt
bilayer has been demonstrated to be dependent on
the peptide sequence and temperature [39]. It should
be noted that the research on the interaction between
peptide mimics of viral fusion proteins and mem-
branes has both theoretical and industrial importance.
For example, the virosomes, the particles of mem-
brane (liposome) reconstituted with viral envelope
proteins such as the fusion protein, have been
developed as a vaccine with enhanced immune
sensitivity [40].
acronym (glutamic acid-alanine-leucine-alanine) for a
membrane destabilizing peptide than was EALA so
vanity won out over science in the naming of this
peptide sequence.
GALA is a synthetic peptide designed to interact
with lipid bilayer preferentially at acidic pH [1]
(Fig. 1). The sequence of GALA is (WEAALAEA-
LAEALAEHLAEALAEALEALAA). The factors
considered for choosing the peptide sequence were
(a) hydrophobicity and conformational preference of
the residues, (b) length of the peptide, (c) type of pH-
titratable amino acids, and (d) topology of the residues
on the peptide when set in an a-helix. The sequence
was chosen to increase the peptide hydrophobicity,
and therefore its interaction with bilayers, at low pH.
In addition, amino acids with a high a-helical pro-
pensity were selected. Apolar leucine residues were
introduced to confer a relatively high hydrophobicity
on the peptide, whereas polar glutamic acid (Glu)
residues were included to add a pH-dependent com-

2.2. GALA and related peptides: simplified synthetic

amphipathic a-helices

Generally, the structure – activity relationship for

naturally occurring membrane active peptides is dif-
ficult to replicate [26,41]. To investigate more specif-
ically the exact role of peptide hydrophobicity, net
charge, amphipathicity and helicity in the membrane-
perturbing activities of amphipathic a-helices, syn-
thetic peptides with highly simplified sequences have
been studied. These peptides generally contain four or
fewer types of amino acids that are selected for their
helix-forming capabilities, as well as their polar/ Fig. 1. Structure representation of the GALA (up) and KALA
charged or apolar character. Our group has extensively (down) peptide [1,12]. The GALA peptide sequence is: WEAA-
investigated a synthetic amphipathic a-helix, desig- LAEALAEALAEHLAEALAEALEALAA; KALA sequence is:
nated GALA, that perturbs model lipid membranes WEAKLAKALAKALAKHLAKALAKALKACEA. The atoms of
oxygen, carbon, nitrogen, hydrogen and sulfur are represented by
and forms aqueous pore [1 –5,7 – 9]. GALA is a mis- red, light blue, dark blue, white and yellow color. Right parts are
nomer for this sequence since the repeat unit is actual- face-view of peptide C-terminals and the left are features along the
ly EALA, however GALA was considered a jazzier peptide helix axis.
 W. Li et al. / Advanced Drug Delivery Reviews 56 (2004) 967–985
ponent to the hydrophobicity and net charge. A
sequence composed of 30 amino acids was chosen
to yield a stable a-helix at low pH that was long
enough to cross a bilayer. GALA can be essentially
described as a repeat of the sequence glutamic acid-
alanine-leucine-alanine. Deviations from this repeat
were made in the peptide sequence to localize the
hydrophobic leucine residues on one side of the helix
and the pH-titratable hydrophilic Glu residues on the
other side. Alanine residues were selected as spacers
between the polar and the apolar faces of the helix. At
neutral pH, electrostatic repulsions between the car-
boxylic acid moieties of the Glus are expected to
destabilize the helix, whereas at pH 5.0 the neutrali-
zation of these groups should promote helix forma-
tion. Moreover, when the Glu residues are protonated
the hydrophobicity of the Glu side chain increases
[42]. The spatial segregation of the polar and apolar
amino acids on opposite sides of the helix confers a
pronounced amphipathicity on the peptide. Trypto-
phan and histidine residues were introduced as fluo-
rescent and NMR probes, respectively. The fact that
GALA was designed from simple principles makes
this peptide a good model to study the interplay of
components in the progression from choosing an
amino acid sequence to its secondary structure and
membrane-perturbing capabilities.
Based on the biophysical study of GALA peptide,
GALA together with other synthetic amphipathic
peptides have been developed as vectors for DNA
or ODN delivery. However, GALA itself can not
bind to DNA or ODN directly for the strong nega-
tive charges on both GALA and DNA backbone
repelling each other. KALA, an alternative choice to
GALA, is a synthetic cationic peptide, which can
bind directly to DNA or ODN [12]. The basic
concept behind the KALA sequence is to replace
negatively charged Glu residue on GALA with
positively charged lysine (Lys) in a similar position
on the peptide backbone while maintaining the other
characteristics (Fig. 1). The strongly apolar and
helix-forming Leu residue was to generate a peptide
incorporated into the sequence of 30 amino acids
with several Leu-to-Lys repeated unit in the sequence
(Fig. 1). The positive charges yield a high binding
affinity to the negatively charged nucleic acid,
whereas the hydrophobicity perturbs the lipid pack-
ing of the membrane.
971
3. Pore formation mechanism of GALA and
related peptides
3.1. GALA properties in aqueous solution
In aqueous solution, GALA was designed to be
unordered at neutral pH and a-helical below pH 6.0
[1,4,43]. Circular dichroism (CD) measurements
demonstrated the induction of a a-helical conforma-
tion as the pH was decreased from 7.5 to 5.0. The a-
helical content at pH 7.5 in a buffer containing 100
mM KCl was 7% and independent of peptide con-
centration below 16 AM. Above this concentration, it
increased and reached 23% at 74 AM, indicative of
GALA self-association [4]. For a peptide concentra-
tion of 16 AM, the percentage of a-helical structure
increased when lowering the pH from 7.5 to 3.6,
reaching a maximal value of 36% at pH 5.0 in a
sodium acetate buffer [1]. The pH at the midpoint of
maximum change of helical content was 6.0, which
is close to the pKa of Glu in polyglutamic acid. This
indicates that the change in a-helical content was
correlated with the protonation of the Glu side
chains. At neutral pH, the alignment of the negative
charges on the peptide indeed prevents stabilization
of the a-helix. The helix was found stable at pH 5.0
from 10 to 40 jC and could be denatured only above
40 jC. In addition, the a-helical content of GALA
conformation was sensitive to the ionic strength. At
pH 6.5, it increased linearly upon increasing the salt
concentration from 50 to 400 mM KCl. This effect
was attributed to the screening of the carboxylates by
the cations [1].
GALA secondary structure was also determined
using the technique of infrared attenuated total-reflec-
tion (ATR) spectroscopy, by analysis of the amide IV
line shapes with Fourier self-deconvolution and curve
fitting [43]. At pH 7.0, it was estimated that 19% of
the amino-acids adopt an a-helical conformation,
whereas 20% are organized in h-sheet structures. At
pH 5.0, the percentage of a-helix increased to 69%,
and h-sheet structure accounted for 28% of the
conformation computed from fourier transform infra-
red (FTIR) spectra. The discrepancies between CD [1]
and FTIR techniques in estimating GALA secondary
structure at pH 5.0 were explained by the light
scattering caused by peptide aggregation in the CD
measurements.
972 W. Li et al. / Advanced Drug Delivery Reviews 56 (2004) 967–985
The carboxylic acid groups of the Glu residues are
close enough to allow formation of divalent cation
bridges connecting adjacent groups. Interaction of
GALA with divalent cations at neutral pH should
therefore result in charge neutralization and promote
helix formation. At pH 7.5 the a-helical content
increased upon raising Ca2 + concentration, from 7%
without cation to 41% at 100 mM Ca2 + [1]. At a
concentration of 15 mM, Mg2 + was slightly more
efficient than Ca2 + in stabilizing the helix, while Zn2 +
was as potent as Ca2 +. At pH 5.0, divalent cations
only slightly increased the helicity, which confirmed
that most of the carboxylic acids are neutralized at this
pH.
GALA1, GALA2, GALA3 and GALA4 are short
GALA analogues that were investigated to demonstrate
the importance of peptide length on the stabilization of
its secondary structure. The sequence of these peptides
is (Glu-Ala-Leu-Ala)n, n = 1 –4, for GALA1 – 4, respec-
tively. The end effects are expected to dominate their
conformation, allowing little stable secondary struc-
ture. They undergo, however, a change in hydropho-
bicity when the carboxylic acid groups are protonated
at low pH. The CD spectra of GALA3 and GALA4 did
not change when the pH of the solution was decreased
from 7.5 to 5.0, showing that they were too short to
have any stable secondary structure [1]. However, the
helicity of a peptide (named as SFP-1) composed
essentially of the first 13 amino-acids of the GALA
sequence was shown by CD measurements to increase
from 3% at neutral pH to 41% at pH 4.6 [44]. Com-
pared to GALA3 or GALA4, the SFP-1 has an addi-
tional Phe residue on the N-terminal. We believe that
the hydrophobicity of Phe might help to stabilize the
helix at low pH.
The sensitivity of Trp-1 to the polarity of its
environment was also used to investigate the solution
properties of GALA. The fluorescence emission spec-
trum of GALA’s tryptophan (1.6 AM peptide) dis-
played a maximum intensity that varied from
kmax = 360 nm at pH 7.5 to kmax = 350 nm at pH 5.0
[1]. This blue shift indicated that the N-terminal
tryptophan was in a more hydrophobic environment
at acidic pH, due to a modification of GALA’s
conformation or to a different state of aggregation.
A gradual blue shift of kmax, concomitant with an
increase in helicity, occurred at pH 7.5 upon increas-
ing the peptide concentration above 20 AM [4]. These
modifications were indicative of oligomer formation.
When the pH was rapidly changed from 7.5 to 5.0, a
drastic variation in Trp-1 fluorescence intensity (1.6
AM peptide) was observed, at the kmax found at pH
5.0. This indicated that the environment of the tryp-
tophan was altered in the 2-s mixing time of the
instrument. These results agree with CD studies [1]
that demonstrated how GALA undergoes a reversible
helix– coil transition as the pH is cycled between 5.0
and 7.5. The influence of divalent cations was also
investigated. In agreement with CD measurements,
the presence of 20 mM Ca2 + or Mg2 + at pH 7.5
induced a blue shift of kmax from 360 to 353 nm,
whereas Zn2 + caused a further alteration to 344 nm.
To evaluate the importance of the amino-acids
topology in the GALA sequence, three amino acids
were transposed. Leu was replaced by Glu at positions
9, 17, and 25, and Leu replaced Glu at positions 11,
19, and 26. These transpositions resulted in a peptide
designated LAGA, with little amphipathic character
when folded into an a-helix [4]. CD studies indicated
that LAGA was random coil at pH 7.5 whereas 22%
of the amino-acids adopted an a-helical conformation
at pH 5.0. The value observed at pH 5.0 was lower
than that of GALA, revealing a higher stability of the
amphipathic helix.
The internal motions of GALA and LAGA at pH
5.0 and 7.5 were studied over a broad range of peptide
concentrations, by observing the anisotropy of the
tryptophan moiety [4]. At pH 7.5, GALA and LAGA
anisotropies (0.019 and 0.016, respectively) did not
change at concentrations lower than 10 AM. At 50 AM
it increased to 0.142 for GALA, which was consistent
with the formation of aggregates at high peptide
concentrations. The anisotropy of GALA at pH 5.0
was much higher than that observed at pH 7.5. At the
lowest concentration tested (0.33 AM), the value
obtained (0.046) was already significantly higher than
that observed at pH 7.5. Increasing the peptide con-
centration at pH 5.0 resulted in a further increase in
anisotropy up to 0.075 at 7 AM, which reflected
changes in the self-association state of GALA. At this
pH the anisotropy of LAGA (0.026) was much lower
and, contrary to GALA, was concentration-indepen-
dent. These results demonstrated that LAGA, because
of the lack of amphipathicity of its helix, does not
self-associate extensively at pH 5.0, whereas GALA
does and in a concentration-dependent manner.
 W. Li et al. / Advanced Drug Delivery Reviews 56 (2004) 967–985
Contrasted to GALA, the KALA peptide under-
goes a pH-dependent a-helix to random coil change
when pH is decreased from 7.5 to 5.0 [12]. The pH-
dependence of this peptide is based on the ionization
properties of residues with charged side chains, in-
cluding the q-amino group of seven Lys, the imidazole
of histidine, and g-carboxyl groups of two Glu resi-
dues. When the pH is decreased from 8 to 4.5, the a-
helix percentage in the peptide will decrease from
45% to 24%. At pH 7– 8, KALA has five net positive
charges ( + 7 from Lys residues and 2 from Glu),
and the peptide keeps the a-helix. When the pH is
decreased to 5, the charge on Glu residue will be
neutralized and the imidazole of histidine will be
protonated. The increased net charge (maximum + 8)
appears to disrupt the a-helix in aqueous solution
[12].
3.2. GALA interactions with model lipid membranes
GALA’s ability to perturb lipid membranes has
been extensively investigated. In particular, GALA
was shown to induce membrane fusion [2], fragmen-
tation [3], and permeabilization by pore formation
[1,5]. In addition, the membrane-binding properties of
GALA have been studied to explain its ability to
perturb lipid bilayers [1– 4,43]. In these studies GA-
LA was always added to the vesicles suspensions from
a stock solution at pH>7.0, to avoid pre-aggregation
of the peptide. Further investigations with fluores-
cence labeled-GALA revealed the orientation of pep-
tide in unilamellar vesicles [8,10]. The state of the
peptide in membranes is influenced by the composi-
tion of the membrane lipids, such as the charge, the
length of acyl chain, the saturation or unsaturation of
acyl chain, and the percentage of cholesterol [7,9].
3.2.1. pH-dependent GALA binding to vesicles made
of unsaturated PC lipids
The apparent association of GALA and LAGA with
vesicles was measured at pH 5.0 and 7.5 from the
separation of 125I-peptide – vesicle mixtures on discon-
tinuous ficoll gradients [4]. When GALA and egg
phosphatidylcholine (PC) large unilamellar vesicles
(LUV) (diameter f100 nm) were mixed at a 5000/1
lipid to peptide molar ratio for 15 min at pH 5.0 and
then applied on a gradient prepared at the same pH, the
fraction of lipid-bound GALA co-migrated with the
973
vesicles to the top of the gradient, while the unbound
fraction remained at the bottom. The apparent distri-
bution coefficient (Ka=(bilayer-bound peptide/peptide
in solution)  ([H2O]/[lipid])) in these conditions (2
AM peptide) was 2.0  105. This distribution coeffi-
cient can also be considered as an apparent association
constant. When incubated at pH 7.5 under similar
conditions, GALA remained largely at the bottom of
the gradient, resulting in a very low apparent partition
coefficient (Ka = 1.8  103). This result reveals a dras-
tic pH-dependence of GALA partitioning between
lipid bilayers and water, and demonstrates the strong
propensity of the amphipathic helix to associate with
lipid vesicles.
When GALA was first incubated with egg PC
liposomes at pH 5.0 and the pH was elevated to 7.5,
the results mimicked the outcome at pH 7.5, indicat-
ing that GALA dissociated from the vesicles as a
result of the pH elevation. Keeping the peptide con-
centration constant at 2 AM while lowering the lipid to
peptide ratio yielded an increase in GALA binding to
egg PC vesicles at pH 5.0, from Ka = 2.0  105 at
5000/1 to 4.5  106 at 50/1. An increase of the
apparent partition coefficient when the lipid concen-
tration is reduced indicates a positive cooperativity of
the binding process. This cooperativity can be attrib-
uted to GALA aggregation in egg PC bilayers at pH
5.0.
When LAGA was mixed with egg PC liposomes at
pH 5.0 at a lipid/peptide ratio of 5000/1 the resulting
apparent association constant was 1.9  104, which is
an order of magnitude lower than that found for
GALA in similar conditions. Since the two peptides
have comparable average hydrophobicity and helical
contents in solution at pH 5.0, the amphipathic helix
and the self-assembly of GALA in membrane is the
reason for its bilayer association.
The wavelength of tryptophan’s maximal emission,
kmax, was also monitored to demonstrate an interac-
tion between GALA and egg PC liposomes [1,4]. At
pH 7.5 the kmax remained constant (360 nm) when the
peptide (1.6 AM) was mixed with egg PC liposomes at
a lipid/peptide molar ratio varying from 50/1 to 500/1.
At pH 5.0, the kmax shifted to 335 nm in the presence
of egg PC vesicles, indicating an association with the
vesicles. The spectral changes observed upon lower-
ing the pH from 7.5 to 5.0 in the presence of egg PC
vesicles were reversed upon elevating the pH to 7.5.
974 W. Li et al. / Advanced Drug Delivery Reviews 56 (2004) 967–985
The secondary structure of GALA when bound to
small unilamellar vesicles (SUV) (diameter f 50 nm)
made of the lipid POPC at pH 5.0 was determined
using ATR spectroscopy, by analysis of the amide IV
line shapes with Fourier self-deconvolution and curve
fitting [43]. A single peptide conformation was
obtained at the two lipid/peptide molar ratios tested,
i.e. 50/1 and 100/1, revealing a a-helical content of
100%. This result demonstrates that the amphipathic
helix is stabilized upon binding to POPC vesicles. The
lower values estimated from CD measurements with
egg PC LUV vesicles were explained by a lack of
correction for adsorption flattening, which is known
to cause a significant underestimation of the a-helical
content of membrane proteins. Polarized FTIR experi-
ments on oriented lipid multibilayers pre-formed with
the peptide GALA indicated that the axis of the helix
was essentially oriented parallel to that of the acyl
chains of the phospholipid, with the helical axis being
possibly slightly tilted (10 – 15j) in respect to the axis
of the hydrocarbon chains [43]. This result confirmed
the interpretation by the tryptophan quenching experi-
ments that GALA exhibits a transverse orientation in
egg PC bilayers. As discussed later, GALA’s ability to
permeabilize egg PC vesicles at pH 5.0 were attribut-
ed to the formation of aqueous pores composed of
transmembrane helices.
3.2.2. GALA induces the fusion of SUV made of
unsaturated PC lipids at low pH
GALA was originally designed to generate a water
soluble pH-triggered peptide fusogen that can mimic
the behavior of the fusogenic sequence of viral
proteins. The influenza virus is internalized into endo-
somes where the N-terminus of its HA protein is
rendered fusion-competent by a conformational
change resulting from pH acidification [45,46]. Here,
the idea was to use the pH-dependence of both GALA
conformation and its binding affinity to PC mem-
branes, to induce vesicle fusion preferentially at low
pH. A lipid mixing assay was employed to monitor
GALA-induced fusion of vesicles [2]. In this assay, a
population of vesicles containing 1 mol% of the two
fluorescence labeled lipids, N-4-nitrobenzo-2-oxa-1,3-
diazole (NBD)-phosphatidylethanolamine (PE) and
rhodamine (Rh)-PE was mixed in a 1:9 molar ratio
with probe-free vesicles. Before fusion of vesicles
occurred, NBD fluorescence was partially quenched
due to the transfer of its energy to the acceptor
fluorophore Rh. Upon addition of peptide to induce
the fusion of labeled and unlabeled vesicles, lipid
mixing resulted in a decrease of the bilayer concen-
trations of the donor and acceptor fluorophores, which
reduced fluorescence energy transfer. The percentage
of lipid mixing could be estimated from the degree of
recovery of NBD fluorescence. When POPC SUV
were used at a concentration of 100 AM, GALA-
induced lipid mixing at pH 5.0 was significant only
above a lipid to peptide molar ratio of 300/1 and was
peptide concentration dependent, reaching a value of
55% at a ratio of 50/1. In addition, the initial rate and
final extent of lipid mixing increased with the lipid
and peptide concentrations, at a constant lipid to
peptide ratio. This behavior suggests that vesicle
contact was required for lipid mixing. At pH 7.5 no
lipid mixing was observed for any of the lipids
studied, at the same lipid to peptide ratios of 50/1
and 100/1. This result confirms the drastic pH-depen-
dence of GALA’s ability to perturb bilayers. Lipid
mixing does not result from vesicle lysis and refor-
mation, since a FITC-detran 4100 Mw pre-encapsu-
lated in egg PC SUV remained entrapped after
addition of the peptide [2].
Lipid mixing at pH 5.0 was correlated with an
increase in light scattering, indicative of vesicle ag-
gregation or fusion [2]. Dynamic light scattering was
used to obtain an average size of egg PC and POPC
vesicles before and after addition of GALA at a 50/1
lipid to peptide ratio. Measurements demonstrated an
increase of the vesicle size at pH 5.0, whereas no
increase was observed at pH 7.5. In the case of egg PC
and POPC vesicles at pH 5.0, the size of the SUV
increased from 50 to 70 and from 50 to 100 nm,
respectively, after the addition of GALA [2]. When
the pH was elevated to 7.5 after incubation of vesicles
with GALA at pH 5.0, the vesicles maintained their
size of 70 nm, which confirmed that the increase in
lipid mixing at pH 5.0 was not due to reversible
aggregation but fusion. In agreement with the fact
that fusion of POPC SUV terminated when a size of
100 nm was reached, LUV of 100 nm diameter did not
show any lipid mixing or size increase at pH 5.0 after
GALA addition.
A short peptide (SFP-3) corresponding to the 14
first amino-acids of the GALA sequence, with an
additional cysteine at the C-terminus, was conjugated
 W. Li et al. / Advanced Drug Delivery Reviews 56 (2004) 967–985
to egg PC/Cholesterol/SMCC-PE (6/3/0.5) liposomes
to enhance the peptide’s ability to induce vesicles
fusion [44]. The fusogenic potency of SFP-3 after
coupling to 200 nm liposomes at a 400/1 lipid/peptide
molar ratio was studied using a lipid mixing assay
based on the fluorescence energy transfer between
NBD and Rh-labeled lipids. No lipid mixing was
observed at pH 7.4 when mixing (SFP-3)-coupled
Rh-labeled liposomes with (SFP-3)-coupled NBD-
labeled liposomes nor at pH 4.5 when mixing an
unconjugated peptide with Rh-labeled and NBD-la-
beled liposomes. However, a fusion yield of 75% was
obtained at pH 4.5 when mixing (SFP-3)-coupled Rh-
labeled liposomes with (SFP-3)-coupled NBD-labeled
liposomes. Light scattering measurements showed a
marked increase in the vesicle diameter from 235 nm
at pH 7.4 to 889 nm after 30 min at pH 4.5 [44].
Compared to the unconjugated SFP-3, the lipid-an-
chor of SFP-3-PE conjugate might facilitate the pep-
tide interaction with two adjacent bilayers and
promote membrane fusion by orienting the peptide
at the interface between the two membranes.
3.2.3. GALA induces the fragmentation of LUV made
of saturated PC lipids at neutral pH
GALA’s interaction with vesicles made of the
unsaturated lipids at neutral pH is very weak [3].
Nevertheless, with liposomes made of the saturated
lipids such as DMPC or DPPC, the situation is
drastically different [3]. Incubation of GALA (33
AM) with DMPC LUV at a lipid/peptide ratio of 30/
1 resulted in a clarification of the liposome suspension
within 30 min at 4 or 26 jC which is below or above
the gel to liquid-crystalline transition temperature
(Tm = 23 jC) of the lipid. A similar behavior was
observed with DPPC vesicles, only when the mixture
was incubated a few degrees above the Tm of the lipid,
i.e. 41 jC. In that case, the mixture remained optically
clear when the temperature was decreased below the
Tm after a pre-incubation above the Tm. Surprisingly,
the negatively-charged GALA (pH 7.5) could also
solubilize vesicles made of the saturated anionic lipid
DMPG.
The GALA/DMPC complex was isolated by gel
filtration chromatography at pH 7.5. Although incu-
bated at a molar ratio of DMPC to GALA of 30/1, the
resulting complex had a stoichiometry of 19/1. The
apparent diameter of the GALA/DMPC complex, as
975
measured by dynamic light scattering, was 30 nm.
Negative stain electron microscopy of the complex
revealed only stacked discoidal structures with an
approximate edge thickness of 44 Å and diameters
between 170 and 350 Å. When the pH was subse-
quently lowered from 7.5 to 5.0, the apparent diameter
of the GALA/DMPC complex increased to 415 nm
[3]. On negative stain electron micrographs, exten-
sively aggregated arrays of discoidal structures with
the same dimensions as those observed at pH 7.5 were
present. However, when GALA was mixed with egg
PC LUV at pH 5.0 at a lipid/peptide ratio of 30/1, only
vesicle profiles could be observed [3].
Thus, GALA could interact with liposomes com-
posed of saturated phospholipids at neutral pH and
form complexes similar to those described for apoli-
poproteins [3]. CD measurements confirmed that
GALA interacted with these vesicles at neutral pH.
Upon incubation of GALA with DMPC vesicles at 26
jC or with DPPC vesicles at 26 or 45 jC, an increase
of the peptide helicity was observed at a lipid/peptide
ratio of 30/1 [3]. Using the technique of FTIR, a a-
helical content of 65% was determined at pH 7.0 with
DMPC vesicles [43]. At 26 jC, the increase in helical
content with DMPC and DPPC vesicles was accom-
panied by a 7 nm blue shift of the tryptophan kmax,
while no shift was observed with DPPC at 45 jC.
In the DMPC/GALA discoidal structures formed at
pH 7.5, GALA helix was oriented parallel to the lipid
acyl chains, which supports the idea of a rim of
GALA surrounding a DMPC bilayer core [3]. The
same parallel orientation occurred when GALA inter-
acted at pH 5.0 with LUV composed of the unsatu-
rated lipids POPC and egg PC, but in this case the
vesicle structure was retained. This behavior suggests
that the amphipathic a-helix forms transmembrane
aggregates, or aqueous pores, in the membrane of
LUV composed of unsaturated lipids. Aggregation of
the discoidal DMPC/GALA particles upon lowering
the pH from 7.5 to 5.0 might reflect a transition
between these two states.
3.2.4. pH-dependent pore formation by GALA in LUV
As described previously, GALA binding to ve-
sicles made of POPC or egg PC is highly pH-depen-
dent. The binding is enhanced at low pH due to a
stabilization of GALA amphipathic a-helix. As dis-
cussed in the second part, it is believed that the ability
976 W. Li et al. / Advanced Drug Delivery Reviews 56 (2004) 967–985
of natural amphipathic helical peptides to permeabi-
lize vesicles or cells is related to their propensity to
adopt such a conformation. Therefore, GALA’s ability
to induce the release of contents from LUV made of
the lipid egg PC was investigated using an ANTS/
DPX leakage assay [1,5]. In this assay, 1-aminonaph-
thalene-3,6,8-trisulfonic acid (ANTS) and p-xylene-
bis-pyridinium bromide (DPX) are encapsulated in
egg PC LUV at concentrations where DPX quenches
ANTS fluorescence (Fig. 2). When a vesicle is per-
meabilized, release of its contents in the external
milieu produces a dilution of ANTS and DPX, which
highly reduces the quenching. The extent of ANTS/
DPX leakage can be estimated from the fluorescence
increase.
GALA’s ability to induce ANTS/DPX leakage
from egg PC LUV was highly dependent on the pH
of the solution from 4.5 to 7.5 [1,5]. At each pH
tested, a decrease in the lipid to peptide molar ratio at
a constant lipid concentration (0.1 mM), i.e. an
increase in the peptide concentration, resulted in a
higher extent of leakage. At pH 4.5 and 5.0, GALA
displayed similar behaviors and was considerably
active, since a lipid/peptide molar ratio of 2500/1
Fig. 2. Schematic kinetics of GALA-induced ANTS/DPX leakage
assay at pH 5.0. In this assay, ANTS and DPX are encapsulated in
vesicle at concentrations where DPX quenches ANTS fluorescence.
When a vesicle is permeabilized by GALA, release of its contents in
the external milieu produces a dilution of ANTS and DPX, which
highly reduces the quenching. The extent of ANTS/DPX leakage
can be estimated from percentage of the fluorescence increase.
was sufficient to induce 100% leakage. The enhanced
GALA activity observed when the pH is decreased
from 7.5 to 5.0 correlates with the increase in peptide
helicity and membrane-binding.
In all conditions tested, the leakage reached a
stable but different plateau of fluorescence value
f 10 min after addition of the peptide to the lip-
osomes [1,5]. If the peptide transferred among the
vesicles, eventually all of the contents would be
released. Thus this analysis revealed that the leakage
induced by GALA proceeded via an ‘‘all or none’’
mechanism, i.e. vesicles either leaked their entire
content in ANTS/DPX or did not leak at all. Thus,
the percent increase in ANTS fluorescence observed
at the plateau corresponded to the percent of vesicles
that had leaked their entire content in ANTS and
DPX. This behavior suggests that there were a critical
number of peptides that needed to be bound to a
vesicle to generate leakage of its contents, and the
peptide did not readily transfer among the vesicles at
low pH.
A model was proposed to describe GALA-in-
duced ANTS/DPX leakage at pH 5.0 [5,8] (Fig. 3).
The model predicts that when GALA is added to a
vesicle suspension, the peptide binds rapidly to the
lipid membrane where it aggregates to form aqueous
pores. In a pore, GALA has a predominantly a-he-
lical secondary structure with the helix axis aligned
perpendicular to the bilayer surface, as confirmed by
FTIR measurements [5,10]. Once a pore is assem-
bled, the entire vesicle content is rapidly released in
the external milieu. It is possible that the channel
assembles by the stepwise addition of monomers or
through the association of multimeric aggregates. In
either case, leakage only occurs when the channel
becomes large enough (M monomers) to permit
diffusion of the entrapped molecules. The fact that
ANTS/DPX leakage proceeded by an ‘‘all or none’’
mechanism suggests that once a pore is formed,
release of vesicle contents is fast. In addition, the
rapid changes in tryptophan emission maximum
observed when the peptide is added to an egg PC
liposome suspension at pH 5.0 confirms that GALA
binding to vesicles is a rapid process. A mathemat-
ical analysis of the percent of leakage obtained at
various lipid to GALA molar ratios at pH 5.0 was
attempted. Since from the lipid and peptide concen-
trations, the fraction of bound peptide at these
 W. Li et al. / Advanced Drug Delivery Reviews 56 (2004) 967–985 977

Fig. 3. Model of pore formation by GALA at acidic pH. The model predicts that when GALA is added to a vesicle suspension, the peptide binds
rapidly to the lipid membrane surface where it forms as planar aggregates parallel to the membrane surface with the apolar side of the helix
slightly embedded in the membrane and the polar side facing water. Along with the increase of peptide concentration in the membrane, the
planar aggregates expand and insert into the membrane to form aqueous pores. In a pore, GALA has a predominantly a-helical secondary
structure with the helix axis aligned perpendicular to the bilayer surface [5,10]. Once a pore is assembled, the entire content encapsulated in
vesicle is rapidly released to the external environment. It is possible that the channel assembles by the stepwise addition of monomers or through
the association of multimeric aggregates. In either case, content leakage only occurs when the channel becomes large enough (M monomers) to
permit diffusion of the entrapped molecules.

concentrations and the size distribution of the lip-
osomes are known, the model can predict the distri-
bution of the number of membrane-bound peptides
per vesicle. The model assumes that each vesicle that
contains i z M peptides bound to its bilayer will
contain a pore that allows leakage of ANTS/DPX
(aggregates composed of i z M monomers), which
corresponds to a situation where GALA aggregation
in the membrane is irreversible at low pH such as
pH 5.0 (Fig. 3). The percent of leakage was pre-
dicted as a function of the lipid to peptide molar
ratio for several M values. Calculated values agreed
with the experimental percents of leakage when the
minimum size of the pore that allows ANTS/DPX
leakage was set to M = 10 monomers. Taking into
account the possible experimental errors on the lip-
osomes size distribution and on the fraction of bound
peptide (0.645), the minimum M value was estimated
to be 10 F 2. The model has been improved to take
into account the possibility of reversible surface
aggregation. Results establish that GALA aggrega-
tion in POPC vesicles is quasi-irreversible at pH
5.0 [5,8].
Leakage experiments with vesicle-entrapped com-
pounds of different molecular weights ranging from
445 to 5200 Mw confirmed that 10 F 2 monomers for
pore formation is consistent with the data [5]. At a
lipid to peptide molar ratio of 2500/1, ANTS (445
Mw), NADH (660 Mw), and acid blue 9 (795 Mw)
were released from the vesicles, whereas AP5A (915
Mw) and inulin (5200 Mw) were retained. The pore
diameter was estimated as a function of the number of
helices. Given the Stokes radius of the vesicle-encap-
sulated compounds, a pore of 8 – 12 monomers could
explain the retention of AP5A and inulin and the
release of ANTS, NADH, and acid blue 9. The ability
of the model to explain and predict the kinetics of
leakage was also tested. It was assumed that the rate-
limiting step for ANTS/DPX leakage is the formation
of an aggregate composed of M = 10 peptides in the
bilayer. When the forward rate constant of aggrega-
tion, was set to 0.002 s 1, and aggregate dissociation
was ignored, the calculated curves were in good
agreement with the experimental kinetics, especially
within the first minute after peptide addition [5].
Using egg PC LUV asymmetrically labeled with
the fluorescent lipid probe NBD-PC, GALA was
shown to catalyze rapid translocation of the lipids
from one bilayer leaflet to the other at pH 5.0 [6]. A
mathematical analysis of the kinetics and final
978 W. Li et al. / Advanced Drug Delivery Reviews 56 (2004) 967–985
extents of NBD-PC flip-flop observed after addition
of GALA to asymmetrically labeled vesicles was
performed over a wide range of lipid to peptide
ratios (from 30/1 to 300,000/1). Two modes of
flip-flop were deconvoluted, one is related to pore
formation and a second arising from peptide aggre-
gates that are too small to form a pore. Analysis of
the final extents of lipid flip-flop confirmed that
GALA forms pores with a minimum size of
M = 10 F 2 monomers. In addition, the kinetic anal-
ysis predicted that the pore-mediated mode of flip-
flop proceeded with a rate constant kpore z 1 s 1,
while the bilayer-perturbing mode was much slower
(kperturbation = 3  10 6). The estimated rate constant
of lipid flip-flop associated with pore formation
corresponded to a situation where the asymmetry
of the probe distribution in the bilayer was relieved
in less than 60 s after a pore had been formed. A
mechanism to account for the rapid flip-flop induced
by GALA pore is that the phospholipid headgroup
resided in the water-filled pore, while the translocat-
ing acyl chains remained within the hydrophobic
interior of the bilayer. However, the peptide LAGA,
which was unable to form discrete pores, catalyzed
flip-flop through bilayer perturbations, with a rate
constant comparable to that of the bilayer-perturbing
mode of GALA [6].
The above investigations on GALA induced pore
formation were done using egg PC as membrane
model. To better understand the influence of phos-
pholipid acyl chain composition on the pore forma-
tion by GALA, the ANTS/DPX leakage assay was
examined at pH 5.0 from LUV vesicles made of PC
and phosphatidylglycerol (PG) with the following
acyl chain compositions: 1-palmitoyl-2-oleoyl (PO),
1,2-dioleoyl (DO), 1,2-dielaidoyl (DE), and 1,2-dipe-
troselinoyl (Dpe) [9]. A mathematical model was
able to predict and simulate the final extents of
GALA-mediated ANTS/DPX leakage assay from
POPC/POPG and DEPC/DEPG liposomes at pH
5.0 as a function of peptide concentration in the
bilayer, by considering that GALA pores responsible
for this leakage have a minimum size of 10 F 2
monomers and are formed by quasiirreversible ag-
gregation of the peptide [9]. With the phospholipid
acyl chain compositions tested, GALA-induced
ANTS/DPX leakage follows the rank order POPC/
POPG c DEPC/DEPG>DPePC/DPePG>DOPC/
DOPG. Results from binding experiments reveal that
this reduced leakage from DOPC/DOPG vesicles
cannot be explained by a reduced binding affinity
of the peptide to these membranes. By monitoring
the leakage of a fluorescent dextran, an increase in
the minimum pore size also does not explain the
reduction in ANTS/DPX leakage. The data suggest
that the bilayers composed of phospholipids contain-
ing a cis unsaturated bond per acyl chain (DO and
DPe) can stabilize surface-associated GALA mono-
mers or aggregates, while the effect of peptide
transbilayer insertion is reduced. GALA-induced
ANTS/DPX leakage is also decreased when the
vesicles contain PE lipid with small hydrophilic head
group [9]. This lends further support to the pore-
forming model that factors stabilizing the surface
state of GALA reduce its insertion and subsequent
pore formation in the bilayer (Fig. 4).
The effect of cholesterol on the pore formation
of GALA in LUV composed of neutral and nega-
tively charged phospholipids was also determined
[7]. Increasing cholesterol content in the membranes
resulted in a reduced efficiency of GALA to induce
membrane leakage. Part of the cholesterol effect
was due to reduced binding of the peptide to
cholesterol-containing membranes. An additional ef-
fect of cholesterol was to increase reversibility of
surface aggregation of the peptide in the membrane.
FRET experiments using NBD-labeled GALA and
Rh-labeled GALA confirmed that the degree of
reversibility of surface aggregation of GALA was
significantly larger in cholesterol-containing lipo-
somes, thus reducing the efficiency of pore forma-
tion [7].
3.2.5. Peptide orientation on membrane
In an attempt to resolve whether, at pH 5.0, the
N-terminus of the membrane-bound GALA is pre-
dominantly interacting with the bilayer surface or
whether it is embedded deeper in the bilayer core,
bromine-labeled lipids were employed as quenchers
of tryptophan fluorescence [4]. The depth of pene-
tration of the tryptophan within the bilayer can be
probed by including brominated lipids that position
the bromine at different locations in the bilayer. For
quenching to occur, bromine must contact or be
within 5 Å of the tryptophan moiety. 5,6-Dibromo-
cholesterol, which positions the bromine near the
 W. Li et al. / Advanced Drug Delivery Reviews 56 (2004) 967–985 979

Fig. 4. Reduced insertion of GALA aggregates with DOPC and DOPE. The bilayers composed of phospholipids containing a cis unsaturated
bond per acyl chain (DOPC) can stabilize surface-associated GALA monomers or aggregates, while the effect of peptide transbilayer insertion is
reduced. Peptide transbilayer insertion is also decreased when the vesicles contain PE lipid with small hydrophilic head group.

lipid headgroup region of the bilayer, and BrPC,
which positions the bromide at the 9,10-position of
the lipid acyl chains, were each incorporated into
egg PC LUV at varying mole percents. Both 10
mol% brominated cholesterol and 12 mol% BrPC
were able to quench tryptophan fluorescence at pH
5.0 at lipid/peptide molar ratios of 50/1 (24% and
31%, respectively) and 500/1 (31% and 33%, re-
spectively). These extents of fluorescence quenching
indicated that the N-terminus of GALA had pene-
trated into the hydrophobic portion of the bilayer. It
was proposed that GALA tryptophan is positioned
closer to the headgroup – hydrocarbon interface than
to the interior of the bilayer, which is consistent
with a transbilayer orientation of GALA in egg PC
liposomes.
We also determined the orientation of a biotiny-
lated version of the pore-forming peptide GALA at
pH 5.0 in LUV by designing a dipyrrometheneboron
difluoride (BODIPY)-avidin fluorescence binding to
a GALA biotinylated at the N- or C-terminus [8]
(Fig. 5). The BODIPY-avidin/biotin binding assay
was developed based on the phenomenon that the
green fluorescence intensity of a BODIPY-modified
avidin, upon its quasi-irreversible binding to D-biotin,
will be enhanced [47]. BODIPY-avidin was either
added externally after vesicle formulation or was
pre-encapsulated in vesicles to determine the fraction
of liposome-bound biotinylated GALA that exposed
its labeled terminus to the external or internal side of
the bilayer, respectively. Under conditions (at low
lipid/peptide molar ratios such as 2500/1) where
most of the membrane-bound peptides were involved
in transmembrane aggregates and formed aqueous
pores, the head-to-tail (N- to C-terminus) orientation
of GALA peptide was that 3/4 of the peptides
exposed their N-terminus on the inside of the vesicle
and their C-terminus on the outside, and 1/4 of the
peptides exposed their N-terminus outside and their
C-terminus inside. Under conditions resulting in
reduced amount of pore formation (at higher lipid/
peptide molar ratios), an increase was found in the
fraction of GALA termini exposed to the outside of
the vesicle when biotinylated GALA was added
externally. These results are consistent with the pore
formation model that requires a critical number of
peptides (M) in an aggregate to form a transbilayer
structure [5]. When the aggregation size of GALA
peptide is smaller than M, the peptide orientation is
mostly parallel to the membrane surface, and both N-
980 W. Li et al. / Advanced Drug Delivery Reviews 56 (2004) 967–985

Fig. 5. GALA-termini targeting using BODIPY-avidin/biotin binding assay. The BODIPY-avidin/biotin binding assay was developed based on
the phenomenon that the green fluorescence intensity of a BODIPY-modified avidin, upon its quasi-irreversible binding to D-biotin, will be
enhanced [47]. BODIPY-avidin was either added externally after vesicle formulation or was pre-encapsulated in vesicles to determine the
fraction of liposome-bound biotinylated GALA that exposed its labeled terminus to the external or internal side of the bilayer, respectively. Both
N- and C-terminus of GALA can be biotinylated.

and C-terminus of the biotinylated peptide are ex-
posed to external BODIPY-avidin [8].
4. Applications for the delivery of plasmid DNA
and oligonucleotide (ODN)
To a large extent, advances on gene therapy depend
on the efficient and safe design of gene delivery
vectors. Non-viral gene delivery systems are devel-
oped as a substitution for viral gene transfer vectors
because they may be safer and easier to manufacture.
Cationic systems that mimic aspects of the viral
structure and its protein/membrane coating, can po-
tentially protect the DNA from blood clearance, and
then transfer the gene into the targeting tissue.
The bilayer-destabilizing properties of GALA at
low pH have been utilized to enhance the delivery of
genes into the nucleus of cells in vitro (Table 1).
Complexes prepared by mixing DNA with polylysine-
conjugated ligands can be efficiently delivered into
cells by receptor-mediated endocytosis [48,49]. How-
ever, such complexes accumulate in the endosome/
lysosome. This reduces the efficiency of DNA deliv-
ery into the nucleus. Addition of a peptide that
destabilizes membranes at low pH can mediate the
release of the DNA from the acidic endocytic com-
partment and enhance gene expression. In that respect,
synthetic pH-sensitive peptides derived from the N-
terminal region of influenza virus HA subunit (HA2)
were shown to be particularly potent [50,51]. GALA
is considerably more active than these peptides in
permeabilizing PC vesicles at low pH but when bound
by electrostatic interactions to a DNA/polylysine-
conjugated ligand (transferrin) complex, the GALA
peptide was less efficient in enhancing gene expres-
sion than the influenza-based peptides [51].
When DNA was complexed to a dendritic poly-
amidoamine cationic polymer (dendrimer) to which
GALA was covalently attached to amines on the
dendrimer, an increased gene expression was ob-
served [18]. This enhanced effect is an indicator that
GALA motif can be covalent conjugated into suit-
able platform such as dendrimer to make an efficient
gene transfer system. We observed that GALA when
simply added to the dendrimer DNA complex, could
also improve gene transfer. We ascribe this to the
formation of a ternary complex between the compo-
 W. Li et al. / Advanced Drug Delivery Reviews 56 (2004) 967–985 981

Table 1
Formulation of DNA or ODN delivery systems based on GALA and related peptides
Fusogenic peptide Vector composition Reference
Nucleic acid condensing reagent Ligand
GALA Polylysine Transferrin [51]
GALA Cationic liposome (DOTAP, or DOTAP/DOPE) Transferrin [52 – 54]
GALA Dendrimer – [18]
EALA Cationic liposome (DOTAP/cholesterol/PEG-PE) Folate [66]
Gramicidin S Gramicidin Sa – [11]
KALA – – [12,67]
KALA PEI – [55]
KALA Polylysine – [57]
KALA (Poly(DMAEMA-NVP))-b-PEG-galactose Galactose [56]
46, Hel 11-7 – – [68]
INF7, JTS-1 K8 peptide K(GalNAc)2 [69]
PpTG1, ppTG20 – – [64]
LAH4 analogues – – [62]
a
Neutral lipid DOPE was added into the complex for transfection enhancement.

nents and GALA’s ability to destabilize membranes
at low pH.
Gene transfection of a formulation based on DNA/
cationic liposome/transferrin complex can be en-
hanced by GALA peptide [52 – 54]. By studying the
transfection mechanism of the complex, the presence
of transferrin is proposed to trigger internalization of
the complex by receptor-mediated endocytosis and the
GALA peptide is used to promote endosomal desta-
bilization and release the genetic material into cyto-
plasm. Since the size of the pores (5– 10 Å) caused by
GALA is too small to allow the direct passage of
DNA through, GALA was suggested to not only
induce the membrane disruption at low pH but also
promote the DNA unpacking [54]. These studies
support the concept that pH-sensitive membrane dis-
rupting peptides such as GALA can improve gene
transfer. To date, the interaction mechanism between
GALA and the DNA condensing reagent (including
polylysine, cationic liposome or dendrimer) is not
well characterized and requires further evaluation if
fusogenic peptide-based gene delivery systems are to
be improved (Table 1).
Compared to the multi-component systems de-
scribed above, the cyclic amphipathic peptide (gram-
icidin S) a cyclic h-turn structure was used to form a
three-component gene delivery system [11]. In this
system, the amphipathic peptide itself is a DNA-
condensing reagent; the only other component in-
volved in, is the fusogenic ‘‘helper’’ lipid, DOPE.
Gramicidin S strongly binds to DNA by charge
interactions and mediates high level of luciferase
transfection activity ( f 109 light units per mg of cell
protein) [11]. More interestingly, the peptide/DNA/
DOPE complex mediates rapid association of DNA
with cells, and lysomotrophic agents do not influence
the extent of transfection, which suggest that DNA
entry into the cell may be directly via the plasma
membrane [11]. Our unpublished in vivo studies with
this complex indicated a substantial level of toxicity
when the gramicidin S/DOPE/DNA complexes were
injected intramuscularly into animals.
KALA is a fusogenic peptide designed to fulfill all
the aspects required for efficient gene delivery without
the aid of other components [12]. To our knowledge,
KALA was the first designed peptide, which can bind
DNA, destabilize membranes and mediate significant
gene delivery. KALA undergoes a pH-dependent
amphipathic a-helix to random coil conformational
change when environmental pH decreased from 7.5 to
5.0. KALA mediates release of entrapped dyes from
lipid vesicles in the presence and absence of ODN,
and the leakage is correlated with interaction of the
peptide with bilayer by KALA tryptophan emission
spectrum [12]. Both ODN and plasmid DNA can be
delivered into cells by KALA. GALA, by itself, on the
other hand did not enhance the delivery due to its
strong negative charge. This demonstrated that the
positively charged Lys residues on KALA are re-
quired for DNA (or ODN) condensing and intracel-
982 W. Li et al. / Advanced Drug Delivery Reviews 56 (2004) 967–985
lular trafficking. However, the mechanism of how
KALA promotes the transfer of DNA across mem-
branes is unknown.
KALA has also be used with other components,
such as polyethylenimine (PEI) [55], (poly
(DMAEMA-NVP))-b-PEG-galactose [56], polylysine
[57] to formulate DNA complex for gene delivery
(Table 1). In the KALA/polylysine/DNA system,
KALA increases the transfection level of luciferase
activity 10,000 times in evaluated conditions, and
further displacement of native polylysine to poly(eth-
ylene glycol) (PEG) modified polylysine keeps the
delivery system at high transfection level but obvi-
ously decreases the toxicity [57]. PEGylated KALA is
used to coat the surface of DNA/PEI complex to
prevent the nonspecific aggregation of particles and
enhance the gene delivery efficacy by membrane
destabilization [55].
5. What are the advantages of GALA-like peptides
as components of a drug and gene delivery system?
We believe that the advantages of synthetic pep-
tides as components of delivery systems are that they:
can be synthesized in high purity, are biocompatible
and have a significant data base for the secondary
structural preference and hydrophobic contribution of
the side chains. Moreover, there is an increasing
availability of interesting side chains that can be
incorporated into a peptide to endow the peptide with
useful characteristics. Finally, combinatorial synthetic
methods and high throughput screening enable the
identification of interesting sequences that improve
cytoplasmic delivery.
Clearly the low pH-sensitive amphipathic a-helical
structural motif lends itself to incorporation into water
soluble complexes or onto the surface of liposomes
precisely because such peptides can be manipulated in
water at neutral pH. The advantage of a titratable
linkage compared to a hydrolytically or an enzymat-
ically activated linkage is that the rate of conversion is
orders of magnitude faster for the titration compared
to the other two mechanisms [58]. These biorespon-
sive peptides exploit the pH-drop as the endosome
acidifies to between pH 5 and 6. Peptides with a more
hydrophobic character, such as the influenza based
fusion peptides have solubility limits. Peptides with a
more hydrophilic character, such as the polyhistidine
peptides [59] may be able to buffer the pH in endo-
somes and promote a lysosomotropic effect [18,60]
but are unable to disrupt membranes unless hydro-
phobic residues are included [13,61,62].
Whether or not the a-helical motif is the preferred
secondary structure for membrane disruption, is not
known. Other structural motifs such as the hydropho-
bic a-helix or the h-sheet have not been well studied
because of the difficulties to make them water soluble
in the case of the hydrophobic a-helix and the prob-
lems associated with the propensity of hydrophobic h-
sheet structures to form insoluble fibers.
A drawback of the peptide membrane destabilizers
is that current sequences range in length from 9 to 30
amino acids. This is not a problem for an experimental
delivery system but if such molecules are to have a
commercial role, the therapeutic agent being delivered
must either be very potent or produce a therapeutic
effect that cannot be achieved using other technolo-
gies. This is because if the peptides are a significant
fraction of the dose, the cost of the components must
be factored into the final cost of the product. Thus less
complicated synthetic molecules might be devised to
perform functions analogous to the peptides.
A variety of groups have synthesized a series of
peptides to optimize gene delivery [62 – 64]. The
highly refined tools of combinational chemistry and
high throughput screening system make it possible to
select suitable peptide-based gene delivery system
from a large library of peptide analogues in the
absence of defined sequence– structure –function re-
lationship [65]. The challenge for the field is to design
screens that will predict how such systems work for in
vivo gene transfer.
6. Conclusions
Studies with the pH-sensitive peptide GALA and
its analogues have been particularly informative, es-
pecially regarding the requirement of a long amphi-
pathic a-helical conformation to efficiently perturb
lipid membranes. This phenomenon raised the possi-
bility to include the GALA peptide for gene delivery
where the pH-sensitive peptide can perturb the cell
membrane system to help the vector-encapsulated
nucleic acid drugs escaping from endosome in low
 W. Li et al. / Advanced Drug Delivery Reviews 56 (2004) 967–985
pH environment. Since GALA has net negative
charges, it is usually used together with other DNA
or ODN condensing reagents to formulate particles for
delivery. Amphipathic peptides other than GALA also
mediate high efficient gene transfection. A partial
replacement of Glu by Lys in the GALA sequence
yields the KALA peptide with a pH-dependent ability
to form pores drastically different from that of GALA.
The cationic peptide KALA also has the membrane-
perturbing capabilities, and its additional ability is to
condense DNA, which can be used to deliver ODN
and plasmid DNA into the nucleus of cells without
other condensing reagent although the conformation
change of KALA is from a-helix to random coil when
the pH decreased from neutral to the acidic range.
The mechanisms of membrane perturbation in-
duced by amphipathic a-helical peptide-based gene
delivery systems are not yet understood and explan-
ations for how such systems function to deliver DNA
into cells is based upon work in synthetic lipid
vesicles. This is not a criticism just of the peptide
systems rather it is a shortcoming of almost all of the
non-viral gene delivery systems. Further work is
necessary to gain insights into the mechanisms of
membrane perturbation by amphipathic a-helices
when they are complexed with DNA or ODN, in
living cells to insure that studies in lipid vesicles are a
good indicator for what is occurring when a delivery
system interacts with cells.
Acknowledgements
Research on GALA in the Szoka laboratory has
been supported by the National Institutes of Health,
most recently by R01 EB003008. Mass spectrometry
analysis of peptide sequences was supported by
NCRR RR01614 (UCSF).
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