Liposomal polyamine-dialkyl phosphate conjugates as effective gene carriers: Chemical structure, morphology, and gene transfer activity

Article abstract of DOI:10.1021/bc900376y

Synthetic cationic lipids are promising transfection agents for gene therapy. We report here that polyamine conjugates of dialkyl phosphates, combined with natural lipids and assembled in the form of liposomes (polycationic liposome: PCL), possess high transfection activity in the COS-1 cell line. Furthermore, we describe the functional morphology of the PCL/DNA complexes as revealed by atomic force microscopy (AFM). The conjugates were synthesized from dialkyl phosphates (with alkyl chain lengths of 12, 14, or 16 carbons) by reaction with the polyamine molecules, spermidine, spermine, or polyethylenimine (PEI(1800)). [Dewa, T., et al. Bioconjugate Chem. 2004, 15, 824]. The PCL composed of the spermidine and C16 conjugate combined with phospholipid and cholesterol (conjugate/phospholipid/cholesterol = 1/1/1 as a molar ratio) exhibited 3.6 times higher activity than that of a popular commercial product. Systematic tests revealed clear correlations of the transgene activity with physical properties of the polyamine, in particular, that longer alkyl chains and the lower molecular weight polyamines (spermidine, spermine) favor high efficacy at the higher nitrogen/phosphate ratio = 24 (N/P, stoichiometric ratio of nitrogen in the conjugate to phosphate in DNA). The low molecular weight polyamine-based PCLs, which formed 150-400 nm particles with plasmid DNA (lipoplexes), exhibited ～3-fold higher gene transfer activity than micellar aggregates (lacking phospholipid and cholesterol) of the corresponding conjugate. In contrast, the PEI-based PCL formed large aggregates (～1 μm), that, like the micellar aggregate form, had low activity. Activity of the low molecular weight polyamine-based PCLs increased linearly with the N/P of the lipoplex up to N/P = 24. Formation of lipoplexes was examined by agarose gel electrophoresis, dynamic light scattering (DLS), and AFM. At the lower N/P = 5, large aggregates of complex (～1 μm), in which DNA molecules were loosely packed, were observed. At higher N/P, lipoplexes were converted into smaller particles (150-400 nm) having a lamellar structure, in which DNA molecules were tightly packed. Such morphological features of the lipoplex correlate with the dependence of transfection on the N/P in that the lamellar structures gave superior transfection. AFM also indicated that the lipoplexes disassembled significantly, releasing DNA, when the lipoplexes were exposed to acidic conditions (pH 4). The significance for transfection activity of the metamorphosis of bilayer lipoplexes is discussed relative to that of the less active micellar aggregate form, which is unresponsive to pH change.

Full text of DOI:10.1021/bc900376y

844
Bioconjugate Chem. 2010, 21, 844–852
Liposomal Polyamine-Dialkyl Phosphate Conjugates as Effective Gene
Carriers: Chemical Structure, Morphology, and Gene Transfer Activity
Takehisa Dewa,*,† Tomohiro Asai,⊥ Yuka Tsunoda,⊥ Kiyoshi Kato,† Daisuke Baba,† Misa Uchida,†
Ayumi Sumino,† Kayoko Niwata,⊥ Takuya Umemoto,⊥ Kouji Iida,# Naoto Oku,⊥ and Mamoru Nango*,†
Department of Life and Materials Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku,
Nagoya 466-8555 Japan, Department of Medical Biochemistry and Global COE, University of Shizuoka School of
Pharmaceutical Sciences, 52-1 Yada, Suruga-ku, Shizuoka, 422-8526 Japan, and Nagoya Municipal Industrial Research Institute,
3-4-41 Rokuban-cho, Atsuta-ku, Nagoya 456-0058 Japan. Received August 24, 2009; Revised Manuscript Received January 5, 2010
Synthetic cationic lipids are promising transfection agents for gene therapy. We report here that polyamine
conjugates of dialkyl phosphates, combined with natural lipids and assembled in the form of liposomes (polycationic
liposome: PCL), possess high transfection activity in the COS-1 cell line. Furthermore, we describe the functional
morphology of the PCL/DNA complexes as revealed by atomic force microscopy (AFM). The conjugates were
synthesized from dialkyl phosphates (with alkyl chain lengths of 12, 14, or 16 carbons) by reaction with the
polyamine molecules, spermidine, spermine, or polyethylenimine (PEI(1800)). [Dewa, T., et al. Bioconjugate
Chem. 2004, 15, 824]. The PCL composed of the spermidine and C16 conjugate combined with phospholipid
and cholesterol (conjugate/phospholipid/cholesterol ) 1/1/1 as a molar ratio) exhibited 3.6 times higher activity
than that of a popular commercial product. Systematic tests revealed clear correlations of the transgene activity
with physical properties of the polyamine, in particular, that longer alkyl chains and the lower molecular weight
polyamines (spermidine, spermine) favor high efficacy at the higher nitrogen/phosphate ratio ) 24 (N/P,
stoichiometric ratio of nitrogen in the conjugate to phosphate in DNA). The low molecular weight polyamine-
based PCLs, which formed 150-400 nm particles with plasmid DNA (lipoplexes), exhibited ∼3-fold higher
gene transfer activity than micellar aggregates (lacking phospholipid and cholesterol) of the corresponding conjugate.
In contrast, the PEI-based PCL formed large aggregates (∼1 µm), that, like the micellar aggregate form, had low
activity. Activity of the low molecular weight polyamine-based PCLs increased linearly with the N/P of the
lipoplex up to N/P ) 24. Formation of lipoplexes was examined by agarose gel electrophoresis, dynamic light
scattering (DLS), and AFM. At the lower N/P ) 5, large aggregates of complex (∼1 µm), in which DNA molecules
were loosely packed, were observed. At higher N/P, lipoplexes were converted into smaller particles (150-400
nm) having a lamellar structure, in which DNA molecules were tightly packed. Such morphological features of
the lipoplex correlate with the dependence of transfection on the N/P in that the lamellar structures gave superior
transfection. AFM also indicated that the lipoplexes disassembled significantly, releasing DNA, when the lipoplexes
were exposed to acidic conditions (pH 4). The significance for transfection activity of the metamorphosis of
bilayer lipoplexes is discussed relative to that of the less active micellar aggregate form, which is unresponsive
to pH change.
for cationic amphiphiles concerning the cationic and hydropho-
bic portions (24-28). Such amphiphiles form self-assembling
micelles and liposomes in an aqueous phase, the structures of
which have been investigated using small-angle X-ray scattering
(SAXS)1, transmission electron microscopy (TEM), and atomic
force microscopy (AFM) to gain knowledge about structure-
activity relationship, particularly those involving ordered struc-
tures (lamellar, inverted hexagonal, and cubic phases) and their
morphological changes (29, 30) as well as about the size of
complexes (31).
INTRODUCTION
Development of more efficient and safer gene carriers using
nonviral compounds is one of the most challenging aspects of
gene therapy (1, 2). Compared to viral carrier systems, nonviral
gene carrier systems have advantages in simplicity of use, lack
of specific immune response, and ease of mass production due
to the low cost of preparation; however, they have the
disadvantage of low transfection efficiency, which needs to be
overcome (3, 4). To improve the efficiency of nonviral carriers,
many synthetic organic compounds, including cationic lipids
(5-9), polycations (10-15), and combinations thereof (16-22),
have been developed as nonviral gene carriers (23). Substantial
research has been reported on structure-activity relationships
The mechanism of gene delivery by such cationic carriers
probably involves an endosomal pathway (32): (i) cellular uptake
via endocytosis, (ii) DNA release from endosome, and (iii) entry
1Abbreviations: SAXS, small-angle X-ray scattering: TEM, transmis-
sion electron microscopy; AFM, atomic force microscopy; NLS, nuclear
localization signal peptide; PCL, polycation liposome; PEI, polyeth-
ylenimine; MALDI-TOF-MS, matrix assisted laser desorption ioniza-
tion-time-of-flight-mass spectroscopy; DOPE, 1,2-dioleoyl-sn-glycero-
3-phosphoethanolamine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phospho-
ethanolamine; PLL, poly(L-lysine); FBS, fetal bovine serum; DMEM,
Dulbecco’s Modified Eagle Medium; N/P, nitrogen/phosphate ratio.
* To whom correspondence should be addressed. Department of Life
and Materials Engineering, Nagoya Institute of Technology, Gokiso-
cho, Showa-ku, Nagoya 466-8555 Japan (T.D.) Tel/Fax: +81-52-735-
5144. (M.N.) Tel/Fax: +81-52-735-5226. E-mail: takedewa@nitech.ac.jp,
nango@nitech.ac.jp.
† Nagoya Institute of Technology.
⊥ University of Shizuoka.
# Nagoya Municipal Industrial Research Institute.
10.1021/bc900376y  2010 American Chemical Society
Published on Web 04/08/2010

Liposomal Polyamine Conjugates as Gene Carriers
Bioconjugate Chem., Vol. 21, No. 5, 2010 845
Scheme 1. Polyamine-Dialkyl Phosphate Conjugates As Gene Carriersa
a NH2-(Polyamine) represents polyamines and their primary amine portion that reacts with the phosphate portion.
into the nucleus. Many researchers have devised cationic
compounds that facilitate the process, for example, ligand-
conjugated molecules targeting a receptor such as integrin (33, 34),
pH-responsive or cleavable molecules that enable escape of
DNA from endosome (13, 35-38), and conjugation of nuclear
localization signal peptides (NLS) (39, 40) for steps (i)-(iii),
respectively. For cationic lipids/DNA complexes (lipoplexes),
it has been proposed that a morphological change from lamellar
to inverted hexagonal phase in the acidic endosomal environ-
mentfacilitatestheendosomalreleaseandescapeofDNA(41,42).
In addition to investigation of intracellular trafficking of
polycation-DNA complexes (polyplexes and lipoplexes), ob-
servation of morphology and metamorphosis of the complexes
is very important to shed light on the mechanism of gene transfer
and provide information for development of novel synthetic
carriers (30, 43, 44).
We have reported that polycationic liposomes (PCL) contain-
ing cetylated polyethylenimine (cetyl-PEI) possess high gene
transfer activity (45-47). The cetyl-PEI molecule is anchored
by the hydrophobic cetyl portion and is distributed over the
liposomal surface. In our previous report, we proposed a possible
mechanism of PCL-mediated gene transfer, in which PCL/DNA
complexes are taken up by the endosomal pathway (based on
tracking of fluorescence-labeled components, PCL lipid, cetyl-
PEI, and DNA), after which the cetyl-PEI/DNA complex is
released and transferred into the nucleus via the cytosol (48).
Compaction of DNA is therefore crucial, and both electrostatic
and hydrophobic interactions in the cetyl-PEI/DNA complex
are responsible for its effective compaction.
We have previously described novel polyamine (spermidine,
spermine, or PEI)-dicetyl phosphate conjugates, prepared via a
facile synthetic route (Scheme 1) (49) in which the polyamine
and phosphate portions are linked through a P-N bond. When
suspended in aqueous solution, they form micellar aggregates
and exhibit moderate gene-transfer activity, the magnitude of
which is relatively insensitive to the modification of the
polyamine portion. In the present report, we present (1) evidence
demonstrating that the transgene activity is dramatically en-
hanced when the conjugates are assembled into liposomes
containing cholesterol and phospholipid and that the activity is
susceptible to the chemical modification of the conjugate both
in the polyamine and in the hydrophobic chain portions (Scheme
1). We show further that (2) gene transfer activity of the
corresponding PCLs strongly depends on the type of polyamine
in the conjugate, with notable differences between the lower
molecular weight polyamines (spermidine and spermine) on one
hand and the polymer type (PEI(1800)) on the other.
lipoplexes and their transfection efficiency. AFM analysis has
a considerable advantage for observation of lipoplex morphol-
ogy, especially for less ordered structures (50); however, until
now little clear evidence has been reported on the relationship
between morphological change and DNA release. In this
research, DNA release as a result of disassociation of the
complex was revealed by AFM. We discuss morphology-activity
relationships on the basis of electrophoresis analysis, dynamic
light scattering (DLS), and AFM observation.
EXPERIMENTAL PROCEDURES
General Methods. Unless stated otherwise, all chemicals and
reagents were obtained commercially and used without further
purification. Dialkyl phosphates, dilauryl phosphate (DLP), dimyri-
styl phosphate (DMP), and dicetyl phosphate (DCP) were synthe-
sized from corresponding alcohols, i.e., lauryl (C12), myristyl
(C14), and cetyl (C16) alcohols, and POCl3 in accordance with
the reported method (51). The polyamine-dicetyl phosphate
conjugates, DCP-spermidine (DCP-spd), DCP-spermine (DCP-
spm), and DCP-PEI(1800) (DCP-PEI) were synthesized as
described previously (49). Other conjugates, DLP-spermidine
(DLP-spd), DLP-spermine (DLP-spm), DLP-PEI(1800) (DLP-
PEI), DMP-spermidine (DMP-spd), DMP-spermine (DMP-
spm), and DMP-PEI(1800) (DMP-PEI), were also synthesized
using analogous procedures. Spectral data are given below.
(Note: The asymmetric molecule, spermidine, has two distin-
guishable primary amines. Products, DLP-spd, DMP-spd, and
DCP-spd, bearing a spermidine moiety, have two isomers,
which, to date, have not been identified spectroscopically.
Similarly, the content of primary amine of PEI(1800) in the
products DLP-PEI, DMP-PEI, and DCP-PEI is 25% in total
amino moieties (on the basis of theoretical calculation); thus,
some structural uncertainty cannot be avoided.)
DLP-spermidine conjugate (DLP-spd). 1H NMR (300 MHz,
CDCl3): δ 0.88 (t, 6H), 1.26 (s, 36H), 1.51-1.95 (br m, 12H),
2.65-3.05 (br m, 6H), 3.98 (br m, 4H), 4.50 (br s, 4H). MALDI-
TOF-MS for (C31H69N3O3P)+: calcd 562.9, found 563.0.
1
DLP-spermine conjugate (DLP-spm). H NMR (300 MHz,
CDCl3): δ 0.88 (t, 6H), 1.26 (s, 36H), 1.49-2.00 (br m, 15H),
2.63-3.05 (br m, 14H), 3.95 (br m, 4H). MALDI-TOF-MS for
(C34H76N4O3P)+: calcd 620.0, found 620.2.
DLP-PEI(1800) conjugate (DLP-PEI). 1H NMR (300 MHz,
CDCl3): δ 0.88 (t, 6H), 1.26 (s, 36H), 1.58-1.65 (br m, 4H),
2.55-3.80 (br m, 209H), 3.95 (br m, 4H).
DMP-spermidine conjugate (DMP-spd). 1H NMR (300 MHz,
CDCl3): δ 0.88 (t, 6H), 1.26 (s, 44H), 1.50-1.71 (br m, 10H),
2.61-3.02 (br m, 12H), 3.98 (br m, 4H). MALDI-TOF-MS for
(C35H77N3O3P)+: calcd 619.0, found 618.9.
We also examined (3) the morphology of the lipoplexes by
AFM and discuss the relationship between the structure of

846 Bioconjugate Chem., Vol. 21, No. 5, 2010
Dewa et al.
DMP-spermine conjugate (DMP-spm). 1H NMR (300 MHz,
CDCl3): δ 0.88 (t, 6H), 1.26 (s, 44H), 1.61-2.00 (br m, 17H),
2.63-3.04 (br m, 12H), 3.97 (br m, 4H). MALDI-TOF-MS for
(C38H84N4O3P)+: calcd 676.1, found 676.1.
DMP-PEI(1800) conjugate (DMP-PEI). 1H NMR (300 MHz,
CDCl3): δ 0.88 (t, 6H), 1.26 (s, 44H), 1.56-1.65 (br m, 4H),
2.55-3.82 (br m, 209H), 3.95 (br m, 4H).
Preparation of Polycation Liposome (PCL). Polycation
liposome suspensions were typically prepared as follows:
polyamine conjugate, phospholipid (either 1,2-dioleoyl-sn-
glycero-3-phosphoethanolamine (DOPE) or 1,2-dipalmitoyl-sn-
glycero-3-phosphocholine (DPPC)), and cholesterol (1/1/1 as
a molar ratio) were dissolved in chloroform/t-butyl alcohol
(2/1). After removal of chloroform under reduced pressure, the
residual solvent was removed by freeze-drying overnight. The
lyophilized powder was hydrated with Tris-HCl buffer (20 mM,
pH 8.0) followed by three freeze-thaw cycles, and the resultant
suspension was then subsequently extruded through polycar-
bonate membranes of 0.4, 0.2, and 0.1 µm pore diameter at
room temperature. For vesicles composed of DPPC, the hydra-
tion and extrusion were carried out at 60 °C. Unless stated
otherwise, PCL refers to the DOPE-based polycation liposome.
A suspension of the polyamine conjugate alone was prepared
in the Tris-HCl buffer (20 mM, pH 8.0) by ultrasonication for
3 min. For convenience, particles so prepared are termed
“micellar aggregates”. Hereafter, polycation liposomes (PCL)
and micellar aggregates (M) composed of the conjugates are
described as “conjugate(PCL)”, such as DCP-spd(PCL), and
“conjugate(M)”, such as DCP-spd(M), respectively.
Lipoplex Formation with Plasmid DNA. A plasmid encod-
ing luciferase gene, pCAG-luc3 (6480 bp, a gift of DNAVEC
Institute, Tsukuba, Japan), was amplified in E. coli JM109
(Nippon Gene, Toyama, Japan) and purified as described before
(47). One microgram of the plasmid DNA in a TE buffer was
added to a suspension of PCL containing 1 mM of polyamine
conjugate so as to give the desired nitrogen/phosphate ratio,
N/P. The mixture was incubated for 20 min at room temperature
when used for transfection. A plasmid, ColE1 DNA (6646 bp,
Nippon Gene), was used for an assay of ethidium bromide
intercalation and morphological observation of lipoplexes with
AFMs (JSPM-4210, JEOL, and PicoPlus, Molecular Imaging),
using the AC mode under ambient air conditions. A sample
was deposited on a mica surface by spin coating, followed by
drying in a desiccator for 1 h under reduced pressure to remove
water from the mica surface. A freshly cleaved mica surface is
negatively charged. As appropriate, the mica was treated with
poly(L-lysine) (PLL) to provide positively charged surface.
Hereafter, the negatively and positively charged mica substrates
are described as bare mica and PLL-mica, respectively.
The sizes of PCLs, micellar aggregates, and their lipoplexes
were analyzed by dynamic light scattering (DLS) with a
NICOMP particle sizing system (model 370, Santa Barbara, CA,
USA). The ꢀ-potentials of the cationic vectors were measured
with ZETA SIZER Nano-ZS (MALVERN, Worcestershire,
U.K.). Agarose-gel electrophoretic analysis was conducted by
using 0.8% agarose (UltraPure agarose, Invitrogen) and 1 kb
DNA ladder marker (Invitrogen). Electrophoresed DNA bands
were stained with ethidium bromide.
Figure 1. Transfection efficacy of polyamine-lipid conjugates, DCP-
spd (A), DCP-spm (B), and DCP-PEI (C), and their constituents, DCP
(A) and polyamines, spermidine (spd, A), spermine (spm, B), and
PEI(1800) (PEI, C), on COS-1 cells. Efficacy was evaluated with the
luciferase activity. The observed values for spermidine and spermine
indicated by the asterisks (*) were apparently negligible on the activity
scale shown. The conjugates DCP-spd(M), DCP-spm(M), and DCP-
PEI(M) represent their micellar aggregate forms, and DCP-spd(PCL),
DCP-spm(PCL), and DCP-PEI(PCL) represent the conjugate-based
PCLs (conjugate/DOPE/cholesterol ) 1/1/1 (mol/mol/mol)). The
nitrogen/phosphate (N/P) ratio was 16/1 for the polyamine conjugate/
DNA complexes. For the monoanionic DCP, the molar ratio, DCP/
nucleotide ) 16/1, was applied as a negative control experiment.
Transfection was conducted in the presence of 10% FBS.
onto each of several 35 mm dishes and incubated overnight in
a CO2 incubator. Then, the cells were washed twice with
DMEM, and a suspension of lipoplex (1 µg DNA) was added
to them in the presence of 10% FBS-DMEM. After 3 h
incubation (37 °C, 5% CO2), the cells were washed twice with
DMEM and cultured for another 48 h in 10% FBS-DMEM.
The cells in the 35 mm dishes were washed twice with
phosphate-buffered saline at 37 °C, and 200 µL of cell lysis
buffer (LC-ꢁ, TOYO B-Net Co. Ltd., Tokyo) was added. After
15 min incubation, the cells were collected with a cell scraper,
frozen at -80 °C, and then thawed at room temperature. The
lysate was centrifuged at 15 000 rpm for 5 min at 4 °C. The
supernatant was subjected to the luciferase assay (Pica Gene,
TOYO B-Net Co. Ltd., Tokyo) using a luminophotometer
(Luminescencer-PSN AB-2200, ATTO). The observed intensity
in instrument light units was normalized to the amount of protein
determined by BCA protein assay kit (PIERCE) to give relative
light units (RLU/mg protein).
RESULTS
Transfection Efficacy of Micellar Aggregates and PCL
Vectors: Comparison of the Dicetyl Phosphate Derivatives
of Spermidine, Spermine, and Polyamine. Figure 1A shows
the transfection efficacy of the polyamine conjugate, DCP-
spd(M), and its constituent molecules, DCP and spermidine
(spd). The conjugate (in this case an aqueous micellar suspen-
sion) shows greatly increased efficacy relative to the constituent
molecules, DCP and spermidine. For these polyamine/DNA
complexes, the nitrogen/phosphorus ratio (N/P) was 16. The
data of the figure indicate that coupling the lipophilic and
cationic portions is essential to obtain gene transfer. Such an
For observation of morphological change of lipoplex by
acidification, to a solution of lipoplex (pH 8.0) was added acetic
acid so as to acidify down to pH 4. The solution was incubated
for 1 h at room temperature.
Transfection Procedure. COS-1 cells were cultured in
Dulbecco’s modified Eagle medium (DMEM) supplemented
with 10% fetal bovine serum (FBS, Japan Bioserum Co. Ltd.)
under a humidified atmosphere of 5% CO2 in air. One day before
a transfection experiment, 1 × 105 COS-1 cells were seeded

Liposomal Polyamine Conjugates as Gene Carriers
Bioconjugate Chem., Vol. 21, No. 5, 2010 847
Figure 2. Transfection efficacy of spermidine and spermine conjugates
(DCP-spd and DCP-spm, respectively) incorporated into DOPE-based
(closed bar) and DPPC-based (open bar) liposomes. Polyamine
conjugate/phospholipid/cholesterol ) 1/1/1 (mol/mol/mol). Transfection
was conducted in the presence of 10% FBS.
effect of conjugating these two moieties was also observed for
the spermine conjugate, DCP-spm(M) (Figure 1B). When the
conjugates were further formulated with DOPE and cholesterol
(conjugate/DOPE/cholesterol ) 1/1/1 (mol/mol/mol)) to gener-
ate polycation liposomes (PCL) (DCP-spd(PCL) and DCP-
spm(PCL)), efficacies were further enhanced by a factor of 2-3
relative to the micellar aggregate suspensions (DCP-spd(M) and
DCP-spm(M)). The polycation PEI(1800) itself showed moder-
ate activity and the conjugate, DCP-PEI(M), exhibited even
greater activity. However, in contrast to the other conjugates,
the activity of the liposomal form, DCP-PEI(PCL), was
comparable to that of the micellar version, DCP-PEI(M). The
cytotoxicity of the conjugates, as both micellar aggregates and
PCLs, was low; the latter vectors, which exhibited higher
activity, also had slightly higher toxicity than the former
(Supporting Information Figure S1).
Figure 3. (A) Effect of polyamine and hydrophobic portions on PCL-
mediated gene transfer efficiency. PCL were composed of polyamine
conjugate/DOPE/cholesterol (1/1/1 (mol/mol/mol)). N/P ratio was 24.
Transfection was done in the presence of 10% FBS. LF represents
Lipofectamine 2000 as a positive control experiment. (B) Transfection
efficiency of DCP-spd(PCL) (dark bar) and DCP-spm(PCL) (open bar)
for various murine cancer cells. In the presence of 10% serum,
lipoplexes (N/P ) 24) were applied to Colon26 NL-17, Lewis lung
carcinoma (LLC), and B16BL6 melanoma cells. Transfection procedure
was carried out as described in the Experimental Section.
Effect of the Phospholipid Components of PCLs on
Their Transfection Efficacy. Figure 2 shows the transfection
efficacy of DCP-spd(PCL) and DCP-spm(PCL) composed of
either DOPE (dark bar) or DPPC (bright bar) in the N/P range
20-30. It is clear that the DOPE-based PCL exhibits signifi-
cantly greater activity than the DPPC-based compound for both
the DCP-spd(PCL) and the DCP-spm(PCL). This is indicative
of a lipid-mediated gene transfer mechanism; DOPE is well-
known as a “helper” lipid, which is believed to facilitate
membrane fusion and endosomal escape of the DNA (52).
DOPE in the present PCL systems presumably also plays a role
in the mechanism to expedite membrane fusion and destabiliza-
tion of the endosomal membrane. DPPC, whose Tm is 41.5 °C
(53), renders the PCL more stable and more rigid than does
DOPE. Thus, it is likely that the fusogenic property of DOPE
is responsible for the enhanced transfection activity of its
complexes relative to those containing DPPC.
Optimal Structure of the Conjugate Molecules for Gene
Transfer. The facile synthetic route provides a variety of
polycationic compounds that can be exploited to examine the
effect of the polycationic and hydrophobic portions on trans-
fection efficiency. We hence examined the effect on transfection
activity of different polyamine conjugates incorporated into
PCLs: C12, C14, or C16 alkyl chain in the lipophilic portion
and spermidine, spermine, or PEI(1800) as the polycationic
headgroup of the conjugate. The data on these compounds are
shown in Figure 3. This result reveals clear tendencies of longer
length of the alkyl group and the lower molecular weight of
the polyamines (spermidine, spermine) to enhance transfection.
When compared with a commercial product, Lipofectamine
2000, the DCP-spd(PCL) possessed 3.6-fold higher activity.
The transfection activity of DCP-spd(PCL) and DCP-spm(PCL)
depends on cell lines tested (Figure 3B): DCP-spd(PCL) <
DCP-spm(PCL) for Lewis lung carcinoma (LLC) and DCP-
spd(PCL) > DCP-spm(PCL) for B16BL6.
Figure 4. Effect of the N/P ratio on the transfection efficacy of DCP-
spm(PCL) (DCP-spm/DOPE/cholesterol ) 1/1/1 (mol/mol/mol)).
Transfection was in the presence of 10% FBS.
N/P-Dependent Efficacy and Complexation of PCL with
DNA. Figure 4 shows the dependence of DCP-spm(PCL)
efficacy on the ratio of the number of nitrogen atoms in the
conjugate to that of phosphate in the DNA (N/P). The efficacy
increases with the N/P ratio essentially linearly up to 24. A
similar N/P dependence has been also observed for DCP-
spd(PCL) (data not shown for clarity), indicating that excess
polyamine relative to DNA is needed for effective gene transfer
by PCLs. In contrast, the transfection activity of micellar
aggregates DCP-spd(M) and DCP-spm(M) reaches plateau
values in the N/P range 11-16 (1-2 × 109 of RLU/mg protein).
This tendency is consistent with our previous data obtained with
the ꢁ-galactosidase expression system (49).
To elucidate the characteristics of N/P dependence, formation
of DCP-spd(PCL)/DNA and DCP-spm(PCL)/DNA lipoplexes
was analyzed by agarose gel electrophoreses (Figure 5A,B) and
DLS analysis (Table 1). In Figure 5, lane 2 of the plasmid DNA
(pCAG-luc3) shows free-DNA bands (supercoiled and open
circular DNAs). Lane 3 represents DCP-spd(PCL) and DCP-
spm(PCL) alone as a control experiment. In the lower N/P range

848 Bioconjugate Chem., Vol. 21, No. 5, 2010
Dewa et al.
Figure 5. Agarose gel electrophoresis of PCL/plasmid DNA (pCAG-
luc3: 6480 bp) lipoplexes. DCP-spd(PCL) (A) and DCP-spm(PCL)
(B) were mixed with the DNA in the N/P range 5-30. Lane 1, 1 kb
DNA ladder marker; lane 2, plasmid DNA (6480 bp) alone; lane 3,
PCL alone. Lanes 4-9 represent PCL/DNA lipoplexes at N/P ) 5,
11, 16, 20, 24, and 30, respectively.
Table 1. DLS Analysis of Various Polyamine-Dialkyl Phosphate
Conjugate/DNA Complexes
size (nm)
entry compound N/P ratio pH
PCL
micelle
1
2
3
4
5
6
7
8
DCP-spd
DCP-spm
DCP-PEI
s
2
5
16
24
24
s
2
8
8
8
8
8
4
8
8
8
8
8
4
8
8
158 ( 56
231 ( 89
651 ( 501
190 ( 83
261 ( 114
249 ( 106
1181 ( 1057
1791 ( 1630
985 ( 1437 1624 ( 1566
159 ( 30
225 ( 102
293 ( 112
764 ( 378
296 ( 168
256 ( 116
9
5
321 ( 181
10
11
12
13
14
16
24
24
s
24
1767 ( 1284
1033 ( 1060 1209 ( 949
214 ( 90
1062 ( 783
275 ( 184
317 ( 243
5-16 (lanes 4-6), the open circular DNA band vanished and
the supercoiled DNA band gradually faded for both of DCP-
spd(PCL) and DCP-spm(PCL). In the higher N/P range
(20-30, lanes 7-9), the latter DNA band totally disappeared,
indicating that the DNA molecules are completely entrapped
within the lipoplex. Ethidium bromide (EtBr) replacement
experiments also reveal condensation of DNA in the N/P range
5-30 (Supporting Information Figure S2).
The particle size of lipoplexes estimated by DLS analysis is
summarized in Table 1. The diameters of the DCP-spd(PCL)
and DCP-spm(PCL) alone are 158 ( 56 and 159 ( 30 nm,
respectively (entries 1 and 7). Lipoplexes were larger and their
size increased with increasing N/P up to 5 (entries 2 and 8 at
N/P ) 2 and entries 3 and 9 at N/P ) 5). At N/P ) 5, the
PCL/DNA lipoplexes became larger with a broad distribution
from 650 nm to over 1 µm (entries 3 and 9). In the higher N/P
range (N/P ) 16-24), sizes were reduced, converging at 261
( 114 nm (DCP-spd(PCL), entry 5) and 256 ( 116 nm (DCP-
spm(PCL), entry 11). ꢀ-potential measurements indicated the
polarity of surface charge of lipoplexes inverts from negative
in the lower N/P (5-11) to positive in the higher N/P (>16)
regions.
AFM images revealed characteristic morphologies of li-
poplexes in the both low and high N/P ranges (Figure 6A-D).
When the DCP-spd(PCL)/DNA and DCP-spm(PCL)/DNA
lipoplexes at N/P ) 5 were put on PLL-treated mica (positively
charged surface), large aggregates in the sub-micrometer size
range (600-1200 nm, Figure 6A,B) were observed. These
structures resembled by bead-like aggregates (54) composed of
small particles (80-120 nm in diameter, 8-20 nm in height)
connected to one another. Such aggregates were not observed
on a negatively charged bare mica. This is understandable since
the lipoplex at N/P ) 5 is negatively charged, and the lipoplex
Figure 6. AFM images of PCL/plasmid DNA (ColE1; 6646 bp)
lipoplexes: (A), DCP-spd(PCL)/DNA (N/P ) 5); (B), DCP-spm(PCL)/
DNA (N/P ) 5); (C), DCP-spd(PCL)/DNA (N/P ) 24); (D), DCP-
spm(PCL)/DNA (N/P ) 24); (E and F), DCP-PEI(PCL)/DNA complex
(N/P ) 24). Scale bars shown in the images are 500 nm (A,B,D) and
1000 nm (C,E,F). The PCL/DNA complexes were applied to PLL-
mica (A,B) and bare mica (C,D,E,F). All images were taken under an
ambient air conditions. Height profiles of the objects (i)-(iii) in C and
(iv)-(v) in D are shown below these images. Arrows in (ii) and (iii)
indicate step-like profiles discussed in the text. Images E and F for
DCP-PEI(PCL)/DNA were taken from a different area of the bare mica
surface.
must be adsorbed on the PLL-mica surface through electrostatic
interaction to be imaged. The outer periphery of the large
aggregates is rich in DNA molecules, presumably those were
so loosely attached that they were liberated from the aggregates
during electrophoresis.
At the high N/P ) 24, spherical complexes were observed
for DCP-spd(PCL) and DCP-spm(PCL) complexes; their
diameters were 200-400 nm for DCP-spd(PCL)/DNA (C) and
150-250 nm for DCP-spm(PCL)/DNA lipoplexes (D), and
their heights were 12-30 and 27-60 nm, respectively. The size
of the complexes is in good agreement with the values observed
by DLS. The detailed topography of these DCP-spd(PCL)/DNA

Liposomal Polyamine Conjugates as Gene Carriers
Bioconjugate Chem., Vol. 21, No. 5, 2010 849
and DCP-spm(PCL)/DNA lipoplexes showed flat-topped spheres
and spherical structures (line profiles (i)-(iii) for (C) and
(iv)-(vi) for (D)). The profiles (ii) and (iii) are characteristically
“step-like” (arrows). The heights indicated in (ii) are 6, 10, and
14 nm. Considering the thickness of lipid bilayer (4 nm) and
the diameter of DNA (2 nm), these three values correspond to
one bilayer (4 nm) + DNA (2 nm) ) 6 nm, a double bilayer (8
nm) + DNA ) 10 nm, and a triple bilayer (12 nm) + DNA )
14 nm, respectively. Thus, the step-like structure is reasonably
indicative of a smectic lamellar assembly, where DNA mol-
ecules are laminated between bilayers.
Compared with DCP-spd(PCL) and DCP-spm(PCL) li-
poplexes, the size of the DCP-PEI(PCL)/DNA lipoplex (N/P
) 24) is large and has a broad distribution (1062 ( 783 nm,
Table 1, entry 14). AFM images of the DCP-PEI(PCL)/DNA
lipoplex show aggregates with heterogeneous and featureless
shapes (Figure 6E,F). EtBr replacement experiments revealed
DNA condensation in a similar manner to that of DCP-
spd(PCL) and DCP-spm(PCL) (Supporting Information Figure
S2).
Disruption of the PCL/DNA Lipoplexes. When the DCP-
spd(PCL)/DNA and DCP-spm(PCL)/DNA lipoplexes (N/P )
24) were incubated in acidic solution (down to pH 4), the particle
sizes measured by DLS became significantly larger and exhib-
ited broad distributions (Table 1, entries 6 and 12). AFM
imaging revealed morphological transformation of the PCL/
DNA complexes upon acidification. When the dispersion of
DCP-spd(PCL)/DNA lipoplexes (N/P ) 24) was acidified at
pH 4 for 1 h by addition of acetic acid, deformed structures
were observed on bare mica (Figure 7A). Relative to the original
structure (Figure 6C), the complex is decisively deformed by
the acid treatment. Although some of the flat-topped sphere
complexes remain, the predominant morphology is particles
connected with strings. The height of the clusters is 25-53 nm
and they are connected with string portions that are 6-10 nm
high. When the acid-treated complex solution was put on PLL-
mica, additional deformed objects appeared on the surface
(Figure 7B). A “beads on a string” deformed structure is
composed of very small particles (50-100 nm in diameter) and
string parts (∼70 nm in width and 2-5 nm in height). The beads
on a string structure observed on the positively charged surface
must consist of DNA-rich fragments associated with some lipid
components. The DCP-spm(PCL)/DNA lipoplex maintains its
spherical structure on bare mica (Figure 7C). On the PLL-mica,
on the other hand, deformed structures were observed as in the
case of DCP-spm(PCL)/DNA lipoplex (Figure 7D). Such
morphological changes upon acidification result from disas-
sembly of the lipoplexes and the accompanying DNA release.
Gel electrophoretic analysis provided evidence for the DNA
release; the released plasmid band increased with the acidifica-
tion from pH 8 to pH 4 (Supporting Information Figure S4). In
sharp contrast, such a morphological change was not observed
for the micellar aggregate DCP-spd(M) (Figure 7E,F). DLS
analysis indicates an insensitivity of the micellar aggregates to
acidification (Table 1, entries 6 and 12).
Figure 7. AFM images of disassembled lipoplexes, DCP-spd(PCL)/
DNA (A and B) and DCP-spm(PCL)/DNA (C and D) by acidification
at pH 4. Images (E) and (F) represent the micellar aggregate DCP-
spd(M)/DNA complex before and after the acid treatment, respectively.
The acid-treated suspensions of complexes were put on bare mica
(A,C,E,F) and PLL-mica (B,D). After removal of the solution, the
images were acquired. Scale bars: 2000 nm (A), 1000 nm (B), 500 nm
(C), 1000 nm (D), 2000 nm (E) and (F).
combinations of dialkyl and polyamine portions for their activity
in gene transfection. The longer alkyl chain exhibited higher
efficiency (Figure 3). The ꢀ-potential of the DOPE-based PCL
increased with alkyl chain length, for example, 27.6, 33.4, and
37.1 mV for DLP-spd, DMP-spd, and DCP-spd, respectively,
showing that the conjugate with the longer chain length provides
the higher positive potential. The hydrophobic interaction results
in stable incorporation of the conjugate into the PCL, consistent
with transfection activity in the order of C16 g C14 > C12.
We found that the micellar aggregate of DCP-PEI conjugate
exhibited slightly higher transfection activity than the low-
molecular-weight amine conjugates, DCP-spd(M) and DCP-
spm(M); however, the activity of DCP-PEI(PCL) was marginal.
The size of DCP-PEI(PCL)/DNA lipoplex is significantly
greater than that of the other lipoplexes (Table 1, entry 14).
The DCP-PEI conjugate is composed of DCP/PEI(1800) ) ∼1/
DISCUSSION
1
1, judged by H NMR (49). As previously reported, the cetyl-
Chemical Structure of the Polyamine Conjugates. The
polyamine-dialkyl phosphate conjugates can be readily synthe-
sized via a two-step reaction: (i) formation of dimerized dialkyl
phosphate anhydride and (ii) its nucleophilic substitution with
polyamines. The synthetic strategy gives access to a wide variety
of polyamine-dialkyl phosphate derivatives. Conjugation of the
polyamine and hydrophobic portions is required for an effective
gene carrier (Figure 1). Such amphiphilicity is essential to
condense DNA molecules, which requires both electrostatic and
hydrophobic interactions (45, 49). We tested a number of
PEI, whose PCL possesses high transfection activity, consists
of 10 cetyl portions in the polymer (47). The cetyl-PEI can attach
to the PCL surface via the anchoring of cetyl portions in the
lipid bilayer. However, that is not the case for DCP-PEI(PCL);
the single hydrophobic portion in the conjugate is not enough
to provide adequate covering of PEI over the PCL surface. This
may cause “PEI-protrusion” from the surface, which gives rise
to the large and heterogeneous aggregation seen upon combina-
tion with DNA molecules. This is likely the reason for the lower
activity of the DCP-PEI(PCL). Taken together, these consid-

850 Bioconjugate Chem., Vol. 21, No. 5, 2010
Dewa et al.
erations suggest that a homogeneous positive charge distribution
on the PCL surface is important to the transfection activity.
accompanying DNA release has been confirmed. In sharp
contrast, the size and shape of micellar aggregate/DNA com-
plexes composed of DCP-spd and DCP-spm are insensitive to
acidification (Figure 7F and Table 1, entries 6 and 12). This
would be due to their amorphous structure, which does not
respond to pH change. Thus, the disassembly of the lipoplex
composed of the bilayer-structured PCL is essential in effective
gene transfer, especially in the process of endosomal escape,
where lipid exchange and flip-flop are involved in disrupting
the membrane and leading to DNA release (42). Although the
acidity of this experimental condition (pH 4) seems higher than
that in endosome (pH 5.5), such a protonation process on the
polyamines should be involved in the endosomal environment,
because the protonation on the polyamines whose pKa values
are >8 may proceed rather gradually in the acidic region,
especially in the self-assembled lamellar structure, where
polyamines are densely packed. This finding suggests a strategy
for molecular designsespecially in the polyamine portionsin
which a morphological transformation of lipoplexes is taken
advantage of for new nonviral transfection strategies.
Effect of Bilayer Structure on the Transfection Activity.
In the series of polyamines tested in this study, the low-
molecular-weight polyamines were found to be more effective
gene carriers when these conjugates were assembled into PCLs.
The transfection activity of the PCL was ∼3 times larger than
as the corresponding micellar aggregate (Figure 1). The effect
of the helper lipid, DOPE, was clearly substantial, as is shown
in Figure 2. DOPE, a predominantly nonlamellar lipid, is thought
to facilitate fusion and destabilization of the endosomal mem-
brane after uptake of cationic lipid/DNA complexes into a cell.
In our previous report, we described intercellular trafficking of
PCL (composed of cetyl-PEI and DOPE)-DNA complexes,
which were taken up into cells by endosomal pathway (48),
followed by endosomal escape. In fact, transfection activity of
the present PCLs was inhibited by nigericin, which is able to
dissipate the pH gradient across the endosomal membranes, by
30-50%, suggesting that endosomal pathway is likely involved
in the mechanism in the present lipoplex system. It appears,
therefore, that the mechanism of the lipofection by the com-
pounds in this study may be similar to that of this and other
agents known to be enhanced by DOPE. The lipoplexes made
from the bilayer-structured PCLs evidently involve lamellar
assemblies, given that AFM images reveal the presence of step-
like profiles ((ii) and (iii) in Figure 6). The step-like profiles
imply a lamellar complex, in which DNA rods (2 nm in
diameter) are laminated between bilayers (4 nm thickness). Such
an intrinsic bilayer structure may predispose lipoplexes to
interact with cell and endosomal membranes. This is not the
case for micellar aggregates, whose morphology is large spheres
(Figure 7E and Table 1 entry 5), in which polyamine conjugate
and DNA molecules likely aggregate randomly. This may be
one of the reasons for the higher activity of PCL-based lipoplex,
whose size is more favorable to transfection.
The linear dependence of activity on N/P (Figure 4) is related
to the morphology of the lipoplex. The electrophoresis experi-
ment (Figure 5) and AFM images (Figure 6A-D) suggest a
reasonable explanation of the dependence, namely, the follow-
ing: In the low N/P range (∼5), the PCLs inadequately condense
DNA molecules, giving the bead-like structures (Figure 6A,B).
The DNA molecules loosely packed in the complex are readily
released during electrophoresis. Such a complex, whose ꢀ-po-
tential is negative, is too large to be introduced into the cell
membrane via endocytosis; therefore, the transfection level is
low. With increasing N/P ratio, the morphology of the lipoplex
transforms from large bead-like structure into smaller particles,
wherein DNA molecules are condensed more tightly (Figure
6C,D). The size of the lipoplexes, whose ꢀ-potential is positive,
is 150-400 nm, more favorable for cellular uptake via endocy-
tosis (30). Given that the lamellar assembly in the lipoplex is
responsible for its effectiveness as a gene carrier (29, 30), the
population of active species for gene transfer would increase
with increasing in the N/P. Although highly positive-charged
carriers are generally toxic, the PCL described here exhibit low
cytotoxicity, an advantage for in vitro and in vivo applications.
CONCLUSION
We have demonstrated that PCL composed of the low-
molecular-weight polyamine conjugates, DCP-spermidine (DCP-
spd) and DCP-spermine (DCP-spm), exhibit much higher
gene transfer activity than PEI(1800) conjugate-based DCP-
PEI(PCL). The former compounds generate 150-400 nm
diameter lipoplexes, whereas the latter gives rise to large
aggregates. In the case of the former compounds, AFM images
clearly reveal a morphological change upon acidification,
indicating DNA release from the lipoplexes, whereas in contrast,
the morphology of micellar aggregates is insensitive to pH
change. A pH-dependent transformation is crucial in gene
transfer, and the chemical structure of the polyamine portion
may therefore play an important role in the acidification-induced
transformation. We have also described the relation between
the N/P dependence of transfection activity and the morphology
of the lipoplexes as revealed by AFM. Morphological study with
AFM provided useful information for understanding the basis
of lipoplexes with superior activity and for design strategies
leading to optimally efficient gene carriers.
ACKNOWLEDGMENT
The authors thank Dr. Robert C. MacDonald for helpful
comments and discussion. The present work was partially
supported by “Grant-in-Aid for Scientific Research on Innova-
tive Areas 2006 (Molecular Science of Fluctuations toward
Biological Functions)” (21107510) from the Ministry of Educa-
tion, Culture, Sports, Science and Technology (MEXT) of the
Japanese Government.
Supporting Information Available: Evaluation of cytotox-
icity polyamine conjugates and their PCLs (Figure S1), changes
in fluorescence intensity of ethidium bromide intercalated into
ColE1 plasmid DNA-PCLs (Figure S2), and electrophoretic
analysis of DNA release from the lipoplex (Figure S3). This
material is available free of charge via the Internet at http://
pubs.acs.org.
Disassembly of the Lipoplexes and DNA Release. Facile
escape from the acidic endosomal compartment is necessary
for efficient gene transfer. Disassembly of the lipoplex associated
with DNA release has been clearly observed by AFM imaging
(Figure 7) and by electrophoretic analyses (Supporting Informa-
tion Figure S3). Upon acidification (down to pH 4), the extent
of protonation of the polyamine portion is increased. Assuming
that the lipoplex forms smectic lamella, electrostatic repulsion
between layers must be increased with such protonation,
resulting in the disruption of the lipoplex. For lipoplexes
composed of DCP-spd(PCL) and DCP-spm(PCL), disruption
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