Synthesis and in vitro testing of new potent polyacridine-melittin gene delivery peptides

Article abstract of DOI:10.1021/bc9003124

The combination of a polyacridine peptide modified with a melittin fusogenic peptide results in a potent gene transfer agent. Polyacridine peptides of the general formula (Acr-X)_n-Cys were prepared by solid-phase peptide synthesis, where Acr is

Full text of DOI:10.1021/bc9003124

74
Bioconjugate Chem. 2010, 21, 74–83
Synthesis and In Vitro Testing of New Potent Polyacridine-Melittin Gene
Delivery Peptides
Nicholas J. Baumhover, Kevin Anderson, Christian A. Fernandez, and Kevin G. Rice*
Divisions of Medicinal and Natural Products Chemistry and Pharmaceutics, College of Pharmacy, University of Iowa, Iowa
City, Iowa 52242. Received July 14, 2009; Revised Manuscript Received October 28, 2009
The combination of a polyacridine peptide modified with a melittin fusogenic peptide results in a potent gene
transfer agent. Polyacridine peptides of the general formula (Acr-X)n-Cys were prepared by solid-phase peptide
synthesis, where Acr is Lys modified on its ε-amine with acridine, X is Arg, Leu, or Lys and n is 2, 3, or 4
repeats. The Cys residue was modified by either a maleimide-melittin or a thiolpyridine-Cys-melittin fusogenic
peptide resulting in reducible or non-reducible polyacridine-melittin peptides. Hemolysis assays established that
polyacridine-melittin peptides retained their membrane lytic potency relative to melittin at pH 7.4 and 5. When
combined with plasmid DNA, the membrane lytic potency of polyacridine-melittin peptides was neutralized.
Gene transfer experiments in multiple cell lines established that polyacridine-melittin peptides mediate expression
as efficiently as PEI. The expression was very dependent upon a disulfide bond linking polyacridine to melittin.
The gene transfer was most efficient when X is Arg and n is 3 or 4 repeats. These studies establish polyacridine
peptides as a novel DNA binding anchor peptide.
An early study by Szoka demonstrated the synthesis of a divalent
acridine neoglycopeptide for delivery of DNA to the cells
expressing the asialoglycoprotein receptor (24). More recently,
polyacridine-nuclear localization sequences (NLS) possessing
up to three acridines were shown to improve in vitro gene
transfer (25, 26).
INTRODUCTION
An important element of many polyplex nonviral gene
delivery systems is a releasable fusogenic peptide that can effect
endosomal lysis (1). The endosomal escape of DNA is especially
essential for receptor-targeted gene delivery systems to avoid
polyplex degradation by the lysosome (2-4).
However, to date, a polyacridine DNA binding peptide has
not been substituted with a fusogenic peptide. The new strategy
describedhereindemonstratesthesynthesisofpolyacridine-melittin
as a DNA binding peptide and establishes its efficacy as a potent
in vitro gene transfer agent. The results demonstrate that short
polyacridine anchor peptides bind to plasmid DNA with
sufficient affinity to deliver melittin in vitro and have the
potential to control DNA polyplex charge to allow improved
in vivo gene delivery.
To improve the endosomal escape of DNA, several prior
studies have incorporated fusogenic peptides, including GALA,
KALA, HA2, JTS-1, and melittin, into a nonviral gene delivery
system (5-11). Most applications in gene delivery have utilized
melittin because of its relative ease of chemical synthesis and
because its mechanism of membrane lysis has been extensively
studied (12-14). Melittin has been covalently attached to
polylysine, PEI, and cationic lipids and even self-polymerized
by terminal disulfide bonds and demonstrated to increase in vitro
gene transfer efficiency (4, 10, 11, 15, 16).
While DNA anchoring polymers such as PEI, polylysine,
dendrimers, and chitosan are known to bind ionically to the
phosphate backbone of plasmid DNA, this strategy leads to the
formation of cationic polyplexes that favor in vitro gene transfer,
but to a much lesser degree in vivo gene transfer (17). This is
partly because positively charged polyplexes bind non-specif-
ically to proteins and cells (18). Attempts at masking the charge
with PEG still results in a residual positive charge as measured
by zeta potential (19, 20). The non-specific binding of PEGy-
lated cation polyplexes in vivo complicates strategies that
attempt to combine a receptor ligand and releasable fusogenic
peptide into a non-viral delivery system (21, 22). Consequently,
it would be beneficial to reversibly bind fusogenic peptides,
receptor ligand, and PEG to DNA while simultaneously control-
ling the charge of polyplexes. Likewise, it would be advanta-
geous if the DNA binding anchor were sufficiently small in size
to allow for its chemical manipulation and specific functional-
ization with PEG, targeting ligand, and fusogenic peptide (22).
Polyacridine peptides have been shown to reversibly bind with
high affinity by intercalating into double-stranded DNA (23).
MATERIALS AND METHODS
Unsubstituted Wang resin for peptide synthesis, 9-hydroxy-
benzotriazole, Fmoc-protected amino acids, O-(benzotriazole)-
N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU),
1,3-diisopropylcarbodiimide(DIC),Fmoc-Lysine-OH,andN-meth-
yl-2-pyrrolidinone (NMP) were obtained from Advanced
ChemTech (Lexington, KY). N,N-Dimethylformamide (DMF),
trifluoroacetic acid (TFA), and acetonitrile were purchased from
Fisher Scientific (Pittsburgh, PA). Diisopropylethylamine, pip-
erdine, acetic anhydride, tris(2-carboxyethyl)-phosphine hydro-
chloride (TCEP), 9-chloroacridine, maleic anhydride, 2,2′-
dithiodipyridine (DTDP), and thiazole orange were obtained
from Sigma Chemical Co. (St. Louis, MO). Polyethylene amine
(PEI 25 KDa) was purchased from Aldrich (Milwaukee, WI).
D-Luciferin and luciferase from Photinus pyralis were obtained
from Roche Applied Science (Indianapolis, IN). HepG2, CHO,
and 3T3 cells were acquired from the American Type Culture
Collection (Manassas, VA). Inactivated qualified fetal bovine
serum (FBS) was from Life Technologies, Inc. (Carlsbad, CA).
BCA reagent was purchased from Pierce (Rockford, IL). pGL3
control vector, a 5.3 kb luciferase plasmid containing a SV40
promoter and enhancer, was obtained from Promega (Madison,
* To whom correspondence should be addressed. E-mail kevin-
rice@uiowa.edu. tel: 319-335-9903; fax: 319-335-8766.
10.1021/bc9003124  2010 American Chemical Society
Published on Web 12/07/2009

Polyacridine-Melittin Gene Delivery Peptides
Bioconjugate Chem., Vol. 21, No. 1, 2010 75
WI). pGL3 was amplified in a DH5R strain of Escherichia coli
mL/min with 0.1 v/v % TFA with an acetonitrile gradient of
20-45 v/v % over 30 min while monitoring tryptophan (Abs
280 nm) or acridine (Abs 409 nm). The major peak was
collected and pooled from multiple runs, concentrated by rotary
evaporation, lyophilized, and stored at -20 °C. Purified peptides
were reconstituted in 0.1% v/v TFA and quantified by absor-
bance (tryptophan ε280nm ) 5600 M-1 cm-1, thiolpyridine, and
tryptophan ε280nm ) 10 915 M-1 cm-1, or acridine ε409nm ) 9266
M-1 cm-1) to determine isolated yield. Purified peptides were
characterized by LC-MS by injecting 2 nmol onto a Vydac C18
analytical column (0.47 × 25 cm) eluted at 0.7 mL/min with
0.1 v/v % TFA and an acetonitrile gradient of 10-55 v/v%
over 30 min while acquiring ESI-MS in the positive ion mode.
Cys-melittin was reacted with 10 equiv of DTDP in 2-pro-
panol/2 N acetic acid (10:3) for 8 h at RT to generate the
thiolpyridine protected peptide. The reaction mixture was
concentrated by rotary evaporation and applied to a Sephadex
G-10 column eluted (2.5 × 50 cm) with 0.1 v/v % TFA while
monitoring absorbance at 280 nm to remove excess DTDP. The
peptide peak fractions were pooled, concentrated, lyophilized,
and purified to homogeneity by RP-HPLC as described
previously.
Poly(Acr-X)n-SS-melittin and poly(Acr-X)n-Mal-melittin pep-
tides were synthesized on a 2 µmol scale based on melittin,
using 1.7 equiv (3.4 µmol) of the reduced poly(Acr-X)n-Cys,
where X equals Arg, Lys, or Leu. Mal-melittin (2 µmol) was
prepared in 8 mL of 10 mM ammonium acetate pH 5 and added
to poly(Acr-X)n-Cys and 2 mL of methanol to facilitate
solubility. The reaction occurred over 12 h at RT as determined
by analyzing aliquots by LC-MS. At completion, the reaction
mixture was concentrated by rotary evaporation and lyophilized
prior to purification of poly(Acr-X)n-Mal-melittin by RP-HPLC
eluted with 0.1 v/v % TFA and acetonitrile gradient (35-45%
over 30 min) while detecting at 409 nm.
Alternatively, thiolpyridine-Cys-melittin (2 µmol) was pre-
pared in 8 mL of 10 mM ammonium acetate pH 6 and added
to 3.4 µmol poly(Acr-X)n-Cys in 2 mL of methanol. After 24 h,
poly(Acr-X)n-SS-melittin was purified and characterized by LC-
MS as described above.
Polyacridine-Melittin Hemolysis Assay. Whole blood was
obtained from male ICR mice by heart puncture with heparinized
22G needles and collected in conical tubes containing 10 mL
of 0.15 M PBS (pH 7.4) prewarmed to 37 °C. Erythrocytes were
immediately separated from plasma by centrifugation at 2000
rpm for 2 min, washed three times with 10 mL of PBS, and
then diluted to 1.5 × 108 cells/mL. Peptides were prepared at
15 µM and serially diluted to 10-0.01 µM, after which 100
µL was pipetted in triplicate into a MultiScreenHTS BV 96-
well plate. Erythrocytes (50 µL, 7.5 × 106 cells) were added to
the peptides and incubated at 37 °C for 1 h followed by filtration
on a Multiscreen vacuum manifold (Millipore Corporation,
Billerica, MA). The filtrate was measured for abs410nm on a
Biotech plate reader (Biotech Instruments Incorporated., Wi-
nooski, VT) and compared to the 100% hemolysis caused by
replacing PBS with water. Peptide DNA polyplexes (1.5 nmols
and 7.5 µg pGL3) were also assayed for hemolysis as described
above.
Formulation and Characterization of Polyacridine-Melittin
Polyplexes. The relative binding affinity of peptides for DNA
was determined by a fluorophore exclusion assay (29). pGL3
(200 µL of 4 µg/mL in 5 mM Hepes pH 7.5 containing 0.1 µM
thiazole orange) was combined with 0, 0.05, 0.1, 0.25, 0.35,
0.5, 1, and 2 nmols of peptide in 300 µL of Hepes and allowed
to bind at RT for 30 min. Thiazole orange fluorescence was
measured using an LS50B fluorometer (Perkin-Elmer, U.K.) by
exciting at 498 nm while monitoring emission at 546 nm with
the slit widths set at 10 nm. A fluorescence blank of thiazole
and purified according to manufacturer’s instructions.
Synthesis of 9-Phenoxyacridine and Fmoc Lysine
(Acridine). 9-Phenoxyacridine was synthesized with modifica-
tion from the methods of Tung et al. (27). Briefly, 12 g of phenol
(127.5 mmol) and 0.72 g of sodium hydroxide (18 mmol) were
heated to 100 °C. To the liquefied phenol, 2.8 g of 9-chloro-
acridine (13.105 mmol) was added and stirred vigorously for
1.5 h. The reaction was quenched by the addition of 100 mL of
2 M sodium hydroxide, then allowed to sit at RT overnight. A
yellow precipitate was filtered, washed with water, and dried
in vacuo (3.4822 g, 12.835 mmol, 97.9%, m.p. 123-124 °C,
TLC 15:5:1:0.5 ethyl acetate/methanol/hexane/acetic acid, Rf
1
) 0.18). H NMR (DMSO-d6) δ 8.23 (d, 2H), 8.04 (d, 2H),
7.88 (t, 2H), 7.59 (t, 2H), 7.32 (t, 2H), 7.08 (t, 1H), 6.88 (d
2H).
Fmoc-Lysine(Acridine)-OH was prepared by adding 2.18 g
of Fmoc-Lys-OH (5.91 mmol) in liquid phenol (6.781 g, 73.01
mmol) to 9-phenoxyacridine (3 g, 11.06 mmol), then heated at
60 °C for 4 h under an argon atmosphere. Diethyl ether (80
mL) was then added while stirring vigorously until a yellow
precipitate formed that was immediately recovered by filtration
and washed repeatedly with diethyl ether. The product was
allowed to dry overnight under vacuum (2.90 g, 5.32 mmol,
90%), m.p. 135-140 °C, TLC 1:1, 0.1 v/v % TFA/acetonitrile,
1
Rf ) 0.75). MS: (M + H+)1+ ) 545.5 m/z. H NMR (DMSO-
d6) δ 6.7-7.8 (m, 17H), 3.6-3.95 (m, 2H), 3.25-3.53 (br, 3H),
2,06 (m, 1H), 0.8-1.5 (m, 6H).
Synthesis of Maleimide Glycine (Mal-Gly-OH). Glycine
(5 g, 66.6 mmol) and maleic anhydride (6.6 g, 66.6 mmol) were
suspended in 80 mL of acetic acid and allowed to react for 3 h
at RT. The resulting white precipitate was collected by filtration,
washed with cold water, and dried (10.95 g, 63.3 mmol, 95%,
m.p. 187-189 °C, TLC 2:1:1:1 isopropyl alcohol/acetic acid/
ethyl acetate/water, Rf ) 0.5). The glycine maleic acid
intermediate was characterized by proton NMR (300 MHz,
DMSO-d6): δ ) 9.2 (s, 1H), 6.397 (d, 1H, j ) 8.57 Hz), 6.310
(d, 2H, j ) 12.86 Hz), 2.0 (d, 2H, j ) 6.86 Hz).
Glycine maleamic acid (5.2 g, 30.04 mmol) and 2.1 equiv of
triethylamine (6.37 g, 63 mmol) were refluxed for 3 h in 500
mL toluene with removal of water with a Dean-Stark apparatus.
Upon reaction completion, the toluene solution was decanted
and dried. The resulting solid was acidified with 2 M HCl,
extracted with ethyl acetate, dried with MgSO4, and evaporated
to yield Mal-Gly-OH (1.7 g, 11 mmol, 36.5%, m.p. 99-110
°C, TLC 2:1:1:1 isopropyl alcohol/acetic acid/ethyl acetate/
water, Rf ) 0.72). The product was characterized by proton
NMR (300 MHz, DMSO-d6): δ ) 7.108 (s, 2H), 4.105 (s, 2H)
(28).
Synthesis and Characterization of Polyacridine-Melittin
Analogues. Melittin analogues, polyacridine peptides, and
control peptides were prepared by solid-phase peptide synthesis
using standard Fmoc procedures with 9-hydroxybenzotriazole
and HBTU double couplings on a 30 µmol scale on an
Advanced ChemTech APEX 396 synthesizer. Mal-melittin was
prepared by coupling Mal-Gly-OH to the N-terminus of side-
chain protected full-length melittin on resin utilizing a 6-fold
excess of Mal-Gly-OH, DIC, and HOBt, reacted for 4 h while
stirring. The resin was filtered; washed with DMF, DCM, and
methanol; then dried. Peptides were removed from resin and
sidechain deprotected using a cleavage cocktail of TFA/
triisoproylsilane/water (95:2.5:2.5 v/v/v) for 3 h followed by
precipitation in cold ether. Precipitates were centrifuged for 10
min at 4000 rpm at 4 °C and the supernatant decanted. Peptides
were then reconstituted with 0.1 v/v % TFA and purified to
homogeneity on RP-HPLC by injecting 0.5-2 µmol onto a
Vydac C18 semipreparative column (2 × 25 cm) eluted at 10

76 Bioconjugate Chem., Vol. 21, No. 1, 2010
Baumhover et al.
orange in the absence of DNA was subtracted from all values
before data analysis. The data are presented as nmol of peptide
per µg of DNA versus the percent fluorescence intensity plus/
minus the standard deviation determined by three independent
measurements.
Particle size and zeta potential were determined by preparing
2 mL of polyplex in 5 mM Hepes pH 7.5 at a DNA
concentration of 30 µg/mL with 15 nmols/mL of peptide (0.5
nmol of peptide per µg of DNA). Particle size was measured
by quasi-elastic light scattering (QELS) at a scatter angle of
90° on a Brookhaven ZetaPlus particle sizer (Brookhaven
Instruments Corporation, NY). The zeta potential was deter-
mined as the mean of ten measurements immediately following
acquisition of the particle size.
Polyacridine-melittin DNA polyplexes were prepared at 0.5
nmol of peptide per µg of DNA at a concentration of 100 µg
per mL of DNA and directly deposited on a freshly cleaved
mica surface and allowed to bind for 10 min prior to washing
with deionized water (30). Alternatively, naked DNA (100 µg
pGL3 per mL) was prepared in 10 mM Tris, 1 mM EDTA pH
8, then diluted to 1 µg per mL in 40 mM Hepes 5 mM nickel
chloride pH 6.7, and deposited on a fresh cleaved mica surface
for 10 min followed by washing with deionized water. An
Asylum atomic force microscope (AFM) MFP3D (Santa
Barbara, CA) was operated in the AC-mode in order to image
either naked DNA or DNA polyplexes using a silicon cantilever
(Ultrasharp NSC15/AIBS, Mikro Masch).
(Berthold Systems, Germany) with 10 s integration after
automatic injection of 100 µL of 0.5 mM D-luciferin. The
relative light units were converted to fmol using a standard curve
generated by adding a known amount of luciferase to 35 mm
wells containing 50% confluent HepG2 cells. The resulting
standard curve had an average slope of 2.6 × 104 RLU/fmol
enzyme. Protein concentrations were measured by a BCA assay
using bovine serum albumin as a standard (32). The amount of
luciferase recovered in each sample was normalized to mg of
protein and reported as the mean and standard deviation obtained
from triplicate transfections. PEI pGL3 polyplexes were pre-
pared by mixing 35 µg of DNA in 525 µL of HBM with 43.8
µg PEI in 525 µL of HBM while vortexing to create DNA
complexes possessing a charge ratio (NH4+/PO4-) of 9:1. Cells
were transfected with 10 µg PEI-DNA polyplexes as previously
described. CHO and 3T3 (5 × 105) cells were plated on 6 ×
35 mm wells and grown to ca. 50% confluency and then
transfected as described above.
RESULTS
Synthetic Strategy for Polyacridine-Melittin. Naturally
occurring melittin isolated from bee venom is a 26 amino acid
peptide amide composed of two R-helices conjoined through
an interrupting proline (12-14). Numerous studies have been
conducted to investigate the mechanism and sequence specificity
of melittin’s membrane lytic activity.
Natural melittin contains a single aromatic Trp at residue 8
and does not contain a Cys residue. To improve the synthesis
of polyacridine-melittin, we replaced Trp 8 with Leu, and
reinstalled a single Trp near the N-terminus in Cys-Trp-Lys-
Lys. The reactivity of the Cys residue was increased by flanking
Lys residues. This also allowed selective chromatographic
detection of full-length melittin at 280 nm. These modifications
resulted in a Cys-melittin analogue, CWKKGIGAVLKVLT-
TGLPALISLIKRKRQQ, that SOPMA analysis (33) predicted
to maintain R-helical character. A similar Mal-melittin analogue
(Mal-GWKKGIGAVLKVLTTGLPALISLIKRKRQQ), possess-
ing an N-terminal maleimide-glycine (Mal-G) in place of Cys,
was also prepared.
Polyacridine and melittin peptides were synthesized using
HBTU/HOBt with increased coupling efficiency compared to
DIC. Early attempts to synthesize polyacridine peptides using
DIC resulted in heterogeneous mixtures of truncated peptides
with minimal recovery of full-length peptide. The identity of
the spacing amino acid between Lys-acridine residue signifi-
cantly influenced the synthetic yield. Bulky amino acid side
chains and protecting groups such as Lys-Boc, Phe-Trt, Leu,
Glu-OBut, and Arg-Pbf were well-tolerated, whereas Gly
surprisingly was not. Polyacridine peptides possess a C-terminal
Cys to accommodate bioconjugation to Cys-melittin or Mal-
melittin. Polyacridine peptides possessing an alternating Lys-
Acr (Acr) spaced by Arg, Lys, or Leu were designed to influence
the charge character of the anchor peptide (Scheme 1 and Table
1). The lengths of polyacridine peptides were systematically
varied to possess 2-4 Acr-Arg repeats to examine the influence
of affinity. Polyacridine nona-peptides were routinely obtained
in 30-35% purified yield with >95% purity.
Polyacridine-Melittin Polyplex Toxicity. The in vitro
toxicity of polyacridine-melittin polyplexes was evaluated by
MTT assay (31). CHO, HepG2, and 3T3 cells were plated on
6 × 35 mm wells at 5 × 105 cells/well and grown to 40-70%
confluency. The media was replaced with 2 mL of fresh MEM
supplemented with 2% FBS and the cells treated with 10 µg of
DNA polyplex (0.5 nmol peptide/µg DNA) in HBM (HBM; 5
mM Hepes, 0.27 M mannitol, pH 7.5). After 6 h, the media
was replaced with fresh culture media and grown an additional
18 h. Alternatively, cells were treated with DNA polyplexes
for 24 h. The media was replaced with 2 mL of fresh media
and 500 µL of 0.5% (w/v) 3-(4,5-dimethylthiazole-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT) in PBS solution and
allowed to incubate at 37 °C for 1 h to let formazan crystals
form. The media containing MTT was removed, and the crystals
dissolved by the addition of 2 mL of dimethyl sulfoxide
(DMSO) and 250 µL Sorenson’s Glycine Buffer, then measured
by absorbance 595 nm on a microplate reader. The percent
viability was determined relative to untreated cells.
In Vitro Gene Transfer of Polyacridine-Melittin DNA
Polyplexes. HepG2 cells (5 × 105) were plated on 6 × 35 mm
wells and grown to approximately 50% confluency. Transfec-
tions were performed in MEM supplemented with either 2% or
10% FBS, sodium pyruvate (1 mM), and penicillin and
streptomycin (100 U and 100 µg/mL). Polyplexes were prepared
at a DNA concentration of 30 µg/mL and a stoichiometry of
0.5 nmol peptide per µg of DNA in HBM (HBM; 5 mM Hepes,
0.27 M mannitol, pH 7.5). Polyplexes (10 µg of DNA in 0.3
mL of HBM) were added dropwise to wells in triplicate and
incubated 24 h. After 24 h, the cells were washed twice with 2
mL of ice-cold phosphate-buffered saline (Ca2+- and Mg2+-free)
and then treated with 0.5 mL of lysis buffer (25 mM Tris
chloride, pH 7.8, 1 mM EDTA, 8 mM magnesium chloride,
and 1% Triton X-100) for 10 min at 4 °C. Cell lysates were
scraped, transferred to 1.5 mL microcentrifuge tubes, and
centrifuged for 10 min at 13,000 g at 4 °C to pellet cell debris.
Lysis buffer (300 µL) and sodium-ATP (4.3 µL of a 165 mM
solution at pH 7, 4 °C) were combined in a test tube, mixed
briefly, and immediately placed in the luminometer. Luciferase
relative light units were measured by a Lumat LB 9501
Polyacridine-melittin peptides were designed to possess
either a reducible dithiol or a nonreducible maleimide linkage
(Scheme 1 and Table 1). The most expedient synthetic route
proved to be cleavage of Cys-melittin from the resin, followed
by modification with dithiol dipyridine to produce a thiolpyr-
melittin. The RP-HPLC monitoring of the reaction of thiolpyr-
melittin with poly(Acr-Arg)4-Cys is illustrated in Figure 1A and
B. The desired poly(Acr-Arg)4-SS-melittin product is formed
with complete consumption of thiolpyr-melittin, along with
formation of a poly(Acr-Arg)4-Cys2 byproduct (Figure 1B). This

Polyacridine-Melittin Gene Delivery Peptides
Bioconjugate Chem., Vol. 21, No. 1, 2010 77
Scheme 1. Synthesis of Polyacridine-Melittin Peptides
Thiolpyr-melittin is reacted with the DNA binding anchor peptide poly(Acr-Arg)4-Cys at pH 6 to yield poly(Acr-Arg)4-SS-melittin as a
reducible gene transfer polyacridine melittin peptide. Alternatively, Mal-melittin is reacted with poly(Acr-Arg)4-Cys at pH 5 to yield a
non-reducible poly(Acr-Arg)4-Mal-melittin gene transfer peptide. In each case, Acr refers to a Lys substituted on its ε-amine with acridine.
The Arg was substituted with Lys and Leu to produce DNA binding anchor peptides with different affinity. The poly(Acr-Arg)n was also
varied from n ) 2-4 repeats.
synthetic route resulted in poly(Acr-X)n-SS-melittin in 35-50%
purified yield at >95% purity as illustrated by LC-MS analysis
of purified poly(Acr-Arg)4-SS-melittin (Figure 1C). ESI-MS was
used to establish structure based on the triple and quadruple
charged positive ions corresponding to the calculated mass of
the desired polyacridine-melittin peptide (Figure 1C, inset).
The release of melittin from poly(Acr-Arg)4-SS-melittin was
established by reaction with TCEP to regenerate poly(Acr-Arg)4-
Cys and Cys-melittin peptide peaks (Figure 1D).
Poly(Acr-X)4-Mal-melittin peptides, where X is Arg, Lys, or
Leu, were synthesized to compare their gene transfer properties
with poly(Acr-Arg)4-SS-melittin (Scheme 1 and Table 1). The
synthetic strategy utilized Gly-maleimide that was coupled to
the N-terminus of the fully protected melittin peptide on resin
(Scheme 1). Upon workup, this yielded Mal-melittin that was
then directly conjugated to poly(Acr-X)n-Cys peptides. The
conjugation of Mal-melittin with poly(Acr-Arg)4-Cys was
monitored by RP-HPLC resulting in the complete consumption
of Mal-melittin and the formation of poly(Acr-Arg)4-Mal-
melittin. Purification of poly(Acr-Arg)4-Mal-melittin was achieved
with a final yield of 35% and >95% purity. Reducible and
nonreducible polyacridine-melittin peptides with Acr-Arg
repeat of 2 and 3 were synthesized with similar yield and purity,
and characterized by LC-MS (Table 1).
Physical Properties of Polyacridine-Melittin DNA
Polyplexes. The binding of polyacridine-melittin peptides to
pGL3 was investigated using a thiazole orange exclusion assay
(29). Polyacridine-melittin peptides were found to compete for
intercalation and displace thiazole orange resulting in a decrease
in fluorescence intensity. When titrated at increasing concentra-
tion, the fluorescence decreased until an asymptote was reached
at an equivalence point (Figure 2A). Analysis of the DNA
binding affinity of poly(Acr-Arg)4-Cys established an equiva-
lence point at approximately 0.4 nmol peptide per µg of DNA.
By comparison, Cys-melittin demonstrated weaker binding and
was not able to fully exclude thiazole orange, only approaching
an equivalence point at approximately 0.5 nmol of peptide per
µg of DNA (Figure 2A). Alternatively, poly(Acr-Arg)4-SS-
melittin demonstrated higher affinity resulting in an equivalence
point at approximately 0.25 nmol of peptide per µg of DNA.
This affinity is comparable to that afforded by a Cys-Trp-Lys18
peptide using the same assay (29).
Comparison of poly(Acr-Arg)2-4-SS-melittin demonstrated
nearly equivalent affinity for Acr-Arg repeats of 3 and 4, but

78 Bioconjugate Chem., Vol. 21, No. 1, 2010
Baumhover et al.
Table 1. Sequence and Hemolytic Potency of Peptide Conjugates
massa (calc/obs)
HL50 (µM)b pH 7.4: 5.0
Polyacridine Anchor Peptides (Acr) ) Lys-ε-acridine, Ac ) acylated ε-amine)
poly(Acr-Arg)2-Cys
poly(Acr-Arg)3-Cys
poly(Acr-Arg)4-Cys
poly(Acr-Lys)4-Cys
poly(Acr-Leu)4-Cys
Trp-(Lys(Ac)-Arg)4-Cys
1044.3/1044.2
1505.8/1505.6
1967.4/1967.0
1855.3/1855.0
1795.3/1795.1
1612.9/1612.7
N.D.c
N.D.
>10/>10
N.D.
N.D.
N.D.
Melittin Analogues (thiolpyr ) thiol pyridine, Mal ) maleimide)
Melittin
GIGAVLKVLTTGLPALISWIKRKRQQ
2847.5/2847.4
3320.1/3320.4
6638.2/6637.6
3429.2/3428.4
3354.0/3454.0
0.9/2.3
1.0/0.7
1.0/1.2
N.D.
Cys-melittin
Dimeric-melittin
Thiolpyr-melittin
Mal-melittin
CWKKGIGAVLKVLTTGLPALISLIKRKRQQ
(CWKKGIGAVLKVLTTGLPALISLIKRKRQQ)2
C(tp)WKKGIGAVLKVLTTGLPALISLIKRKRQQ
(m)GWKKGIGAVLKVLTTGLPALISLIKRKRQQ
3.84/4.8
Polyacridine-Melittin (SS ) reducible, Mal ) nonreducible)
poly(Acr-Arg)2-SS-melittin
poly(Acr-Arg)3-SS-melittin
poly(Acr-Arg)4-SS-melittin
poly(Acr-Arg)4-Mal-melttin
poly(Lys(Ac)-Arg)4-SS-melttin
(Acr-Arg)2-C-CWKKGIGAVLKVLTTGLPALISLIKRKRQQ
4359.5/4362.3
4823.9/4823.2
5285.5/5285.2
5321.4/5320.8
4931.0/4930.4
2.5/3.9
1.4/2.2
1.7/1.9
2.1/1.7
5.6/7.7
(Acr-Arg)3-C-CWKKGIGAVLKVLTTGLPALISLIKRKRQQ
(Acr-Arg)4-C-CWKKGIGAVLKVLTTGLPALISLIKRKRQQ
(Acr-Arg)4-C(m)GWKKGIGAVLKVLTTGLPALISLIKRKRQQ
W(Lys(Ac)-Arg)4-C-CWKKGIGAVLKVLTTGLPALISLIKRKRQQ
poly(Acr-Lys)4-SS-melittin
poly(Acr-Lys)4-Mal-melittin
(Acr-Lys)4-C-CWK KG IGAVLKVLTTGLPALISLIKRKRQQ
(Acr-Lys)4-C(m)GWKKGIGAVLKVLTTGLPALISLIKRKRQQ
5173.4/5173.2
5209.4/5208.8
1.0/2.8
1.5/1.9
poly(Acr-Leu)4-SS-melittin
poly(Acr-Leu)4-Mal-melittin
(Acr-Leu)4-C-CWK KG IGAVLKVLTTGLPALISLIKRKRQQ
(Acr-Leu)4-C(m)GWKKGIGAVLKVLTTGLPALISLIKRKRQQ
5113.4/5112.8
5149.3/5149.2
2.2/1.6
2.4/1.2
a Calculated and observed mass as determined by ESI LC-MS. b RBC hemolysis at pH 7.4 and 5.0. c N.D. ) not determined.
less affinity afforded for a repeat of 2 (Figure 2B). Direct
comparison of the DNA binding affinity of poly(Acr-Arg)4-SS-
melittin with poly(Acr-Lys)4-SS-melittin and poly(Acr-Leu)4-
SS-melittin established that there was a negligible difference,
with each producing fully complexed polyplexes at 0.3 nmol
per µg of pGL3 (Figure 2C).
The particle size and zeta potential of polyacridine-melittin
polyplexes were determined by QELS analysis (Table 2).
Poly(Acr-Arg)4-SS-melittin and poly(Acr-Arg)4-Mal-melittin
produced polyplexes of 79-84 nm average diameter, respec-
tively. Comparison of the size of polyplexes prepared with
poly(Acr-Arg)3-SS-melittin (126 nm) and poly(Acr-Arg)2-SS-
melittin (737 nm) established that shorter repeats produced larger
particle size (Table 2). This result is consistent with earlier
findings that indicated that shorter, low-affinity polylysine
peptides (<13 residues) produced larger polyplexes, whereas
polylysine of 18 repeats or longer produced polyplexes of
approximately 80 nm in diameter.
Substitution of the spacing Arg for Lys or Leu in Acr-Arg
repeats resulted in a minimal change in particle size (Table 2).
This supports the hypothesis that polyintercalation, and not ionic
binding, is primarily responsible for the binding affinity to DNA.
To test this hypothesis, an analogue was prepared that replaced
the acridine modified Lys with an acetyl on the ε-amine of lysine
resulting in poly(Lys(Ac)-Arg)4-SS-melittin. This analogue
produced polyplexes of average diameter of 177 nm, suggesting
a significantly lower affinity (Table 2).
To further validate the change in particle size determined by
QELS, polyplexes were analyzed by atomic force microscopy
(AFM) (30). Analysis of pGL3 by AFM demonstrated an open
structure occupying 0.5-1 µm with no single morphology
(Figure 3A). Polyplexes prepared with poly(Acr-Arg)2-SS-
melittin appeared to form clusters composed of several indi-
vidual particles that account for the larger size determined by
QELS (Figure 3B). In contrast, the AFM images for polyplexes
prepared with poly(Acr-Arg)4-SS-melttin (Figure 3C), poly(Acr-
Leu)4-SS-melittin (Figure 3D), and poly(Acr-Lys)4-SS-melittin
(Figure 3E) all appeared to be less aggregated resulting in overall
smaller particle size. It is interesting to note that the zeta
potentials of polyacridine-melittin polyplexes containing a
spacing Arg, Lys, or Leu were approximately +25 mV (Table
2). This result indicated that the charge on melittin strongly
influenced the overall charge of polyplexes prepared with
different anchoring peptides.
Biological Activity of Polyacridine-Melittin DNA
Polyplexes. The membrane lytic potency of polyacridine-melittin
peptides were investigated using a RBC hemolysis assay (9).
As anticipated, poly(Acr-X)4-Cys anchor peptides alone were
inactive in membrane lysis. Alternatively, modification of the
N-terminus of natural melittin with Cys-Trp-Lys-Lys and
substitution of Leu for Trp 8 to generate Cys-melittin, resulted
in retention of RBC hemolytic (HL50) potency at pH 7.4 relative
to natural melittin (Figure 4A). Likewise, the hemolytic potency
of poly(Acr-Arg)4-SS-melittin was comparable to Cys-melittin,
indicating the attachment of the polyacridine anchor had a
negligible influence on the membrane lytic activity of Cys-
melittin. The results for polyacridine-melittin peptides pos-
sessing either a reducible or non-reducible linkage are summa-
rized in Table 1, establishing their nearly equivalent hemolytic
potency at pH 7.4.
The hemolytic potency of polyacridine melittin peptides was
also determined at pH 5 to simulate the pH of the endosome.
In contrast to an earlier finding that C and N-terminal modifica-
tion of natural melittin with Cys-(Lys)4 resulted in the complete
loss of hemolytic activity at pH 5 (9), Cys-melittin possessed
an improved pH 5 RBC lytic potency relative to that of natural
melittin (Figure 4B). Consequently, poly(Acr-Arg)4-SS-melittin
and poly(Acr-Arg)4-Mal-melittin both possess potent hemolytic
activity at pH 7.4 and 5 (Table 1). When combined with pGL3,
Cys-melittin and Mal-melittin maintained their hemolytic activ-
ity at pH 7.4, demonstrating their weak association with DNA
(Figure 4C). In contrast, the hemolytic activity of poly(Acr-
Arg)4-SS-melittin DNA polyplexes was completely neutralized
due to the strong binding of the polyacridine peptide with DNA
(Figure 4C). Reduction of the disulfide bond would decrease
the binding affinity to DNA and thereby release hemolytic Cys-
melittin.

Polyacridine-Melittin Gene Delivery Peptides
Bioconjugate Chem., Vol. 21, No. 1, 2010 79
Figure 1. RP-HPLC analysis of polyacridine-melittin peptide synthesis.
The reaction monitoring of poly(Acr-Arg)4-Cys with thiolpyr-Cys-
melittin at time 0 at 1.7:1 mol ratio is illustrated in panel A. The RP-
HPLC chromatograms were produced by injecting 2 nmol then eluting
with 0.1% TFA (v/v) and an acetonitrile gradient of 10-55% over 30
min while monitoring absorbance at 280 nm. The reaction monitoring
at 12 h established the complete consumption of thiolpyr-Cys-melittin
with formation of the product poly(Acr-Arg)4-SS-melittin and by-
products of dimeric poly(Acr-Arg)4-Cys2 and thiolphene (panel B). LC-
ESI-MS of purified poly(Acr-Arg)4-SS-melittin produced ions of 1762.7
m/z and 1322.3 m/z, respectively (inset), corresponding to a mass of
5285.2 amu (panel C). Reduction of poly(Acr-Arg)4-SS-melittin with
TCEP resulted in formation of equimolar amounts of poly(Acr-Arg)4-
Cys and Cys-melittin (panel D).
Figure 2. Relative binding affinity of polyacridine-melittin peptides
to DNA. The concentration-dependent displacement of the thiazole
orange from DNA by polyacridine-melittin peptides was used to
establish relative affinity. Cys-melittin bound weakly to DNA resulting
in an asymptote in the fluorescence intensity at approximately 0.5 nmol
of peptide per µg of DNA, compared to 0.35 for (Acr-Arg)4-Cys and
0.25 for poly(Acr-Arg)4-SS-melittin (panel A). Comparison of poly(Acr-
Arg)2-4-SS-melittin established that poly(Acr-Arg)2-SS-melittin was the
weakest binding resulting in an asymptote at 0.35 nmols of peptide
per µg of DNA, whereas poly(Acr-Arg)3and4-SS-melittin were nearly
equivalent in displacing thiazole orange (panel B). An equal DNA
binding affinity was demonstrated for poly(Acr-Arg)4-SS-melittin,
poly(Acr-Leu)4-SS-melittin, and poly(Acr-Lys)4-SS-melittin, each re-
sulting in polyplex formation at 0.25 nmol of peptide per µg of DNA
(panel C).
To establish a relationship among peptide anchor length,
reducibility, and peptide DNA stoichiometry, CHO cells were
transfected with polyplexes formed with poly(Acr-Arg)2-SS-
melittin, poly(Acr-Arg)3-SS-melittin, poly(Acr-Arg)4-SS-melit-
tin, and poly(Acr-Arg)4-Mal-melittin while varying the peptide
to DNA ratio from 0.1, 0.3, 0.5, and 0.75 nmols of peptide per
µg of DNA (Figure 5). Luciferase reporter gene expression for
polyplexes formed with 0.1 nmol of peptide per µg DNA
resulted in expression comparable to a WK18/DNA control
polyplex. Increasing the stoichiometry to 0.35 nmols of peptide
per µg of DNA or higher increased the gene expression
approximately 100-fold relative to 0.1 nmol of peptide per µg
of DNA. Within this series, poly(Acr-Arg)4-SS-melittin pro-
duced the highest expression at a stoichiometry of 0.3 and 0.5.
The expression mediated by poly(Acr-Arg)4-SS-melittin was
approximately 1000-fold greater than that of poly(Acr-Arg)4-
Mal-melittin, which differed only by the reducibility of the
linkage between the anchor and melittin (Figure 5).
A broader panel of polyacridine-melittin peptides were
combined with pGL3 at a fixed stoichiometry of 0.5 nmol per
µg of pGL3 and used to mediate gene expression in HepG2,
3T3, and CHO cells. As illustrated in Figure 6, polyacridine-
melittin peptides of the poly(Acr-Arg)2-4-SS-melittin series
produced the highest gene transfection in all three cell lines,
with levels similar to that of PEI (9:1 N:P) when performed
with either 2% or 10% FBS. By comparison, poly(Acr-Lys)4-
SS-melittin and poly(Acr-Leu)4-SS-melittin were approximately
10-100-fold less active in gene transfer than poly(Acr-Arg)4-
SS-melittin in all three cell lines. Reducible poly(Acr-Arg)2-4
-
SS-melittin peptides were 100-1000-fold more active in gene
transfer compared to poly(Acr-Arg)4-Mal-melittin in all three
cell lines. The importance of the reductive release of melittin
was also determined for poly(Acr-Lys)4-SS-melittin and poly-
(Acr-Leu)4-SS-melittin in all three cell lines.
To establish the contribution of poly-acridine to DNA binding
affinity and gene transfer, the ε-amine of lysine was modified
with an acetyl group to prepare poly(Lys(Ac)-Arg)4-SS-melittin.
Gene transfer mediated by poly(Lys(Ac)-Arg)4-SS-melittin was
approximately 100-1000-fold less relative to poly(Acr-Arg)4-
SS-melittin in all three cell lines (Figure 6). To further establish
the importance of the anchor peptides, Cys-melittin and dimeric-
melittin were used to mediate gene transfer (Figure 6). Both
mediated gene transfer that was 10-100-fold lower than
poly(Acr-Arg)2-4-SS-melittin, demonstrating the importance of
the polyacridine anchor in this gene transfer system. Alterna-

80 Bioconjugate Chem., Vol. 21, No. 1, 2010
Baumhover et al.
Table 2. Physical Properties and Toxicity of DNA Polyplexes
MTT assayc
HepG2
peptide
particle size (nm)a
zeta potential (+mV)b
CHO
3T3
poly(Acr-Arg)2-SS-melittin
poly(Acr-Arg)3-SS-melittin
poly(Acr-Arg)4-SS-melittin
poly(Acr-Arg)4-Mal-melittin
poly(Lys(Ac)-Arg)4-SS-melittin
poly(Acr-Lys)4-SS-melittin
poly(Acr-Lys)4-Mal-melittin
poly(Acr-Leu)4-SS-melittin
poly(Acr-Leu)4-Mal-melittin
737 ( 46.8
126 ( 4.5
84 ( 2.0
79 ( 1.3
177 ( 6.2
89 ( 3.5
86 ( 2.4
119 ( 1.4
121 ( 2.5
25 ( 2.2
29 ( 4.5
29 ( 2.6
28 ( 2.5
20 ( 2.9
23 ( 1.6
26 ( 2.0
20 ( 2.9
22 ( 2.1
99 ( 7
103 ( 7
70 ( 5
69 ( 5
55 ( 4
76 ( 5
87 ( 8
46 ( 4
90 ( 3
92 ( 7
97 ( 13
67 ( 9
88 ( 13
64 ( 9
66 ( 5
79 ( 6
49 ( 4
117 ( 5
85 ( 2
91 ( 2
58 ( 4
82 ( 1
33 ( 4
72 ( 6
82 ( 2
50 ( 4
74 ( 3
a Mean particle size of peptide-DNA polyplex (0.5 nmol of peptide per µg of DNA) as determined by QELS. b Mean zeta potential determined in 5
mM Hepes, pH 7.4. c Percent viability of peptide-DNA polyplexes determined by MTT assay in CHO, HepG2, and 3T3 cells at 10 µg DNA dose (0.5
nmol of peptide per µg DNA) applied to 5 × 105 cells for 6 h.
Figure 3. Atomic force microscopy of polyacridine-melittin polyplexes. The size and shape of pGL3 (A) or polyacridine-melittin DNA polyplexes
(B-E) were determined by atomic force microscope. Poly(Acr-Arg)2-SS-melittin (B), poly(Acr-Leu)4-SS-melittin (C), poly(Acr-Arg)4-SS-melittin
(D), and poly(Acr-Lys)4-Mal-melittin (E) pGL3 polyplexes are compared. The results establish that the weaker DNA binding of poly(Acr-Arg)2-
SS-melittin results in aggregated polyplexes.
tively, poly(Acr-Arg)nCys peptides produced negligible gene
transfer on their own, establishing the importance of both the
anchor peptide and melittin.
compared to poly(Acr-Leu)4-SS-melittin polyplexes, which
produced approximately 50% viability (Table 2).
DISCUSSION
The toxicity of polyacridine DNA polyplexes was assessed
by MTT assay on CHO, HepG2, and 3T3 cells (Table 2).
Poly(Acr-Arg)2-3-SS-melittin DNA polyplexes display very
little toxicity in all three cell lines. However, poly(Acr-Arg)4-
SS-melittin and poly(Acr-Arg)4-Mal-melittin polyplexes re-
sulted in approximately 70% cell viability whereas control
peptide poly(Lys(Ac)-Arg)4-SS-melittin polyplexes were
more toxic, resulting in 55% viability. Treatment of CHO,
3T3, and HepG2 cells with 10 µg of poly(Acr-Lys)4-SS-
melittin and poly(Acr-Lys)4-Mal-melittin polyplexes for either
6 or 24 h resulted in the same degree of toxicity, whereas
lowering the DNA polyplex dose to either 1 or 5 µg resulted
in 100% viability. Despite its lower gene transfer efficiency,
poly(Acr-Leu)4-Mal-melittin polyplexes were less toxic
The endosome has been proposed as one of the major barriers
that limits the gene transfer efficiency of nonviral gene delivery
systems (34). To increase the endosomal escape of plasmid
DNA, many prior studies have incorporated fusogenic peptides
into experimental delivery systems (4, 8, 16, 35-40). Most
often, a fusogenic peptide is linked to a PEI or polylysine to
allow for multivalent reversible ionic binding with the phosphate
backbone of DNA (4, 10, 11). While this approach often leads
to significant increases in gene transfer efficiency in vitro, ionic
interactions are relatively weak leading to premature dissociation
of the carrier in vivo and degradation of the DNA (41). To
overcome weak binding, higher molecular weight polycations
are used. These can be prepared with reducible linkages that
revert to low molecular weight, lower affinity polycations and

Polyacridine-Melittin Gene Delivery Peptides
Bioconjugate Chem., Vol. 21, No. 1, 2010 81
Figure 4. Membrane lytic potency of polyacridine-melittin peptides.
The hemolytic activity of melittin and polyacridine-melittin peptides
were compared at pH 7.4 and 5 using a RBC hemolysis assay. The
results establish Cys-melittin is equally potent as natural melittin at
pH 7.4 (panel A), but more potent at pH 5 (panel B). In comparison,
poly(Acr-Arg)4-SS-melittin retains nearly full hemolytic potency at pH
7.4 (panel A) but is less potent at pH 5. However, when combined
with pGL3, the pH 7.4 hemolytic activity of both Cys-melittin and
melittin is nearly unchanged, whereas poly(Acr-Arg)4-SS-melittin is
non hemolytic (panel C). These results establish the masking of
poly(Acr-Arg)4-SS-melittin membrane lytic activity while bound to
DNA.
Figure6.Cell-dependentinvitrogenetransferpotencyofpolyacridine-melittin
polyplexes. The relative gene transfer efficiency of DNA polyplexes
was determined in CHO (A), 3T3 (B), and HepG2 (C) cells. Trans-
fections were performed with 10 µg of pGL3 polyplex prepared with
0.5 nmol of peptide per µg of DNA. The gene transfer efficiency
mediated by polyacridine-melittin peptides was compared to PEI (N/P
9:1) DNA polyplexes and WK18 DNA polyplexes. The bars indicate
transfection pGL3 combined with (1) PEI, (2)WK18, (3) dimeric-
melittin, (4) Cys-melittin, (5) poly(Lys(Ac)-Arg)4-SS-melittin, (6)
poly(Acr-Arg)2-SS-melittin, (7) poly(Acr-Arg)3-SS-melittin, (8) poly-
(Acr-Arg)4-SS-melittin, (9) poly(Acr-Arg)4-Mal-melittin, (10) poly(Acr-
Lys)4-SS-melittin, (11) poly(Acr-Lys)4-Mal-melittin, (12) poly(Acr-
Leu)4-SS-melittin, and (13) poly(Acr-Leu)4-Mal-melittin. The luciferase
expression was determined at 24 h. The results represent the mean and
standard deviation for three independent transfections.
DNA polyplexes are unable to produce therapeutic levels of
gene expression (22).
We have therefore explored an alternative method of binding
fusogenic peptides to plasmid DNA by reversible intercalation.
Preliminary studies suggested that a single acridine binds to
DNA through intercalation but with relatively weak affinity.
Consequently, we sought to increase the binding affinity by
preparing polyacridines. Considering the pioneering studies of
Szoka who developed a synthetic scheme to prepare a diacridine
glycopeptide (24), Vierling with a single acridine modified
nuclear localizing peptide (26), and Nielsen who prepared and
Figure5.Comparativeinvitrogenetransferpotencyofpolyacridine-melittin
polyplexes. CHO cells were transfected with increasing amounts of
peptide (0.1, 0.35, 0.5, and 0.75 nmol peptide per microgram DNA)
combined with 10 µg pGL3. The luciferase expression determined at
24 h represents the mean and standard deviation for three independent
transfections.
trigger the release of DNA inside the cell (21, 22, 42-45).
However, despite numerous attempts, i.v. dosed polycationic

82 Bioconjugate Chem., Vol. 21, No. 1, 2010
Baumhover et al.
tested di and triacridine nuclear localization peptides (25), we
sought a simplified approach that would allow us to extend the
valency of polyacridine to four or more acridine units and
control the charge of polyacridine DNA polyplexes. We
therefore adopted a strategy reported by Ueyama et al. (23),
who demonstrated that polyacridine peptides could be prepared
from Fmoc-Lys(Acr) using solid-phase synthesis. Several early
attempts at synthesis established that polyacridine peptides could
be prepared in good yield (>30%) provided that coupling was
conducted with HBTU and that a spacing amino acid was used
that had a bulky side chain or protecting group. Using Gly as
a spacing amino acid resulted in poor yields.
The synthetic design of polyacridine peptides took into
account the relative DNA binding affinity by varying polyacri-
dine repeat, the influence of the spacing amino acid on gene
transfer and linkage between polyacridine and a melittin
fusogenic peptide. As indicated in Figures 5 and 6, there is a
strong dependency on the nature of the linkage between
polyacridine and melittin in relationship to the gene transfer
efficiency. A reducible disulfide bond provided a means for the
triggered release of melittin either at or inside the cell. The
reduction of the disulfide bond greatly diminishes the affinity
of melittin for DNA, releasing it and unmasking its membrane
lytic activity (Figure 2C). The released melittin apparently
enhances gene transfer by lysis of endosomal membranes that
then allows the DNA to more efficiently reach the cytosol and
the nucleus, resulting in gene expression that equals or surpasses
that of PEI in multiple cell lines (Figure 6). In support of this
mechanism, polyacridine linked to melittin by a non-reducible
maleimide is 1000-fold less active in gene transfer, despite its
equivalent membrane lysis potency against RBCs (Table 1).
These results agree with previously observed improved gene
transfer mediated by a reductively triggered release of a
fusogenic peptide from a gene delivery system (10, 46).
The length of the polyacridine repeat appears to be less
important for in vitro gene transfer, since repeats of 2, 3, and 4
poly(Acr-Arg)-SS-melittin all produced nearly equivalent gene
transfer in three cell lines (Figure 6). However, it is clear that
poly(Acr-Arg)2-SS-melittin has lower affinity for DNA as
indicated by its ability to displace an intercalator dye (Figure
2B). The lower affinity results in larger DNA polyplexes as
determined by QELS (Table 2) and AFM (Figure 3B). However,
larger polyplexes also sediment more efficiently, facilitating in
vitro gene transfer.
The spacing amino acid of Arg, Lys, or Leu within a
poly(Acr-X)4-SS-melittin only slightly influences their affinity
for binding DNA (Figure 2C). Each of the resulting polyplexes
possessed comparable size, charge, and shape as determined
by QELS, zeta potential, and AFM (Table 2, Figure 3).
However, the gene transfer efficiency was influenced signifi-
cantly by the identity of the spacing amino acid in the order
Arg > Lys > Leu (Figure 6). In each case, and in each cell line,
the reducible polyacridine-melittin peptide mediated greater
expression than the nonreducible analogue. The mechanism by
which the spacing amino acid influences gene transfer efficiency
is unclear. We speculate that the polyacridine and spacing amino
acid collaborate to provide DNA binding affinity. It is likely
that, following disulfide bond reduction to release the melittin,
the anchoring peptide binds to DNA with less affinity, and the
charge character of the polyplex changes significantly.
The lytic potency of melittin on cells in culture has been well
documented (12, 14). However, this toxicity can be overcome
by polymerization of melittin through disulfide bonds resulting
in its neutralization due to high-affinity binding to DNA (9). It
is apparent that even dimeric-melittin can bind to DNA and
mediate moderate gene transfer (Figure 6). The MTT assay did
establish that polyacridine-melittin peptides are moderately
toxic (approx 50-70% viable) when added to DNA at 0.5 nmol
of peptide per microgram of DNA or higher (Table 2). However,
this toxicity is the result of saturation of the DNA with
polyacridine peptide resulting in unbound peptide that can lyse
cells. Lower stoichiometries of 0.3 nmol of peptide per
microgram of DNA or lower DNA doses of 5 and 1 µg result
in negligible toxicity on all three cell types.
In conclusion, we have described a new polyacridine DNA
binding anchor that, when linked with melittin through disulfide
bond, produces potent in vitro gene transfer. The polyacridine
anchor is designed to be optimized for either in vitro or in vivo
gene transfer by controlling the binding affinity to DNA and
charge of resulting DNA polyplexes. Furthermore, the Cys
residue allows coupling of fusogenic peptides, ligands, and PEG.
These attributes should allow polyacridine several unique
advantages compared to other polycationic DNA binding
peptides and polymers.
ACKNOWLEDGMENT
We gratefully acknowledge support for this work from the
NIH Grant DK066212.
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