Improvement of targeted gene delivery to human cancer cells by a novel trifunctional crosslinker

Article abstract of DOI:10.1002/asia.200900129

A facile method for the construction of an immunoconjugate which displays targeting ligands, such as antibody fragments, with a high density is reported. For this purpose, we synthesized a novel trifunctional crosslinking reagent. By the use of this reage

Full text of DOI:10.1002/asia.200900129

FULL PAPERS
DOI: 10.1002/asia.200900129
Improvement of Targeted Gene Delivery to Human Cancer Cells by a Novel
Trifunctional Crosslinker
Maki Shiota, Lahman Shamsur, Shun-ichi Kawahara, Renu Wadhwa, and Yutaka Ikeda*[a]
Abstract: A facile method for the con-
struction of an immunoconjugate which
displays targeting ligands, such as anti-
body fragments, with a high density is
reported. For this purpose, we synthe-
sized a novel trifunctional crosslinking
reagent. By the use of this reagent, li-
gands targeting the specific cell can be
displayed on the surface of the drug
carrier with a high density. In this
study, we display HER2 (human epi-
dermal growth-factor receptor-2) bind-
ing ligands on branched polyethyleni-
mine (PEI), which can form polyplexes
with plasmid DNA. Kinetic analysis of
the binding to the extracellular domain
of HER2 show the PEI displaying a
high density of ligands binds to the
target more strongly compared to the
PEI displaying ligands at a low density.
The increased density of HER2 ligands
displayed on the gene carrier contrib-
utes to the improved transfection effi-
ciency. This approach can be applied to
other drug delivery systems, including
liposome, micelle, and so on.
Keywords: antibodies · cell recog-
nition · conjugation · drug delivery ·
immunoconjugate
Introduction
of antibody-mediated therapeutics has several problems, in
particular, the high cost of preparation of intact antibodies.
To construct alternatives to antibodies, antibody fragments
Targeted delivery is a critical issue in gene therapy to
reduce the amounts of drugs for the effective treatments
and, accordingly, reduce side effects. In recent years, various
gene delivery systems have been developed aiming for tar-
geted-delivery, including the use of viral and non-viral vec-
tors.[1] In case of non-viral vectors, a variety of cell-targeting
ligands, such as small molecules, peptides, proteins, and anti-
bodies, promote them into a wide range of applications in
the field of gene therapy. Natural ligands such as saccharide,
folate, and transferrin are inexpensive and they are easy to
prepare and handle. However, these natural ligands also
bind to some non-target cells, and compete for binding with
native molecules in bodily fluids. Antibodies are favorable
for targeted delivery because of their high affinity and spe-
cificity against their target antigen. However, the application
such as FACTHNUTRGENNG(U ab’)2 and Fab, which can be prepared easily in bac-
teria, have been developed. Moreover, with recent advances
in engineering antibodies, many novel binding proteins that
bind their target antigens with high specificity and affinity
can now be easily generated.[2]
An antibody with two antigen-binding sites, which interact
with its antigen bivalently, achieves high affinity against its
antigen. In contrast, most of these antibody fragments and
binding proteins are monovalent and, consequently, their af-
finities are relatively weak compared to an intact antibody.
Therefore, dimeric and more complicated forms have been
generated by genetic and chemical methods to improve
their binding affinity. For example, by genetic methods, dia-
bodies and minibodies have been developed and achieved
comparable antigen binding affinity to the parental IgG.[3]
Dimeric or multimeric forms have also been generated by
chemical cross-linking methods. Dimeric and trimeric anti-
body fragments have been developed by using bis-malei-
mide and tri-maleimide derivative cross-linkers, respective-
ly.[2] For application of the multimeric fragments or ligands
for ligand-targeted therapeutics, it is desirable for these mul-
timers to modify functional molecules, such as radioisotopes,
cytotoxins, and nanocarriers. The chemical cross-linking
method also enables site-specific modification with these
functional molecules, not only generation of dimeric or mul-
[a] Dr. M. Shiota, Dr. L. Shamsur, Dr. S.-i. Kawahara, Dr. R. Wadhwa,
Dr. Y. Ikeda+
National Institute of Advanced Industrial Science and Technology
(AIST)
Central 4, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan)
[+] Current address:
Graduate School of Pure And Applied Science
University of Tsukuba
1-1-1 Ten-noudai, Tsukuba 305-8573 (Japan)
Fax : (+81)29-853-5749
E-mail: ikeda@ims.tsukuba.ac.jp
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timeric forms. King et al. designed their cross-linkers con-
taining a 12-N-4-macrocycle to allow stable radiolabelling of
the fragments with 90Y.[4] Wilbur et al. conjugated (Fab’)2
with diagnostic and therapeutic agents by using the trifunc-
tional equilibrium transfer alkylation cross-linking reagent.[5]
This cross-linking reagent also permitted the site-specific
PEGylation of native disulfide bonds in therapeutic proteins
without destroying their tertiary structure or abolishing their
biological activity.[6]
In this study, we synthesize a novel trifunctional crosslink-
ing reagent which facilitates the dimerization of ligands such
as antibody fragments and also enables modifications of
these ligands with another functional molecule such as a
drug, chemical probe, and nanocarrier.
(Figure 1B). SDS-PAGE analysis clearly showed that the
fluorescent probe (Dansyl moiety) was covalently conjugat-
ed with the dimerized ligands (Figure 1C). This result indi-
cated the feasibility of the approach in which by using our
trifunctional crosslinker, dimerized antibody fragments or li-
gands can be modified with functional molecules, such as cy-
totoxins, prodrugs, nucleic acids, nanocarriers, and so on.
Furthermore, by using this trifunctional crosslinker we
conjugated Her2ligand with branched polyethylenimine as a
gene carrier for targeted delivery (Figure 2). The cationic
polymer polyethylenimine (PEI) has been successfully used
as non-viral gene carriers both in vitro and in vivo.[8] We
compared the efficiency of the gene delivery by two kinds
of PEI–Her2ligand conjugates which were synthesized by
using
a commercially available bifunctional crosslinker
(EMCS) and our trifunctional crosslinker (bMNHS).
First, purified Her2ligand, PEI–Her2ligand (EMCS), and
PEI–Her2ligand (bMNHS) were analyzed for HER2–ECD
Results and Discussion
We synthesized a novel trifunc-
tional crosslinker (Scheme 1).
This reagent possesses three
functional groups consisting of
two maleimide residues, which
react with the thiol groups, and
an N-hydroxysuccinimidyl ester,
which reacts with the amino
groups. This crosslinker could
facilitate dimerization of li-
gands which have thiol groups
and further modification at the
hinge of dimerized ligands by
an activated ester. It is worth
noting that only recrystalliza-
tion is needed for the purifica-
tion of this reagent, accordingly,
this scheme is suitable for
large-scale synthesis (see Ex-
perimental Section).
We attempted to dimerize
HER2
(human
epidermal
Scheme 1. Reagents and conditions: a) Maleicanhydride, DMF 95%; b) Diglycolicanhydride, DMF;
c) NaOAc., aceticanhydride, 50% from 2; d) DCC, N-hydroxysuccinimide, methoxyethylether, 83%. DMF=
N,N-dimethylformamide, DCC=Dicyclohexylcarbodiimide
growth factor receptor-2)-bind-
ing ligands[7] and modify the
dimer with a fluorescent probe
binding by real-time biospecific interaction analysis using a
BIAcore biosensor instrument. These molecules were inject-
ed over the NTA sensor chip flow-cell surface containing
the immobilized target protein His-HER2-ECD and the
control flow-cell.
Abstract in Japanese:
Her2ligand, PEI–Her2ligand (EMCS), and PEI–Her2li-
gand (bMNHS) were all shown to bind to HER2–ECD.
Upon evaluation of the binding curves of these molecules,
the dissociation equilibrium constant (KD) was determined
to be 53 nm for Her2ligand, 4.7 nm for PEI–Her2ligand
(EMCS), and 0.97 nm for PEI–Her2ligand (bMNHS)
(Table 1). As expected, by using the trifunctional crosslinker
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Y. Ikeda et al.
FULL PAPERS
expression with PEI–Her2li-
gand (bMNHS) compared to
with
the
PEI–Her2ligand
(EMCS). To confirm HER2
specificity of these polyplexes,
HER2 positive SKBR3 cells
were preincubated with free
Her2ligand, which effectively
depressed subsequent transfec-
tion with both PEI–Her2ligand
(EMCS) and PEI–Her2ligand
(bMNHS). These results strong-
ly imply, in this targeting
system, the increased density of
Her2ligand displayed on the
gene carrier contributed to the
improved targeting efficiency,
resulting in an improved trans-
fection efficiency.
Figure 1. a) Structure of the novel trifunctional crosslinker (bMNHS). b) The modification of dimerized Her2-
ligands with Dansyl probe by using the trifunctional crosslinker. c) SDS-PAGE analysis of the dimerized li-
gands conjugated with fluorescent probe.
Conclusions
Multivalent interactions have
been extensively investigated to
promote targeting to specific
cells. In the case of intact anti-
bodies, they can recognize the
specific antigen with two bind-
ing sites. Our novel crosslinker
facilitates dimerization of li-
gands and modification with an-
other functional molecule in-
cluding probe and drug. Our
Figure 2. Schematic presentation of the synthesis of polyethylneimine–Her2ligand conjugates by using the bi-
functional crosslinker (EMCS) or our trifunctional crosslinker (bMNHS). PEI–Her2ligand conjugates could
form polyplexes with plasmid DNA.
Table 1. Kinetic analysis of the binding of Her2ligand, PEI–Her2ligand
(EMCS) and PEI–Her2ligand (bMNHS) to HER2-ECD by biosensor
binding studies.
Ligand
KD(nm)
Ka [mÀ1 sÀ1
]
Kd [sÀ1
]
Her2ligand
PEI–Her2ligand
PEI–Her2ligandACHTUNGTRENNUNG
53
4.7
0.97
6.4ꢁ103
4.8ꢁ103
3.2ꢁ104
1.1ꢁ10À5
9.3ꢁ10À6
2.2ꢁ10À5
G
for conjugation of PEI with Her2ligand, the affinity for
HER2–ECD was improved.
Figure 3. Delivery and expression of a luciferase gene using PEI–Her2li-
gand (EMCS or bMNHS)/Plasmid DNA polyplexes in HER2 positive
SKBR3 cells. Values were normalized against the protein content of the
sample.
The PEI–Her2ligand (EMCS or bMNHS)/DNA polyplex-
es were evaluated for in vitro transfection efficiency in
HER2 positive SKBR3 cells. Before transfection, we
checked the interaction and complex formation between
plasmid DNA and positively charged PEI–Her2ligand con-
jugates by gel retardation assays (data not shown). On the
basis of the cell viability assay by Alamar Blue assay (Trek
Diagnostic Systems, Inc.), we prepared the polyplexes at an
N/P (nitrogen of polyethylenimine/phosphate of DNA) ratio
of 7.5 because polyplexes have increased toxicities above
this point. Figure 3 shows about a fivefold higher luciferase
system exploits another strategy to construct an immuno-
conjugate.
In this study, we also demonstrated the enhancement of
gene delivery by a densely ligand-displayed polyethyleni-
mine. Analysis by real-time biospecific interaction showed
that densely ligand-displayed polyethylenimine bound to the
targeting antigen more strongly. Accordingly, it was consid-
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Improvement of Targeted Gene Delivery to Human Cancer Cells
2,5-dioxopyrrolidin-1-yl-2-(2-(bis(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)ethyl)amino)-2-oxoethoxy)acetate (5).
ered that the improved affinity to the cell gave rise to the
transfection efficiency.
Dicyclohexylcarbodiimide (33 mg) and N-hydroxysuccinimide ware
added to 4 (100 mg) in methoxyethylether (1.3 mL) and stirred at 08C
for 2 h. The reaction mixture was brought to room temperature and fur-
ther stirred for 3 h. A few drops of acetic acid was added to the reaction
mixture at 08C and stirred for 1 h. Resulting white precipitate was re-
moved by filtration and the filtrate was concentrated in vacuum. The res-
On the other hand, especially in the case of solid tumor-
targeted delivery, it became clear that the improved affinity
did not always contribute to ligand-targeted delivery. When
the binding affinity is too high, there are several reports that
penetration of the targeting molecules into solid tumors de-
creases because of the “binding-site barrier”, whereby the
molecule binds strongly to the first targets encountered but
fails to diffuse further into the tumor.[9,10] Adam et al.
showed high affinity restricted the localization and tumor
penetration of single-chain Fv antibody molecules (scFv)
when compared to scFvs with a different affinity to the
same antigen, indicating the existence of a threshold affinity
for the improved delivery.[10] Recently, Zhou et al. showed
the importance of the density of ligands on the nanoparticle
for efficient uptake.[11] They suggest ultrahigh affinity of
ligand may be unnecessary for targeted delivery if low affin-
ity ligands are displayed at high densities.
For further developments of gene delivery in terms of
specificity and efficiency, controlling the affinity and density
of ligands and the choice of a proper set of ligands are de-
manded. By the use of our reagent, several types of ligands,
which can target different antigens at the same time, can be
displayed at high density, enabling the control of the affinity
of the gene carrier. Research into this is currently being un-
dertaken by our group.
idue was recrystallized in 2-propanol to yield
(400 MHz, CDCl3) d=2.83 (s, 4H), 3.40 (t, 2H, J=7.2 Hz), 3. 57 (m,
5
(83%). 1H NMR
2H), 3.67 (t, 2H, J=7.2 Hz), 3. 71 (m, 2H), 4.24 (s, 2H), 4.53ACHTUNGTRENNUNG(s, 2H),
6.68 (s, 2H), 6.71 ppm (s, 2H); 13C NMR d=25.80, 35.23, 35.66, 44.02,
44.71, 67.89, 69.25, 134.54, 134.59, 165.76, 168.95, 169.01, 170.52,
170.59 ppm.
Plasmid Construction and Expression of Her2ligand Peptides
A plasmid vector, which expressed Her2ligand with a cysteine residue at
the N-terminus, was constructed as follows.
A 1 mmol amount of each synthetic DNA primer was mixed, then PCRs
were carried out with Ex Taq DNA polymerase (Takara, Shiga, Japan) to
construct a Her2ligand coding region. The DNA primers were 5’- CAT
GCC ATG GTG GAT AAC AAA TTC AAC AAA GAA CTG CGC
CAG GCG TAC TGG GAA ATC CAG GCG CTG CCG AAC CTG
AAC TG -3’, 5’- CCG AAC CTG AAC TGG ACC CAG AGC CGC
GCG TTC ATC CGC AGC CTG TAC GAT GAT CCG AGC CAG
AGC GCG AAC CTG CTG -3’, and 5’- CCG GAA TTC GCT GCC
ACC GCT GCC ACC TTT CGG CGC CTG CGC ATC GTT CAG
TTT TTT CGC TTC CGC CAG CAG GTT CGC GC -3’. To introduce
the cysteine residue at N-terminus and restriction enzyme sites, a 1 mmol
amount of each synthetic DNA primer was mixed with the above PCR
product and PCRs performed similarly. The DNA primers were 5’- CAT
GCC ATG GGC TGC GTT GAT AAC AAA TTC AAC AAA GAA
CTG -3’ and 5’- CGC GGA TCC TTA TTT CGG CGC CTG CGC ATC
G -3’. Purified PCR product was digested with NcoI and EcoRI restric-
tion enzymes and cloned into pTWIN2 vector (New England Biolabs) to
obtain the pTWIN2Her2ligand vector.
Experimental Section
The E.coli strain ER2566 (New England Biolabs) transformed with
pTWINHer2ligand vector was cultured in LB medium containing ampi-
cillin at 258C until the turbidity at 600 nm reached 0.4. Induction was
performed by adding isopropyl-b-d-thiogalactopyranoside (IPTG, final
1 mm) and the bacteria were cultured for an additional 4 h at 258C, then,
harvested by centrifugation at 4000 g for 15 min. The bacterial pellet was
resuspended in 20 mm HEPES buffer (pH 8.5) containing 500 mm NaCl,
1 mm EDTA and 0.5% TritonX-100 and sonicated on ice. The lysate was
centrifuged at 12000 g for 5 min and the Her2ligand protein in the super-
natant was then purified using chitin beads as described in manufactureꢂs
protocol. Purified Her2ligand was dialyzed into PBS.
Synthesis
ACHTUNGTRENNUNG(2Z,2Z’)-4,4’-(2,2’-azanediylbis(ethane-2,1-diyl)bis(azanediyl))bis(4-
oxobut-2-enoic acid) (2).
Maleicunhydride (10 g) in dimethylformamide (DMF) (30 mL) and dieth-
ylenetriamine 1 (5.4 mL) in DMF (30 mL) were added slowly to DMF
(30 mL) at room temperature and further stirred for 3 h. The resulting
white precipitate was filtered and washed with DMF and chloroform to
afford 2 (14.2 g, 95%). 1H NMR (400 MHz, CDCl3): d=3.06 (t, 4H, J=
5.6 Hz), 3.43 (t, 4H, J=5.6 Hz), 6.07 (d, 2H, J=12.4 Hz), 6.31 ppm (d,
2H, J=12.4 Hz); 13C NMR: d=36.45, 47.50, 131.51, 133.66, 167.59,
168.30 ppm.
Modification of Dimerized Her2ligand with Dansyl
2-(2-bis(2-(2,5-dioxo-2,5-dihidro-1H-pyrrol-1-yl)ethyl)amino)-2-oxo-
ACHTUNGTRENNUNGethoxy)acetic acid (3).
1 mg bMNHS freshly dissolved in dimethylformamide was added to 1 mg
Dansyl-cadaverine and incubated at room temperature with rotating.
After 12 h, 1 mm activated Dansyl was prepared in 0.1m phosphate
buffer (pH 7.0). Ligands were reduced by incubation with 2 mm dithio-
threitol and 2 mm EDTA in 0.1m phosphate buffer (pH 7.0) for 30 min at
room temperature without agitation. DTT and EDTA were eliminated by
gel chromatography (PD-10 column, GE Healthcare). The concentration
of thiol group was measured by the Ellman test.
Diglycolicanhydride (7.17 g) in DMF (25 mL) was added into solution of
2 (14.2 g) in DMF (475 mL) and stirred at room temperature for 2 h. The
reaction mixture was concentrated in vacuum to afford 3.
ACHTUNGTRENNUNG(2Z,2Z’)-4,4’-(2,2’-(2-(carboxymethoxy)acetylazanediyl)bis(ethane-2,1-
diyl)bis(azanediyl))bis(4-oxobut-2-enoic acid) (4).
Compound 3 was dissolved in acetic anhydride. Sodium acetate (2.1 g)
was added to the solution and stirred at 908C for 3 h. The reaction mix-
ture was filtered. Water (30 mL) was added to the filtrate and stirred for
3 h. The solution was evaporated and the residue was dissolved in chloro-
form and washed with brine. The organic layer was dried over anhydrous
sodium sulfate and concentrated in vacuum. The residue was recrystal-
1 mm activated Dansyl was added to reduced ligands at a molar ratio of 1
and incubated with rotating at room temperature for 12 h. Dansyl-la-
belled Her2ligands were fractionated on sodium dodecyl sulfate (SDS)
polyacrylamide (18%) gels. The fluorescence of the Dansyl moiety was
detected by using a UV transilluminater and the ligands were detected
by following Coomasie Brilliant Blue staining.
1
lized in methanol to yield 4 (50%). H NMR (400 MHz, CDCl3): d=3.36
(t, 2H, J=6.8 Hz), 3. 63 (m, 2H), 3.70 (t, 2H, J=6.8 Hz), 3. 75 (m, 2H),
4.15 (s, 2H), 4.31 (s, 2H). 6.70 (s, 2H), 6.78 ppm (s, 2H).); 13C NMR: d=
35.34, 35.64, 44.47, 44.81, 70.64, 71.45, 134.58, 134.67, 170.44, 172.67,
175.85 ppm; Anal. Calcd for C16H18N4O6: C 50.65, H 4.48, N 11.08;
found: C 50.58, H 4.43, N 10.87.
Generation of Polyethylenimine–Her2ligand(EMCS or bMNHS)
In order to generate a gene delivery carrier, branched 25 kDa polyethyle-
nimine (PEI, Aldrich, St. Louis, USA) was dissolved in PBS to a final
concentration of 2 mgmLÀ1. To 1 mg branched PEI, the crosslinker [N-
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Y. Ikeda et al.
FULL PAPERS
(6-Maleimidocaproyloxy)succinimide (EMCS, Dojindo, Japan) or trifun-
cational crosslinker (bMNHS)] freshly dissolved in dimethylformamide
was added (final 4 mm) and incubated at room temperature with gentle
shaking. After 4 h, unconjugated crosslinkers were removed by ultrafil-
tration with a 10 kDa cut-off membrane (VIVASPIN, Sartorius, UK).
100 mg Her2ligand was reduced by incubation with 5 mm Tris(2-carboxye-
thyl)phosphine (TCEP) in PBS for 30 min at room temperature without
agitation. The concentration of Her2ligand was measured using Protein
Assay reagent (Bio-Rad). In order to generate Her2ligand–PEI conju-
gate, the EMCS-activated PEI or bMNHS-activated PEI dissolved in
PBS was added to the reduced Her2ligand and incubated overnight at
room temperature with gentle shaking. The sample complex was purified
by ultrafiltaration on a 30 kDa cut-off membrane (Microcon YM-30, Mil-
lipore). The sample was washed twice with 20 mm MOPS buffer (pH 7.3)
containing 1.5m NaCl. The PEI–Her2ligand conjugate was dissolved in
0.1m MOPS buffer (pH 7.3) containing 150 mm NaCl. The concentration
of PEI was determined by copper complexation.
HEPES buffer (pH 7.4) containing 150 mm NaCl, 50 mm EDTA and
0.005% Surfactant P20, by injecting 20 mL of the solution at a flow rate
of 2 mLminÀ1
.
For the kinetic analysis, each of Her2ligand and PEI–Her2ligand conju-
gates were injected at final concentrations ranging from 25 to 500 nm.
Ackowledgements
We thank Dr. Penmetcha K.R. Kumar and Dr. Subash C.B. Gopinath
for obtaining BIAcore data.
[1] M. E. Davis, Z. G. Chen, D. M. Shin, Nat. Rev. Drug Discovery
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Cell Viability Assay
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Cells (2ꢁ104 cells per well) were cultured in 96-well plates. PEI conju-
gates were mixed with 125 ng plasmid DNA at N/P ratios of 0, 1, 2, 5,
7.5, 10, 12, and 15 in PBS and incubated for 20 min at room temperature.
Polyplexes were then added to the cell culture medium and incubated at
378C. After 24 h, Alamar Blue assay was performed as described in the
manufactureꢂs protocol. The viability of untreated cells was defined as
100%.
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Gene Transfer by PEI–Her2ligand Conjugates
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Cells (2ꢁ104 cells per well) were cultured in 96-well plates. PEI–Her2li-
gand with plasmid DNA was generated as follows. Plasmid DNA encodes
Luciferase. DNA (125 ng) was added to 25 mL of DMEM. Subsequently,
PEI–Her2ligand (EMCS) or PEI–Her2ligand (bMNHS) was added to
the DNA solution to yield an N/P ratio of 7.5. The mixture was mixed by
pipetting and incubated at room temperature for 30 min. Culture
medium of the adherent cells were replaced with 100 mL of fresh culture
medium and the above transfection mixture was added. Cells were incu-
bated for 48 h at 378C.
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2008, 15, 2826–2839.
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Binding Analysis of PEI–Her2ligand Conjugates with HER2-ECD using
SPR (Surface Plasmon Resonance)
The binding analyses of PEI–Her2ligand conjugates with HER2-ECD
(extracellular domain of human epidermal growth factor receptor-2)
were conducted using the SPR method on a Biacore 2000, with a NTA
sensor chip (Biacore, Uppsala, Sweden). Initially, the sensor chip was
coated with 200 nm His-HER2-ECD (R&D systems), dissolved in 10 mm
[11] Y. Zhou, D. C. Drummond, H. Zou, M. E. Hayes, G. P. Adams, D. B.
kirpotin, J. D. Marks, J. Mol. Biol. 2007, 371, 934–947.
Received: April 8, 2009
Published online: July 3, 2009
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