Star-like oligo-arginyl-maltotriosyl derivatives as novel cell-penetrating enhancers for the intracellular delivery of colloidal therapeutic systems

Article abstract of DOI:10.1021/bc200666u

A novel nonpeptide, multiarmed oligo-arginyl derivative was engineered as a cell-penetration enhancer for the delivery of bioactive macromolecules and colloidal drug systems. Hepta-arginyl-maltotriosylamido-N-acetyl-dodecanoyl acid (Arg₇-Malt-NAcC₁₂ acid) was synthesized through a carefully designed multistep chemical protocol, as follows: (1) maltotriose derivatization with 12-amino-dodecanoic acid and acetylation of the free amino group; (2) esterification of the maltotriosyl hydroxyl groups with 2-bromo-isobutyryl bromide; and (3) synthesis of star-like oligomer bearing multiple copies of arginine moieties under atom transfer radical polymerization (ATRP) conditions. The intermediates and final product were characterized by ¹H NMR, IR, mass spectrometry, colorimetric assays, and elemental analysis. Cytotoxicity studies on the final polymeric material showed that this novel cell-penetrating enhancer does not have significant toxic effects on MCF-7 and MC3T3-E1 cell lines. The IC₅₀ was greater than 100 μM with both cell lines, while the polyethylenimine with similar average molecular mass (M_n) that was used as a reference showed an IC₅₀ of 30 and 40 μM, for MCF-7 and MC3T3-E1, respectively. The biological properties of the novel bioconjugate were investigated using a fluorescein-labeled bovine serum albumin (FITC-BSA) as a hydrophilic cargo model. MCF-7 and MC3T3-E1 cells were incubated for 60 min with the Arg₇-Malt-NAcC₁₂- conjugated FITC-BSA [(Arg₇-Malt-NAcC₁₂) ₂-FITC-BSA] or FITC-BSA, and the intracellular fluorescence level was analyzed by spectrofluorimetric analysis of cell lysate, cytofluorimetry, and confocal microscopy. The fluorescence of the lysate of MCF-7 and MC3T3-E1 cells that were incubated with (Arg₇-Malt-NAcC₁₂) ₂-FITC-BSA at 37 °C was approximately 4.5 times higher than the fluorescence obtained with cells incubated with FITC-BSA. At 4 °C, the cell uptake of (Arg₇-Malt-NAcC₁₂)₂-FITC-BSA was only 2 times higher than that of FITC-BSA. Cytofluorimetric studies showed that, after (Arg₇-Malt-NAcC₁₂)₂-FITC-BSA treatment, over 80% of MCF-7 cells and over 95% of MC3T3-E1 cells displayed enhanced fluorescence. Confocal investigations showed punctuated fluorescence within the cytosol in both cell lines, indicating that (Arg₇-Malt-NAcC ₁₂)₂-FITC-BSA was confined to endosomes, with no fluorescence observed in the nucleus.

Full text of DOI:10.1021/bc200666u

Article
pubs.acs.org/bc
Star-Like Oligo-Arginyl-Maltotriosyl Derivatives as Novel Cell-
Penetrating Enhancers for the Intracellular Delivery of Colloidal
Therapeutic Systems
Sara Bersani, Stefano Salmaso, Francesca Mastrotto, Elena Ravazzolo, Alessandra Semenzato,
and Paolo Caliceti*
Deprtament of Pharmaceutical and Pharmacological Sciences, University of Padova, Via F. Marzolo, 5 35131 Padova, Italy
ABSTRACT: A novel nonpeptide, multiarmed oligo-arginyl derivative was engineered
as a cell-penetration enhancer for the delivery of bioactive macromolecules and colloidal
drug systems. Hepta-arginyl-maltotriosylamido-N-acetyl-dodecanoyl acid (Arg -Malt-
7
NAcC acid) was synthesized through a carefully designed multistep chemical protocol,
12
as follows: (1) maltotriose derivatization with 12-amino-dodecanoic acid and acetylation
of the free amino group; (2) esterification of the maltotriosyl hydroxyl groups with 2-
bromo-isobutyryl bromide; and (3) synthesis of star-like oligomer bearing multiple
copies of arginine moieties under atom transfer radical polymerization (ATRP)
1
conditions. The intermediates and final product were characterized by H NMR, IR,
mass spectrometry, colorimetric assays, and elemental analysis. Cytotoxicity studies on
the final polymeric material showed that this novel cell-penetrating enhancer does not
have significant toxic effects on MCF-7 and MC3T3-E1 cell lines. The IC50 was greater
than 100 μM with both cell lines, while the polyethylenimine with similar average
molecular mass (M ) that was used as a reference showed an IC50 of 30 and 40 μM, for MCF-7 and MC3T3-E1, respectively.
n
The biological properties of the novel bioconjugate were investigated using a fluorescein-labeled bovine serum albumin (FITC-
BSA) as a hydrophilic cargo model. MCF-7 and MC3T3-E1 cells were incubated for 60 min with the Arg -Malt-NAcC -
7
12
conjugated FITC-BSA [(Arg -Malt-NAcC ) -FITC-BSA] or FITC-BSA, and the intracellular fluorescence level was analyzed by
7
12 2
spectrofluorimetric analysis of cell lysate, cytofluorimetry, and confocal microscopy. The fluorescence of the lysate of MCF-7 and
MC3T3-E1 cells that were incubated with (Arg -Malt-NAcC ) -FITC-BSA at 37 °C was approximately 4.5 times higher than
7
12 2
the fluorescence obtained with cells incubated with FITC-BSA. At 4 °C, the cell uptake of (Arg -Malt-NAcC ) -FITC-BSA was
7
12 2
only 2 times higher than that of FITC-BSA. Cytofluorimetric studies showed that, after (Arg -Malt-NAcC ) -FITC-BSA
7
12 2
treatment, over 80% of MCF-7 cells and over 95% of MC3T3-E1 cells displayed enhanced fluorescence. Confocal investigations
showed punctuated fluorescence within the cytosol in both cell lines, indicating that (Arg -Malt-NAcC ) -FITC-BSA was
7
12 2
confined to endosomes, with no fluorescence observed in the nucleus.
INTRODUCTION
chimeric peptides. Most of the recognized CPPs contain
cationic, amphiphilic, or hydrophobic functions that promote
cell surface interaction and translocation.
■
The impermeable nature of large hydrophilic structures to
biological barriers is one of the primary causes for the poor
absorption and inefficient intracellular delivery of proteins,
oligonucleotides, and colloidal supramolecular systems. Poor
transmembrane permeability strongly limits the development of
efficient, nonparenteral formulations, and the intracellular
delivery of bioactive macromolecules and colloidal therapeutic
Tat-derived peptides have been demonstrated to be very
efficient penetrating agents. Synthetic peptides have been
designed to mimic the HIV-1 Tat structure, an 86-amino-acid
nuclear transcription-activating protein comprising three func-
tional domains. The region responsible for cell uptake and
nuclear import contains 6 arginine and 2 lysine residues within
a short linear sequence, and the amino acids are in random
1
systems, such as polymer conjugates and nanoparticles. The
development of technologies that enable low-membrane-
permeability therapeutics to effectively penetrate biological
membranes is currently one of the greatest challenges in
7
conformation.
The presence of short peptide sequences containing basic
residues, such as arginines and lysines, is a structural requisite to
enabling cell-penetrating activity and nucleus translocation of
2
pharmaceutical development.
Recently, small regions of proteins, termed protein trans-
duction domains (PTDs) or cell-penetrating peptides (CPPs),
have been demonstrated to possess cell-penetrating properties
that can be exploited to enhance the performance of colloidal
8
,9
these oligo amino acids. Accordingly, model peptides that
contain repeat motifs or poly residues, such as homopolymers
3
−6
therapeutics.
Received: January 3, 2012
Revised: May 4, 2012
CPPs include Tat (trans-activator of transcription)-derived
peptides, signal sequence-derived peptides, and synthetic or
©
XXXX American Chemical Society
A
dx.doi.org/10.1021/bc200666u | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
of arginine, have been synthesized and investigated. Studies
have demonstrated that their internalization efficiency depends
on the length of the peptide backbone. Stretches of six (R6) to
eight (R8) arginine residues showed the highest cytoplasmic
cargo model. The cell-penetration studies were performed
using two cell lines and fluorescence spectroscopy, flow
cytofluorimetry, and confocal microscopy.
10
internalization ability.
MATERIALS AND METHODS
■
The exact mechanism by which cell translocation occurs is
still a matter of debate, as either passive or active pathways, or
receptor- or absorptive-mediated endocytosis, have been
proposed. In addition, the correlation between the structural
features of these peptides and the cell uptake behavior is still
Maltotriose [O-α-D-glucopyranosyl-(1,4)-O-α-D-glucopyrano-
syl-(1,4)- α-D-glucopyranose], 12-amino-dodecanoic acid, ace-
tic anhydride, 2-bromo-isobutyryl bromide, triethylamine,
ascorbic acid, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDC), polyethylenimine (PEI, 1.8 kDa Mn),
fetal bovine serum (FBS), RPMI-1640 medium, Dulbecco’s
modified essential medium (D-MEM), penicillin (10 000 IU/
mL)−streptomycin (10 mg/mL)−amphotericin B (25 μg/mL)
stabilized solution, L-glutamine (200 mM), Dulbecco’s
phosphate buffered saline (DPBS), trypsin−EDTA solution
11,12
under investigation.
Although many aspects of CPP activity have not been fully
elucidated, and a number of contrasting data have been
generated, it is anticipated that these molecules will have a
broad relevance in the pharmaceutical and medical fields, and
they may find applications either for enhancing transmucosal
absorption, mostly for nasal and intestinal delivery, or for
(
2.5 w/v %), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide (MTT) were purchased from Sigma-Aldrich
St. Louis, MO, USA). L-Arginine hydrochloride was furnished
1
3,14
intracellular drug delivery and targeting.
Covalent linkages
(
or simple physical interactions of CPPs have been demon-
strated to promote the cell entry of a variety of hydrophilic
compounds ranging from small molecules to plasmid DNA and
by SERVA Electrophoresis (Heidelberg, Germany). Fluorescein
isothiocyanate (FITC), 2-picolyl hydrochloride, 2-picolylamine,
copper(I) bromide (CuBr), bovine serum albumin−fraction V
1
5,16
17,18
19−21
oligonucleotides,
peptides,
proteins,
nanocarriers,
(
BSA), Triton X-100, and paraformaldehyde were purchased
2
2
23,24
such as liposomes and micelles, and quantum dots.
Nevertheless, many studies have highlighted pitfalls in using
CPPs in drug delivery, including poor cell uptake, unspecific
cell delivery, inconvenient in vivo performance, low stability,
and intrinsic biological activity. Therefore, the use of these
molecules in combination with colloidal therapeutic systems
may result in ineffective systems and/or entail risks of
from Fluka (Buchs SG, Switzerland). Vectashield Mounting
Medium with DAPI was from Vector Laboratories (Peter-
borough, UK). All other reagents were obtained from Fluka
Analytical or Sigma-Aldrich. The solvents of analytical or
HPLC grade were furnished by Carlo Erba (Milan, Italy), VWR
International (Lutterworth, UK), and Sigma-Aldrich (St. Louis,
MO, USA). Cell culture plates and flask, 96-well flat bottomed
microassay plate and chambered slides were purchased from
BD Falcon (BD Bioscience, NJ, USA).
2
5
toxicity.
These drawbacks could be overcome by designing novel
nonpeptide molecules with cell-penetrating activity. These
novel structures could display higher stability and lower toxicity
compared to CPPs and may be devoid of intrinsic biological
activity. Recently, high-molecular-weight polymers bearing
oligo-Arg as pendant functions have been demonstrated to
Synthesis of Maltotriosylamido-N-Acetyl-Dodecanoic
Acid (Malt-NAcC12 acid). 12-Amino-dodecanoic acid (1.0 g,
4.6 mmol) was dissolved in 15 mL of methanol/acetic acid
(95:5 vol/vol). The solution was maintained at 60 °C, and 1.2 g
of maltotriose (2.4 mmol) was added. After 24 h, 7.5 mL of
acetic anhydride (79.4 mmol) was added, and the resulting
solution was stirred for 96 h at room temperature. The volume
was reduced to ca. 4 mL under vacuum, and then 3 mL of 30%
ammonia aqueous solution was added. After 12 h, the solvent
was removed under reduced pressure, and the crude product
was redissolved in 3 mL of methanol and added dropwise to
200 mL of diethyl ether. The precipitate was collected by
filtration and desiccated under vacuum. The recovered product
mass was 1.2 g. The product was analyzed by RP-HPLC using a
Luna C18 column (250 × 4.6 mm, Phenomenex, Torrance, CA,
26
enhance the nasal absorption of insulin. Oligo-Lys and oligo-
Arg hydrophobic-core polysaccharide dendrons have been
27
investigated for the oral delivery of heparin. A novel class
of Arg-rich dendrimers has been successfully developed for the
conjugation and delivery of PNA705 splice-redirecting
2
8
oligonucleotides, while a myristoylated hendecaarginine
derivative has been found to possess promising cell-penetration
29
properties for pharmaceutical applications.
Intrigued by the evidence that nonpeptide cell-penetrating
enhancers may be advantageously exploited for drug delivery,
we designed a novel class of cell-penetrating enhancers with
unusual star-like structures that can be covalently or physically
combined with colloidal therapeutic systems to promote their
cellular uptake. The new enhancers were designed with regard
to the oligo-Arg structure of the Tat-penetrating domain 49−57
and the evidence that the guanidinium headgroup of Arg are
crucial for cellular uptake due to their interactions with
USA) eluted with a gradient of H O/0.05% trifluoroacetic acid
2
(A) and acetonitrile/0.05% trifluoroacetic acid (B) (0−3 min
10% B, 3−27 min from 10% to 80% B). The UV detector was
set at 220 nm. The product was also characterized by elemental
analysis, mass spectrometry (ESI-MS; ESI-TOF Applied
1
Biosystems Mariner Foster City, CA, USA), H NMR (AMX
300 MHz, Bruker Spectrospin, Fallanden, Switzerland), and
FT-IR (FT-IR 1600, Perkin-Elmer, Norwalk, CT, USA).
RP-HPLC (C18): 16.3 min retention time. Elemental
analysis: C, 51.2%; H, 7.9%; N, 1.7% (O, 39.2%) [calcd for
Malt-NAcC12 acid (C H NO ): C, 51.7%; H, 7.7%; N, 1.8%
30
membrane phospholipids. Guanidinium-rich structures, in
fact, undergo cell translocation three times faster than the
transduction domain of Tat, as their insertion into lipid bilayers
produces local membrane distortion leading to transient pore
32
57
18
31
+ 1‑
formation on the membranes.
(O, 39.8%)]. ESI-MS [m/z]: 742.34 (M-H ) [calcd for
1
In this paper, we report an efficient, multistep protocol for
the synthesis of a novel nonlinear multi-Arg-armed molecule
starting from a short oligosaccharide. The cell-penetration
properties of the multiarmed derivative were investigated using
fluorescein-labeled bovine serum albumin as a hydrophilic
C H NO : 743.36]. H NMR (D O): δ ppm 5.40 (d, J = 3.3
32
57
18
2
Hz, 2H, anomeric), 5.23 (d, J = 3.8 Hz, 1H, anomeric), 4.20−
3.60 (m, 18H - sugar region), 3.55 (t, J = 9.3 Hz, 2H, λ-CH −),
2
2.33 (t, J = 7.2 Hz, 2H, α-CH −), 2.18 (s, 3H, CH −CO−),
2
3
1.59 (bm, 4H, ι-CH − and β-CH − of alkyl chain), 1.29 (bs,
2
2
B
dx.doi.org/10.1021/bc200666u | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
4H, −CH − of alkyl chain). NMR signals were indicated as
Article
1
Synthesis of Tris[(2-pyridyl)methyl]amine (TPMA).
Tris[(2-pyridyl)methyl]amine (TPMA) was prepared accord-
2
32
singlet (s), broad singlet, doublet (d), triplet (t), multiplet
m), and broad multiplet (bm); coupling constants of peak
multiplicities (J) have been expressed in Hz. FT-IR (KBr): ν
32
(
ing to a modified protocol described in the literature. First, 2-
picolyl chloride (10.0 g, 61.0 mmol) was dissolved in 25 mL of
distilled water, and the mixture was cooled to 0 °C and added
to 12 mL of 5 N NaOH. This solution was then added to 2-
(aminomethyl)pyridine (3.2 mL, 30.5 mmol) and 50 mL of
dichloromethane. The reaction mixture was allowed to reach
room temperature under vigorous stirring and the pH was
maintained at 9.5 by the addition of 5.0 N NaOH. After 48 h,
the organic phase was washed three times with 20 mL of 2.5 N
−
1
(
(
cm ) 3396 (OH), 2929 (CH), 1717 (CO−OH), 1612
acetyl CO−N).
Synthesis of Hepta(2-bromoisobutyryl)-Maltotriosyla-
mido-N-Acetyl-Dodecanoic Acid (BrIsoBut -Malt-NAcC
7
12
acid). Hepta(2-bromoisobutyryl)-maltotriosylamido-N-acetyl-
dodecanoic acid (BrIsoBut -Malt-NAcC acid) was synthe-
7
12
sized according to a modified procedure reported in the
3
3
NaOH and then dried with anhydrous Na SO and filtered.
literature. Briefly, 1.0 g of Malt-NAcC acid (1.35 mmol) was
2
4
1
2
The organic solvent was removed under reduced pressure.
TPMA was recovered by extracting the resulting waxy, red−
brown residue three times with 25 mL of boiling petroleum
ether. The volatiles were removed under vacuum to afford the
desired TPMA ligand. The mass of the final product was 5.6 g.
suspended in 20 mL of anhydrous chloroform containing
triethylamine (3.7 mL, 26.7 mmol). The suspension was cooled
to 0 °C, and 2-bromo-isobutyryl bromide (3.3 mL, 26.7 mmol)
was added over 30 min. The solution was stirred for 72 h. The
mixture was filtered, and the organic solution was washed three
times with 30 mL of cold water, three times with 20 mL of 0.1
N NaOH, and again three times with 30 mL of cold water. The
organic phase was then dried over anhydrous Na SO , filtered,
+
1+
+ 1+
ESI-MS [m/z]: 291.15 (M+H ) and 313.14 (M+Na )
1
[
calcd for TPMA (C H N ): 290.15]. H NMR (CDCl ): δ
18 18
4
3
ppm 8.53 (d, J = 4.2 Hz, 3H, H-α), 7.65 (t, J = 7.6 Hz, 3H, H-
2
4
βδ), 7.6 (d, 3H, H-βδ), 7.1 (t, 3H, H-γ), 3.9 (s, 6H, −CH −).
2
and concentrated under reduced pressure. The resulting red−
brown solid was dissolved in 5 mL of acetone and added to 100
mL of cold water under vigorous stirring. Acetone was removed
under reduced pressure, and the aqueous suspension was
lyophilized. The crude product (1.47 g) was analyzed by
Synthesis of Hepta-Arginyl-Maltotriosylamido-N-Ace-
tyl-Dodecanoic Acid (Arg -Malt-NAcC acid). TPMA
7
12
(
1.31 g, 4.5 mmol), CuBr (0.65 g, 4.5 mmol), and ascorbic
acid (7.9 mg, 0.045 mmol) were dissolved in 5 mL of DMSO
that had been previously purged with nitrogen. This solution
was then added to 8.5 mL of 0.18 mg/mL Arg-methacryloy-
lamide solution (6.3 mmol) in DMSO while stirring at 55 °C.
Next, the solution was added to 3 mL of 0.27 mg/mL
1
elemental analysis, H NMR, and FT-IR.
Elemental analysis: C, 40.8%; H, 5.1%; N, 1.0%; Br, 30.4%
(
(
(
O, 22.7%) [calcd for BrIsoBut -Malt-NAcC12 acid
7
C H Br N O ): C, 40.3%; H, 5.2%; N, 0.8%; Br, 31.3%
6
0
92
7
1
25
1
BrIsoBut -Malt-NAcC12 acid (0.45 mmol) in DMSO. The
7
O, 22.4%)]. H NMR (CDCl ): δ ppm 4.60−3.30 (m, 18H
3
reaction mixture was maintained under an atmosphere of
nitrogen for 12 h and then was exposed to air. Next, the
solution was added dropwise to 200 mL of an ice-cold 1:1
acetone/diethyl ether mixture containing 1% acetic acid. The
precipitate was desiccated under reduced pressure, redissolved
in 5 mL of warm methanol, and reprecipitated by the addition
of the resulting solution into 200 mL of the acetone/diethyl
ether/acetic acid mixture. Using this procedure, we obtained
sugar region), 3.29 (m, 2H, λ-CH −), 2.15 (m, 2H, α-CH −),
1
2
2
.96 (bs, 42H, CH ), 1.94 (s, 3H, CH −COO−), 1.59 (m, 4H,
3
3
ι-CH − and β-CH − of alkyl chain), 1.33−1.22 (bm, 14H,
2
2
−1
−
CH − of alkyl chain). FT-IR: ν (cm ) 3437 (OH), 2932
2
(
CH), 1743 (isobutyryl CO-O), 1619 (acetyl CO-N).
Synthesis of Arginine-Methacryloylamide (Arg-meth-
acryloylamide). Arginine-methacryloylamide (Arg-methacry-
loylamide) was synthesized according to a procedure described
1
.54 g (98% product yield) of Arg -Malt-NAcC acid. The
3
4
7 12
in literature. Arginine hydrochloride (2.1 g, 10 mmol) was
arginine content in the final product was quantified by
dissolved in 20 mL of saturated NaHCO solution and was
35
3
Sakaguchi assay. The experimental data were plotted on a
then cooled to 0 °C. Next, 970 μL of methacryloyl chloride (10
mmol) was added dropwise under vigorous stirring. After 1 h,
the pH of the mixture was adjusted to 1 with hydrochloric acid,
and the solution was saturated with sodium chloride, filtered,
washed three times with 30 mL of ethyl acetate, and finally
extracted three times with 20 mL of a 1:1 vol/vol mixture of
ethyl acetate/isopropanol. The ethyl acetate/isopropanol
fractions were pooled, concentrated under reduced pressure,
and filtered. The resulting colorless oil was added to 50 mL of
water, and the resulting solution was freeze−dried. The mass of
the final product was 2.4 g. The product was analyzed by ESI-
calibration curve obtained with standard solutions of 0−0.6 μM
2
guanidinium content (y = 7.3955x − 0.0386, R = 0.99). The
final product was also analyzed by elemental analysis, FT-IR,
1
and H NMR (D O).
2
Elemental analysis: found C, 39.3%; H, 5.9%; N, 10.0%; Br,
2
8.1% (O, 16.7%); [calcd for Arg -Malt-NAcC acid HBr salt
7 12
(
2
C130H218Br N O ·7HBr): C, 38.6%; H, 5.6%; N, 10.0%; Br,
7 29 46
−1
7.6% (O, 18.2%)]. FT-IR: ν (cm ) 3373 and 3182
+
[NHC(NH ) ], 2938 (CH), 1735 (CO), 1657 (CN).
2
2
Conjugation of Arg -Malt-NAcC Acid to Fluorescein-
7 12
Labeled Bovine Serum Albumin. Bovine serum albumin
BSA, 10 mg, 0.15 μmol) was dissolved in 250 μL of 0.1 M
1
MS, H NMR, and FT-IR.
ESI-MS [m/z]: 243.13 (M+H ) [calcd for Arg-methacryloy-
(
+
borate buffer, pH 8.3, and then added to 58 μL of a 10 mg/mL
solution of FITC (0.15 μmol) in DMSO. The protein solution
was left at room temperature under mild stirring for 1 h and
then was added to 5.0 μL of ethanolamine (5 mg, 80 μmoles).
The FITC-labeled protein (BSA-FITC) was purified by fast
protein liquid chromatography (FPLC) using a Superose 12
gel-filtration column (Amersham-Pharmacia Biotech, Uppsala,
Sweden) and was isocratically eluted with 0.5 mL/min of 100
mM phosphate buffer, pH 7.0. The fractions containing the
fluorescent protein were pooled, lyophilized, and analyzed by
1
lamide (C H N O ): 242.14]. H NMR (D O): δ ppm 5.69
10
18
4
3
2
(
5
s, 1H, HHCC), 5.47 (s, 1H, HHCC), 4.41 (dd, J = 9.2,
.1 Hz, 1H, α-CH), 3.20 (t, J = 6.8, 2H, δ-CH ), 1.91 (s, 3H,
2
CH ), 1.85−1.72 (m, 2H, γ-CH ), 1.72−1.58 (m, 2H, β-CH ).
3
2
2
−1
+
FT-IR (KBr): ν (cm ) 3368 (OH), 3150 (NHC(NH ) ),
2
2
+
1
654 (CC, CO, and CN), 1528 (CNH or CNH ).
2
3
The purity of the compound was assessed by the integration
1
of the H NMR triplet at 3.20 ppm compared with
methacryloyl singlets at 5.69 and 5.47 ppm (99%).
C
dx.doi.org/10.1021/bc200666u | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
a
Scheme 1. Synthesis of Oligo-Arginyl-N-Acetyl-Maltotriosylamido-Dodecanoic Acid (Arg -Malt-NAcC12 acid)
7
a
(
1) Maltotriose derivatization with 12-amino-dodecanoic acid; (2) esterification of the maltotriosyl hydroxyl groups with 2-bromoisobutyryl
bromide; (3) Arg-mathacryloylamide conjugation by ATRP reaction.
RP-HPLC using a Jupiter C4 column (250 × 4.6 mm,
Phenomenex, Torrance, CA, USA) eluted with a gradient of
analyzed by RP-HPLC, as reported above. The number of Arg7-
Malt-NAcC12 moieties per FITC-BSA molecule was deter-
mined by a Sakaguchi assay, using FITC-BSA as reference.
Cell Lines and Culture. The human MCF-7 breast
adenocarcinoma and murine MC3T3-E1 embryonic fibroblast
cell lines were grown in RPMI 1640 and DMEM medium,
respectively, and supplemented with a mixture of 10% heat-
inactivated fetal bovine serum (FBS), 1% L-glutamine, and a 1%
penicillin−streptomycin−amphotericin B mixture. All cells
H O/0.05% trifluoroacetic acid (A) and acetonitrile/0.05%
2
trifluoroacetic acid (B) (0−3 min 5% B, 3−23 min from 5% to
5
5% B). The UV detector was set at 280 nm. The
bioconjugation degree (number of FITC molecules per BSA
molecule) was assessed by spectrophotometric determination
of FITC at 494 nm (Abs0.1% p/v = 184.9), and the protein was
detected at 280 nm {Abs0.1% p/v = 0.667; [BSA] = [Abs280 nm
Abs494 nm. 0.30) ]/0.667}.
Following this, 100 μL of a solution of 5.5 mg/mL Arg7-
Malt-NAcC acid (0.16 μmol) in DMSO was added to 1 mL of
−
(
were maintained at 37 °C in a humidified 5% CO atmosphere
2
and split every 2−3 days.
Cytotoxicity Assay. MCF-7 and MC3T3-E1 cells were
12
4
water containing 0.12 mg/mL EDC (0.63 μmol). After 30 min,
plated in 96-well flat-bottomed microassay plates (1 × 10
the solution was added to 1.0 mL of 5.2 mg/mL FITC-BSA
cells/well) and grown for 24 h. After complete adhesion, Arg7-
Malt-NAcC acid or polyethylenimine (PEI) was added to the
(0.08 μmol) in 0.1 M 2-(N-morpholino)ethane sulfonic acid,
12
pH 4.6. The reaction mixture was stirred overnight and then
ultrafiltered using an Amicon System equipped with a 10 kDa
cutoff Ultrafree-CL membrane (Millipore Amicon, Bedford,
MA, USA). The bioconjugate was finally purified by ion
exchange chromatography using a TSK-GEL Chelate-5PW
column eluted with a gradient of (A) 20 mM phosphate buffer,
culture media at increasing concentrations from 0 to 100 μM.
The cells were incubated for 24 h at 37 °C, and then the
medium was discarded by aspiration and replaced with 100 μL
of fresh medium. The cells were grown for a further 48 h. Next,
20 μL of a 5 mg/mL solution of 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) in 20 mM phosphate
buffer and 0.15 M NaCl, pH 7.2, was added to each well. After
4 h, the MTT-containing medium was removed and 200 μL of
DMSO was added to each well to dissolve the formazan crystals
0
.1 M NaCl, pH 6.5; and (B) 20 mM phosphate buffer, 0.7 M
NaCl, pH 6.5. The elution profile was analyzed online by UV at
80 nm and by fluorescence (λ 494 nm; λ 520 nm). The
2
ex
em
36
peak detected by UV and fluorescence [(Arg -Malt-NAcC ) -
formed by live cells. The plates were gently shaken for 30 min
and the absorbance was measured at 570 nm. The cell viability
7
12 2
FITC-BSA] was collected, concentrated by ultrafiltration, and
D
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was expressed as a percent viability of treated cells with
untreated cells used as a control.
was carried out according to a three-step procedure described
in Scheme 1: (1) synthesis of maltotriosylamido-dodecanoic
acid (Malt-C12 acid) and N-acetylation of the amino group of
Malt-C12 acid (Malt-NAcC12 acid); (2) synthesis of hepta-(2-
bromoisobutyryl)-N-acetyl-maltotriosylamido-dodecanoic acid
(BrIsoBut -Malt-NAcC acid); (3) synthesis of hepta-arginyl-
Fluorescence Analysis. MCF-7 and MC3T3-E1 cells were
5
seeded in 12-well plates (5 × 10 cells/well). After 48 h (90%
confluence), the medium was removed and the cells were
incubated with FITC-BSA or (Arg -Malt-NAcC ) -FITC-BSA
7
12 2
7
12
(
0.2 μM in culture medium supplemented with 10% FBS) at 37
N-acetyl-maltotriosylamido-dodecanoic acid (Arg -Malt-
NAcC12 acid).
7
or 4 °C. After 60 min, the medium was removed and the cells
were washed three times with DPBS and lysed in 1 mL of 0.1%
Triton X-100 solution. The plates were gently shaken for 2 h
and the lysates were centrifuged at 12 000 rpm (10 625 g) for 5
min. The fluorescence intensity was measured at 520 nm by a
FP-6500 Jasco Fluorescence Spectrophotometer (Kyoto,
Japan) using 0.1% Triton X-100 as the blank. A similar
procedure was carried out with unseeded plates to quantify the
fluorescence intensity of the background. The relative
fluorescence (fluorescence/cell number) was calculated by
determining the cell number on the basis of the BCA protein
assay (Pierce, Celbio, Pero, Italy). The optical density (O.D.)
data at 570 nm were plotted on a standard curve (O.D. values
The synthesis of Malt-C12 acid was carried out through the
reaction of the amino group of 12-amino-dodecanoic acid with
the reducible anomeric carbon of maltotriose. The resulting
amino group was stabilized by acetylation at the nitrogen to
avoid the detachment of the 12-amino-dodecanoic acid residue.
The synthesis and purification process yielded an approximate
70% molar yield (Malt-C acid/maltotriose, mol/mol %). The
12
HPLC analysis of Malt-C12 acid showed a single sharp peak,
which indicated that the product was pure. The chemical
composition of the conjugate was confirmed by elemental
1
analysis, FT-IR, H NMR, and ESI-MS (see Materials and
Methods). FT-IR showed the presence of characteristic signals
corresponding to the hydroxyl groups of the maltotriosyl
residue, C−H bonds and the terminal carboxyl group of the
−6
2
vs cells/mL; y = 10 x + 0.024, R = 0.996).
Fluorescence-Activated Cell Sorting (FACS) Analysis.
1
MCF-7 and MC3T3-E1 cells were seeded in six-well plates (1
alkyl moiety and the acetyl functionality. The H NMR showed
6
×
10 cells/well, 1.8 mL culture medium/well). After 48 h, the
the presence of typical signals of Malt-NAcC12 acid. Mass
spectrometry analysis showed the correspondence of the final
product mass with the expected value. These results confirmed
the chemical identity of the monoacetylated conjugate.
medium was removed and the cells were washed with 1.8 mL of
DPBS and incubated with FITC-BSA or (Arg -Malt-NAcC ) -
7
12 2
FITC-BSA (0.2 μM in culture medium supplied with 10%
FBS), as described above. After 60 min of incubation at 37 °C,
the cells were washed three times with DPBS and treated for 2
min with trypsin/EDTA. Trypsin-treated cells were recovered
by centrifugation at 1500 rpm (166 g) for 2 min and were
washed twice with DPBS. The cells were fixed with 1% (w/v)
paraformaldehyde in phosphate buffer and stored overnight at 4
The carbohydrate hydroxyl groups of Malt-NAcC acid were
12
esterified with 2-bromo-isobutyryl bromide to obtain a
multiarmed oligosaccharide initiator for ATRP reaction. The
FT-IR analysis showed the nearly complete disappearance of
−1
the glycosidic OH signal at 3437 cm , which indicates the
extensive functionalization of the hydroxyl groups with 2-
bromo-isobutyryl bromide. The intensity of the signals of the
acetyl and alkyl terminating carbonyl groups at 1741 and 1619
°
C, protected from light. Afterward, the cell samples (100 000
cells in average count) were analyzed by a FACSCalibur flow
cytometer using the CellQuest software package (BD
Bioscience, Heidelberg, Germany) and then gated using
forward vs side scatter to exclude debris and dead cells.
Confocal Microscopy. MCF-7 and MC3T3-E1 cells were
−1
1
cm were unaffected by the conjugation reaction. The H
NMR showed the presence of the typical signals corresponding
to the oligo-2-bromoisobutyryl derivative (see Materials and
Methods). The elemental analysis showed that the composition
5
plated on four-well chamber slides at a density of 1 × 10 cells
per chamber and incubated at 37 °C in a humidified 5% CO2
atmosphere. After 24 h of adhesion, the cells were incubated
with FITC-BSA or (Arg -Malt-NAcC ) -FITC-BSA (0.2 μM
of the multiarmed initiator corresponded to BrIsoBut -Malt-
7
NAcC12 acid indicating that 7 out of the 10 available hydroxyl
groups were modified with 2-bromo-isobutyryl-bromide. On
the basis of these data, the calculated molecular weight of the
novel bioconjugate was 3482 Da. The synthesis of BrIsoBut7-
Malt-NAcC acid resulted in approximately 60% product yield
7
12 2
in culture medium supplemented with 10% FBS), as described
above. After 60 min incubation at 37 °C, the cells were
thoroughly washed with DPBS and fixed with 1% (w/v)
paraformaldehyde in phosphate buffer for 1 h at 4 °C.
Afterward, the fixed cells were rinsed twice with DPBS, and the
samples were mounted with Vectashield containing 1.5 μg/mL
DAPI and kept at 4 °C. The cells were protected from light
until microscopic examination. Confocal imaging was per-
formed using a Leica TCS SP5 confocal laser-scanning
microscope (Leica Microsystems, Mannheim, Germany)
equipped with a Leica HCX PL APO 63×/1.40 oil immersion
12
[
BrIsoBut -Malt-NAcC acid/Malt-NAcC acid, mol/mol %].
The conjugation of the arginyl residues to the BrIsoBut7-
7 12 12
Malt-NAcC12 acid was carried out under ATRP conditions
using arginine-methacryloylamide (Arg-methacryloylamide) as
the monomer and tris[(2-pyridyl)methyl]amine (TPMA) as
+
the ligand for Cu(I) . Both Arg-methacryloylamide and TPMA
were prepared with high product yields, 99% and 64%,
respectively, and in high purity.
The reaction was carried out under an atmosphere of
nitrogen to avoid the catalyst inactivation by oxidation of Cu(I)
to Cu(II). The reaction also used a 2:1 molar excess of Arg-
methacryloylamide with respect to the 2-bromo-isobutyryl
residues to obtain complete 2-bromo-isobutyryl derivatization.
objective. The blue argon laser was set at 351 nm (blue) and
4
fluorescein, respectively.
9
88 nm to produce the excitation wavelengths for DAPI and
RESULTS
1
■
The H NMR spectrum of the purified final product (Figure
Synthesis and Characterization of Hepta-Arginyl-N-
1A) was complex, but did show the presence of the triplet at
Acetyl-Maltotriosylamido-Dodecanoic Acid (Arg -Malt-
3.35 ppm (J = 6.8 Hz) corresponding to the δ-CH of arginyl
7
2
NAcC12 acid). The synthesis of hepta-arginyl-N-acetyl-
moieties. The two singlets at 5.69 and 5.47 ppm, corresponding
maltotriosylamido-dodecanoic acid (Arg -Malt-NAcC acid)
to the methacrylic functionality, were also absent, confirming
7
12
E
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Figure 2. Cytotoxicity profiles obtained by MCF-7 (A) and MC3T3-
E1 (B) incubation with increasing concentrations of polyethylenimine
(
(
●) and hepta-arginyl-N-acetyl-maltotriosylamido-dodecanoic acid
○). The mean values and standard deviations were calculated on
1
Figure 1. H NMR spectrum (A) and IR spectrum (B) of hepta-
the basis of values obtained from six experiments.
arginyl-N-acetyl-maltotriosylamido-undecanoyl acid (Arg -Malt-
7
NAcC12 acid).
Fluorimetric Assays. In vitro studies were undertaken to
evaluate the ability of Arg -Malt-NAcC acid to transport
7
12
the absence of unreacted monomer. The IR spectrum (Figure
hydrophilic macromolecules into cells. MCF-7 and MC3T3-E1
cells were incubated with FITC-BSA or (Arg -Malt-NAcC ) -
1B) confirmed the presence of the guanidine functions. These
7
12 2
data indicated the conjugation of the Arg-methacryloylamide to
the oligosaccharide structure, and elemental analysis suggested
the hepta-arginyl derivative. This result was confirmed by the
colorimetric determination of the arginyl groups by the
Sakaguchi assay.
FITC-BSA, and the protein uptake was evaluated by
spectrofluorimetric analysis, flow cytometry, and confocal
microscopy.
Since studies reported in the literature showed that tumor
cells, namely, MCF-7 cells, can internalize serum proteins
3
7,38
Cytotoxicity Assay. The cytotoxicity of Arg -Malt-NAcC12
including serm albumin,
FITC-BSA was used as control.
7
acid was investigated by incubation for 24 h in the presence of
MCF-7 and MC3T3-E1 cells. A comparative cell viability assay
was carried out using polyethylenimine (PEI) with a similar
average molecular weight of Arg -Malt-NAcC acid.
Before the fluorimetric analyses, the cells were extensively
washed with buffer to eliminate the FITC-BSA or (Arg -Malt-
7
NAcC ) -FITC-BSA that was not taken up by the cells.
12 2
Spectrofluorimetric Analysis. The cell-associated relative
fluorescence (RF) was examined after MCF-7 and MC3T3-E1
7
12
The cytotoxicity profiles reported in Figure 2 show that the
Arg -Malt-NAcC acid has a very low toxicity toward both of
incubation at 37 and 4 °C with FITC-BSA and (Arg -Malt-
7
12
7
the examined cell lines compared to PEI. The IC50 measured
for PEI was 40 and 30 μM for MCF-7 and MC3T3-E1,
respectively, while for the Arg -Malt-NAcC acid, the obtained
NAcC ) -FITC-BSA and cell lysis with Triton X/100. To
12 2
compare the results obtained under different conditions, the
6
relative fluorescence (RI) values were normalized to 10 cells.
7
12
IC50 with both cell lines was higher than 100 μM.
Preparation of Hepta-Arginyl-N-Acetyl-Maltotriosyla-
Figure 3 reports the RF obtained by cell incubation at 37 °C
with buffer (self-fluorescence), FITC-BSA, and (Arg -Malt-
7
mido-Dodecanoyl-FITC-BSA [(Arg -Malt-NAcC ) -FITC-
NAcC ) -FITC-BSA. The RF associated with the MCF-7 and
7
12 2
12 2
BSA]. Bovine serum albumin (BSA, 66 kDa, isoelectric point
.7) was used as a cargo model to assess the ability of Arg7-
MC3T3-E1 cells incubated with FITC-BSA was in agreement
with the evidence reported in the literature that serum albumin
4
37,38
Malt-NAcC12 acid to act as an intracellular carrier for
hydrophilic macromolecules.
can be taken up by tumor cells.
The fluorescence intensity
values obtained by MCF-7 and MC3T3-E1 cell incubation with
(Arg -Malt-NAcC ) -FITC-BSA were approximately 4.5 times
The conjugation of Arg -Malt-NAcC12 acid to fluorescein-
7
7
12 2
labeled BSA (FITC-BSA) (Scheme 2) was carried out by EDC-
mediated condensation. The chromatographic and spectro-
scopic analyses of the purified product showed the conjugation
of FITC-BSA to Arg -Malt-NAcC acid. The Sakaguchi assay
higher than those obtained with FITC-BSA-treated cells.
Figure 4 shows the RF values obtained by MCF-7 and
MC3T3-E1 incubation with buffer, FITC-BSA, and (Arg -Malt-
7
NAcC ) -FITC-BSA at 4 °C. The basal cell RF of untreated
7
12
12 2
showed that the Arg -Malt-NAcC acid-derivatized FITC-BSA
MCF-7 and MC3T3-E1 cells was similar to that obtained at
7
12
contained a mean of two Arg -Malt-NAcC moieties per
37°, while the RF obtained with FITC-BSA and (Arg -Malt-
7
12
7
FITC-BSA molecule, yielding (Arg -Malt-NAcC ) -FITC-
NAcC ) -FITC-BSA was significantly lower. The incubation
7
12 2
12 2
BSA.
with FITC-BSA resulted in an approximate 2-fold RF increase
F
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Scheme 2. Synthesis of Fluorescent Hepta-Arginyl-N-Acetyl-Maltotriosylamido-Undecanoyl-BSA [(Arg -Malt-NAcC ) -FITC-
7
12 2
BSA]
Figure 3. Relative fluorescence of lysated MCF-7 cells (white) and
MC3T3-E1 cells (gray) incubated at 37 °C with buffer (control),
FITC-BSA, and hepta-arginyl-N-acetyl-maltotriosylamido-dodecanoyl-
FITC-BSA. The mean values and standard deviations were calculated
on the basis of values obtained from five experiments.
Figure 4. Relative fluorescence of lysated MCF-7 cells (white) and
MC3T3-E1 cells (gray) incubated at 4 °C with buffer (control), FITC-
BSA, and hepta-arginyl-N-acetyl-maltotriosylamido-dodecanoyl-FITC-
BSA. The mean values and standard deviations were calculated on the
basis of values obtained from five experiments.
compared to the basal cell RF, and the RF of MCF-7 and
MC3T3-E1 cells incubated with (Arg -Malt-NAcC ) -FITC-
BSA was 2 times higher than that obtained with the cells
incubated with FITC-BSA.
with (Arg -Malt-NAcC ) -FITC-BSA significantly increases
the cell-associated fluorescence. Over 85% and 95% of the
7
12 2
7
12 2
(Arg -Malt-NAcC ) -FITC-BSA treated MCF-7 and MC3T3-
7
12 2
E1 cells, respectively, displayed higher fluorescence compared
Flow Cytometry Analysis. Figure 5 reports the dot plots of
side scatter (SSC-H) vs fluorescence intensity (FL1-H)
obtained by fluorescence-activated cell sorting. The plots
reported in panels A2 and B2 show that the percentage of
FITC-BSA-treated MCF-7 and MC3T3-E1 cells with higher
fluorescence were lower than 8% and 11%, respectively,
compared to the untreated cells (panels A1 and B1). The dot
plots reported in panels A3 and B3 show that the incubation
to the self-fluorescence of untreated cells.
Confocal Microscopy. The confocal images obtained with
MCF-7 and MC3T3-E1 cells incubated with FITC-BSA and
(Arg -Malt-NAcC ) -FITC-BSA are reported in Figures 6 and
7
12 2
7, respectively. Figures 6A and 7A report the images obtained
after 60 min cell incubation at 37 °C, while Figures 6B and 7B
report the images obtained after cell incubation at 4 °C. In
order to evidence the fluorescence of (Arg -Malt-NAcC ) -
7
12 2
G
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Figure 5. Side scatter (SSC-H) versus fluorescence (FL1-H) plots obtained by flow-activated cytometry sorting analysis of MCF-7 (A) and MC3T3-
E1 (B) cells after incubation with plain buffer (A1 and B1), FITC-BSA (A2 and B2), and hepta-arginyl-N-acetyl-maltotriosylamido-dodecanoyl-
FITC-BSA (A3 and B3).
Figure 6. Confocal microscopy images (63×) obtained with MCF-7
incubated for 60 min with (A) (Arg -Malt-NAcC ) -FITC-BSA at 37
7
12 2
°
C (A1, transmission image; A2, reflection image) and FITC-BSA
(
A3); (B) (Arg -Malt-NAcC ) -FITC-BSA at 4 °C (B1, transmission
7
12 2
image; B2, reflection image) and FITC-BSA (B3). The green
fluorescence obtained with FITC-BSA was set as basal level.
Figure 7. Confocal microscopy images (63×) obtained with MC3T3-
E1 incubated for 60 min with (A) (Arg -Malt-NAcC ) -FITC-BSA at
7
12 2
3
7 °C (A1) and FITC-BSA (A3); (B) (Arg -Malt-NAcC ) -FITC-
7 12 2
BSA at 4 °C (B1) and FITC-BSA (B3). The green fluorescence
obtained with FITC-BSA was set as basal level.
FITC-BSA internalized by the cells. the fluorescence of FITC-
BSA treated MCF-7 (Figure 6A3,B3) and MC3T3-E1 cells
DISCUSSION
■
(
and Figure 7A2,B2) has been set as basal level. Therefore, the
images obtained with the control cells (Figures 6A3,B3 and
A2,B2) show the intense blue fluorescence corresponding to
The nonlinear oligo-arginyl cell-penetrating enhancer Arg -
7
Malt-NAcC12 acid was designed for conjugation to large
systems, namely, proteins, oligonucleotides, or colloidal drug
carriers, to promote their cell entry. The nonconventional
architecture of this bioconjugate was obtained by introducing
arginyl moieties, which are the key functions of the cell-
penetrating 49−57 Tat, to a core anchoring structure. The
synthesis protocol was originally designed to obtain a series of
derivatives with a nonpeptidic structure containing a variable
number of Arg functions attached to an anchoring structure
that could perform as a cell-penetration enhancer.
7
DAPI-stained nuclei only. The images obtained with (Arg7-
Malt-NAcC ) -FITC-BSA-treated MCF-7 (Figure 6A2,B2)
12 2
and MC3T3-E1 cells (Figure 7A1,B1) show intense green
fluorescence in the cytosol. The inset of Figures 6A2 and 7A1
show the punctuated fluorescence distribution in the cytosol.
The images obtained with cells incubated at 4 °C (Figures 6B
and 7B) show that (Arg -Malt-NAcC ) -FITC-BSA is taken up
7
12 2
by the cells at a lower extent. This qualitative result confirms
the data obtained by spectrofluorimetry and FACS. Finally,
green fluorescence was never found in the nuclei.
Maltotriose was selected as the anchoring molecule. The
hydroxyl groups of this molecule can provide for multiple
H
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Article
derivatizations with arginyl residues through simple chemical
protocols to obtain a nonpeptide, star-like construct. In
addition, the peculiar reactivity of the reducible anomeric
carbon of maltotriose toward amines can be exploited to
suggest that this product is potentially safe for pharmaceutical
applications. However, since the nonlinear hepta-arginyl
derivative has been designed for conjugation to macro-
molecules or supramolecular drug vehicles, the short- and
long-term toxicity of each colloidal bioconjugate containing the
star-like oligo-arginyl-maltotriosyl derivative must be inves-
tigated in detail.
introduce functional groups, namely, −COOH, −NH , −SH,
2
and so forth, for conjugation to macromolecules or supra-
molecular drug delivery systems.
According to the chemical features of maltotriose, the
anomeric carbon of the oligosaccharide was functionalized with
linear alkyl chains of 12-amino-dodecanoic acid, and the
resulting amino group was stabilized by N-acetylation.
Spectrophotometric and elemental analysis showed that the
acetylation reaction involved only the amino group of the
conjugate, while the hydroxyl functions of maltotriose were not
acetylated and remained available for the following modification
steps.
The most common way to confirm the cell-penetrating
efficiency of different CPPs has been through labeling with
fluorophores, such as fluorescein or rhodamine. Therefore, the
cell-penetrating properties of the Arg -Malt-NAcC derivative
were evaluated using BSA as a cargo molecule labeled with
fluorescein isothiocyanate (FITC).
7
12
The spectrofluorimetric analyses, fluorescence activated cell
sorting, and confocal microscopy showed that, as with other
43
Arg rich macromolecules described in the literature, Arg7-
Malt-NAcC12 can bestow promising cell-penetration properties
on macromolecular bioconjugates.
The conjugation of arginine functions to Malt-NAcC12 acid
was carried out under ATRP conditions, a technique that yields
macromolecular products with defined composition under
The quantitative fluorescence studies and the cytofluori-
metric analyses showed that both the MC3T3-E1 and the
MCF-7 cell lines underwent (Arg -Malt-NAcC ) -FITC-BSA
3
9,40
controlled reaction conditions.
Accordingly, Malt-NAcC12
acid was first converted into a multiarmed initiator by reaction
with 2-bromo-isobutyryl bromide. The acid was then reacted in
the presence of Arg-methacryloylamide, using TPMA/Cu(I)Br
as the catalytic system.
The incomplete maltotriose hydroxyl substitution with 2-
bromo-isobutyryl bromide (7 out of the 10 available OH) that
occurred despite the use of excess 2-bromo-isobutyryl bro-
mide/Malt-NAcC12 acid molar ratios may be ascribed to steric
hindrance that prevents the 2-bromo-isobutyryl-bromide from
reacting with the two vicinal hydroxyl groups of the
oligosaccharide.
7
12 2
uptake. The slight differences observed between the MC3T3-
E1 and the MCF-7 cells appear to indicate that the
translocation process may depend on surface cell-specific
properties, such as specific membrane biological activity or
membrane composition.
Confocal microscopy confirmed the cell uptake of (Arg7-
Malt-NAcC ) -FITC-BSA. The punctuated fluorescence dis-
12 2
tribution in the cytosol obtained with the cells treated with
Arg -Malt-NAcC ) -FITC-BSA and the lack of fluorescence
(
7
12 2
in the nucleus indicate that the cell uptake of (Arg -Malt-
7
Under ATRP conditions, 7 Arg-methacryloylamido moieties
were attached to the BrIsoBut -Malt-NAcC . Preliminary
NAcC ) -FITC-BSA was limited to the cytoplasm compart-
12 2
7
12
ment. This result is in agreement with the data reported in the
literature, which showed that branching membrane transloca-
tional peptides promote gene delivery in the cytoplasm, but not
studies showed that the derivatization of maltotriose bearing
two and four 2-bromo-isobutyryl functions resulted in the
maximal conjugation of two and four Arg-methacryolylamido
moieties, respectively (data not shown). These results seem to
suggest that, under the operative conditions, the 2-bromo-
isobutyryl-bromide groups are completely functionalized with
one Arg-methacryloylamido moiety. Indeed, even the use of
44
in the nucleus.
The cell trafficking of arginine-rich CPPs is still controversial.
The cell uptake of cargo-CCPs has been found to occur
according to mechanisms and kinetics that depend on the
chemical nature of the cell-penetrating function and assay
conditions, namely, temperature, pH, cargo-CCP dose, and so
high molar ratios of Arg-methacryloylamide/BrIsoBut -Malt-
7
NAcC12 (14:1 and 21:1) did not yield higher Arg-
methacryloylamido functionalization. Therefore, for bioconju-
gates bearing short Arg-methacryloylamido chains, the Arg-
methacryloylamide oligomerization could not be obtained. In
this regard, studies reported in the literature have demonstrated
that the inefficient polymerization of (meth)acrylamide
monomers may be ascribed to various phenomena, including
catalyst deactivation by the amido groups and the strong bond
between the (meth)acrylamide units and the bromide
45
forth. The arginine-rich CPPs were found to destabilize the
plasma membrane, creating transient pores for cellular
penetration that resulted in cytosol or nucleus localization
without organelle confinement. Nevertheless, the confocal
results obtained with the cells incubated with (Arg -Malt-
7
NAcC ) -FITC-BSA indicate that active mechanisms may be
12 2
involved in or contribute to the cell uptake of the bioconjugate.
The punctuated fluorescence obtained by the confocal analysis
4
1,42
of cells incubated with (Arg -Malt-NAcC ) -FITC-BSA at 37
atom.
7
12 2
°
C seems, in fact, to corroborate the hypothesis that the star-
The bioconjugate containing 7 Arg residues that is described
in the present study displays composition analogies with cell-
penetration peptides, such as the 49−57 Tat domain and
oligoarginine peptides. Therefore, it was considered a good
candidate as a penetration enhancer for further biological
investigations.
like penetration enhancer promotes the macromolecular cargo
46
uptake by endocytosis or macropinocytosis. This hypothesis
is also confirmed by the data obtained at 4 °C. Under these
conditions, the active cell uptake is minimized and the cell
translocation of (Arg -Malt-NAcC12) -FITC-BSA was signifi-
7 2
Cell culture studies showed that the novel nonlinear hepta-
arginyl derivative does not exhibit significant cytotoxicity, which
is a requisite for a new product that is expected to undergo
intracellular localization. The high IC (>100 μM) and the fact
cantly lower than that obtained at 37 °C. However, the lower
FITC-BSA uptake observed at 4 °C compared to that obtained
at 37 °C may be ascribed to the temperature-related
permeability impairment of the cell membrane. Therefore,
the lower cell uptake of (Arg -Malt-NAcC ) -FITC-BSA at 4
5
0
that only a limited amount of enhancer may be required for
7
12 2
promoting the cell translocation of colloidal formulations
°C could be related to both the inhibition of the active
I
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Bioconjugate Chemistry
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translocation and the lower passive diffusion due to decreased
membrane permeability.
(8) Kalderon, D., Roberts, B. L., Richardson, W. D., and Smith, A. E.
1984) A short amino acid sequence able to specify nuclear location.
(
Cell 39, 499−509.
The combined biological data emphasize the complexity of
the cell uptake process that is mediated by the novel
penetrating agent. Currently, studies have been undertaken to
elucidate the cellular mechanisms involved in the translocation
of Arg -Malt-NAcC functions. These studies are aimed at
(9) Green, M., and Loewenstein, P. M. (1988) Autonomous
functional domains of chemically synthesized human immunodefi-
ciency virus tat trans-activator protein. Cell 55, 1179−1188.
(10) Futaki, S., Suzuki, T., Ohashi, W., Yagami, T., Tanaka, S., Ueda,
7
12
K., and Sugiura, Y. (2001) Arginine-rich Peptides. J. Biol. Chem. 276,
836−5840.
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comparatively examining the cell-penetrating properties of the
corona-like derivative and linear TAT-like sequences. Addi-
tional investigations will also be performed using different cell
lines and experimental conditions, namely, time, pH, and
temperature.
5
(
Design and cellular-uptake mechanisms. Pept. Sci. 84, 241−249.
(12) Patel, L., Zaro, J., and Shen, W.-C. (2007) Cell penetrating
peptides: intracellular pathways and pharmaceutical perspectives.
Pharm. Res. 24, 1977−1992.
(13) Wagstaff, K. M., and Jans, D. A. (2006) Protein transduction:
CONCLUSIONS
■
cell penetrating peptides and their therapeutic applications. Curr. Med.
Chem. 13, 1371−87.
The results reported in the present work show that a novel class
of cell-penetration enhancers with unusual chemical structures
can be easily obtained via simple and reproducible synthetic
steps. In principle, the proposed chemical synthesis protocol is
flexible and can be properly managed to obtain products that,
by virtue of their unconventional structural properties, open
new possibilities in drug delivery.
(14) Okuyama, M., Laman, H., Kingsbury, S. R., Visintin, C., Leo, E.,
Eward, K. L., Stoeber, K., Boshoff, C., Williams, G. H., and Selwood, D.
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oligonucleotide conjugates. J. Biol. Chem. 277, 22980−22984.
A wide array of cell-penetration enhancers can be obtained
by slight modifications of the main structure of these enhancers
to provide for their conjugation or physical combination with a
variety of therapeutic systems. These molecules can be
designed for conjugation to proteins or polymer therapeutics,
or for surface decoration of liposomes or nanoparticles. The
cationic features and the penetration-enhancing properties of
the star-like oligo-arginyl can also be exploited for oligonucleo-
tide, namely, siRNA, or gene delivery.
Finally, a combination of residues, such as histidines and
cysteines, that have been found to possess endosomolytic
properties or short peptides that increase the gene expression of
DNAs, can be introduced within the core structure.
(16) Veldhoen, S., Laufer, S., and Restle, T. (2008) Recent
developments in peptide-based nucleic acid delivery. Int. J. Mol. Sci.
, 1276−1320.
17) Dostmann, W. R. G., Taylor, M. S., Nickl, C. K., Brayden, J. E.,
9
(
Frank, R., and Tegge, W. J. (2000) Highly specific, membrane-
permeant peptide blockers of cgmp-dependent protein kinase iα
inhibit no-induced cerebral dilation. Proc. Natl. Acad. Sci. U.S.A. 97,
1
(
4772−14777.
18) Bonny, C., Oberson, A., Negri, S., Sauser, C., and Schorderet, D.
F. (2001) Cell-permeable peptide inhibitors of JNK. Diabetes 50, 77−
2.
19) Patsch, C., and Edenhofer, F. (2007) , Conditional Mutagenesis
8
(
by Cell-Permeable Proteins: Potential Limitations and Prospects Condi-
tional Mutagenesis: An Approach to Disease Models (Feil, R., and
Metzger, D., Eds.) pp 203−232, Springer, Berlin.
AUTHOR INFORMATION
̈ ̈
(20) Saalik, P., Elmquist, A., Hansen, M., Padari, K., Saar, K., Viht, K.,
■
̈
Langel, U., and Pooga, M. (2004) Protein cargo delivery properties of
Corresponding Author
cell-penetrating peptides. a comparative study. Bioconjugate Chem. 15,
*
E-mail: paolo.caliceti@unipd.it; phone +39 049 8275695; fax
1
(
246−1253.
+39 049 8275366.
21) Albarran, B., To, R., and Stayton, P. S. (2005) A TAT−
streptavidin fusion protein directs uptake of biotinylated cargo into
Notes
mammalian cells. Protein Eng., Des. Sel. 18, 147−152.
The authors declare no competing financial interest.
(22) Torchilin, V. P. (2008) Tat peptide-mediated intracellular
delivery of pharmaceutical nanocarriers. Adv. Drug Delivery Rev. 60,
48−558.
23) Medintz, I. L., Pons, T., Delehanty, J. B., Susumu, K., Brunel, F.
M., Dawson, P. E., and Mattoussi, H. (2008) Intracellular delivery of
quantum dot−protein cargos mediated by cell penetrating peptides.
Bioconjugate Chem. 19, 1785−1795.
(24) Liu, B. R., Huang, Y.-w., Winiarz, J. G., Chiang, H.-J., and Lee,
H.-J. (2011) Intracellular delivery of quantum dots mediated by a
histidine- and arginine-rich HR9 cell-penetrating peptide through the
direct membrane translocation mechanism. Biomaterials 32, 3520−
3537.
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