Acid degradable cross-linked micelles for the delivery of cisplatin: A comparison with nondegradable cross-linker

Article abstract of DOI:10.1021/cm301556b

Well-defined and nontoxic cross-linked polymeric micelles, containing either permanent or acid degradable cross-linkers, were employed for efficient intracellular delivery of cisplatin. The self-assembled structures were generated from triblock copolymers of poly(oligo(ethylene glycol) methylether methacrylate)-block-poly(N-hydroxysuccinic methacrylate)-block-poly(1,1-di-tert-butyl 3-(2-(methacryloyloxy)ethyl) butane-1,1,3-tricarboxylate) (POEGMEMA-b-PNHSMA-b-PMAETC) loaded with cisplatinum. The polymeric micelles were subsequently cross-linked via a reaction between pendant activated esters at the nexus core of the triblock copolymer using acid degrdabale ketal diamino cross-linkers. An in vitro study confirmed that both uncross-linked and cross-linked micelles prior to the loading of the platinum drug were nontoxic against OVCAR-3 cells even at high polymer concentration (around 300 ?g mL?1). The drug loaded cross-linked platinum polymeric micelles were superior to the uncross-linked platinum polymeric micelles in terms of cytotoxicity against OVCAR-3, due to a higher cellular uptake. Although there was no significant difference in cytotoxicity of cross-linked platinum polymeric micelles using different cross-linkers (permanent and acid cleavable) after 72 h of exposure, the difference was noticeable after 24 h of incubation, highlighting a much higher activity for acid degradable crosslinked micelles with conjugated platinum drugs. Moreover, the clonogenic assay suggested that cross-linked micelle loaded platinum drugs, in contrast to uncross-linked micelles, can effectively inhibit the OVCAR-3 cell regrowth for an extended period of time (10 days), even at very low micellar concentrations. In summary, acid degradable linkers ensure high cellular uptake compared to uncross-linked micelles but also lead to a faster drug action in comparison to a permanently cross-linked micelle.

Full text of DOI:10.1021/cm301556b

Article
pubs.acs.org/cm
Acid Degradable Cross-Linked Micelles for the Delivery of Cisplatin:
A Comparison with Nondegradable Cross-Linker
Vien T. Huynh,†,‡ Sandra Binauld, Paul L. de Souza, and Martina H. Stenzel *
†
‡
†,
†
Centre for Advanced Macromolecular Design (CAMD), The University of New South Wales, Sydney NSW 2052, Australia
‡
Liverpool Hospital Clinical School and Molecular Medicine Research Group, University of Western Sydney, Sydney NSW 2170,
Australia
*
S Supporting Information
ABSTRACT: Well-defined and nontoxic cross-linked poly-
meric micelles, containing either permanent or acid degradable
cross-linkers, were employed for efficient intracellular delivery
of cisplatin. The self-assembled structures were generated from
triblock copolymers of poly(oligo(ethylene glycol) methyl-
ether methacrylate)-block-poly(N-hydroxysuccinic methacry-
late)-block-poly(1,1-di-tert-butyl 3-(2-(methacryloyloxy)ethyl)
butane-1,1,3-tricarboxylate) (POEGMEMA-b-PNHSMA-b-
PMAETC) loaded with cisplatinum. The polymeric micelles
were subsequently cross-linked via a reaction between pendant
activated esters at the nexus core of the triblock copolymer
using acid degrdabale ketal diamino cross-linkers. An in vitro study confirmed that both uncross-linked and cross-linked micelles
prior to the loading of the platinum drug were nontoxic against OVCAR-3 cells even at high polymer concentration (around 300
−1
μg mL ). The drug loaded cross-linked platinum polymeric micelles were superior to the uncross-linked platinum polymeric
micelles in terms of cytotoxicity against OVCAR-3, due to a higher cellular uptake. Although there was no significant difference in
cytotoxicity of cross-linked platinum polymeric micelles using different cross-linkers (permanent and acid cleavable) after 72 h of
exposure, the difference was noticeable after 24 h of incubation, highlighting a much higher activity for acid degradable cross-
linked micelles with conjugated platinum drugs. Moreover, the clonogenic assay suggested that cross-linked micelle loaded
platinum drugs, in contrast to uncross-linked micelles, can effectively inhibit the OVCAR-3 cell regrowth for an extended period
of time (10 days), even at very low micellar concentrations. In summary, acid degradable linkers ensure high cellular uptake
compared to uncross-linked micelles but also lead to a faster drug action in comparison to a permanently cross-linked micelle.
KEYWORDS: platinum drugs, polymeric micelles, cross-linked micelles, acid degradable nanoparticles, cellular uptake
INTRODUCTION
accumulation in vascularized solid tumors due to enhanced
■
19
permeability and retention (EPR) effect. Furthermore, the
surface of nanoparticles can be modified to prevent the
recognition by the reticuloendothelial system (RES) even
cis-Diaminedichloroplatinum(II) (cisplatin, CDDP) is one of
the oldest chemotherapy drugs available and has been in
widespread use to treat a wide range of solid tumors including
ovarian, cervical, head and neck, and non-small-cell lung
though the RES can detect such polymeric micelles and
1
−8
19−21
cancer. Treatment is usually limited, however, by side-effects
eliminate them from blood circulation.
An ideal drug
1
,9
such as nephrotoxicity, emetogenesis, and neurotoxicity.
Inside cells, cisplatin undergoes aquation to form [Pt(NH ) Cl-
delivery system should have advantages such as high stability in
the bloodstream, effective protection of drugs from unwanted
and nonspecific binding, and triggered release of loaded drugs
inside targeting cells. On the other hand, polymeric micelles are
based on a dynamic equilibrium, with a tendency to dissociate
3
2
+
2+
(
OH )] and [Pt(NH ) (OH ) ] . Its platinum atom binds
2 3 2 2 2
7
covalently to the N position of purines to form 1,2- or 1,3-
intrastrand cross-links and interstrand cross-links. These
cisplatin−DNA adducts result in various cellular responses
including replication arrest, transcription inhibition, cell-cycle
at low concentrations, especially upon intravenous admin-
22,23
istration.
This leads to the low delivery efficiency in
1
,10
arrest, DNA repair, and apoptosis.
targeting sites and loss of EPR effects. Therefore, stabilization
Recent advances in functional nanoparticles self-assembled
from amphiphilic block copolymers have led to the develop-
ment of smart nanocarriers that can enhance the delivery
efficiency of anticancer drugs, proteins, genes, and imaging
of polymeric micelles becomes an important strategy that has
1
8,24−29
recently attracted a vast number of studies.
7
,11−18
agents.
Polymeric micelles encapsulation of drugs, in
Received: May 20, 2012
Revised: July 16, 2012
Published: July 20, 2012
contrast to administration of free drugs, allows an increased
circulation time in the bloodstream and an effective
©
2012 American Chemical Society
3197
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Chemistry of Materials
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Scheme 1. Synthetic Scheme of Acid Degradable Cross-Linked Nanoparticles Containing Cisplatinum Drugs
Although it has been reported in our previous studies that
more stable polymeric micelles were observed with longer
Numerous different cross-linking techniques have been
2
8,31
investigated using a range of different chemistries.
Although
13
hydrophobic block length or with longer alkyl spacer chain
micelles are commonly cross-linked via the addition of a
bifunctional reactive agent, cross-linking can in addition be
achieved using a radical pathway following the addition of a
11
(
distance between polymer backbone and cisplatinum drugs),
they are not stable enough to tolerate the massive dilution
involved by intravenous administration of drugs. In this
scenario, cross-linking is a powerful tool to stabilize self-
assembled structures. The high structural integrity can ensure
high cellular uptake even at the lowest concentration, resulting
1
6,18,32,33
divinyl cross-linker.
Cross-linking of micelles can be
26
carried out by different ways such as core cross-linking, shell
3
4,35
36,37
cross-linking,
and nexus cross-linking.
Of these
techniques, cross-linking on the nexus, that is, at the interface
between core and shell, is more versatile since it allows the use
of either hydrophilic or hydrophobic cross-linkers. In addition,
it does not limit the loading capacity of the core while the
possibility of intermicellar cross-linking is reduced.
30
in improved IC50 values of the drug. However, cross-linkers
should be able to degrade in the targeted sites because
permanent cross-linking can limit drug release and potentially
the clearance of the drug carrier.
3
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Chemistry of Materials
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Scheme 2. Synthesis of Diamine Acid Degradable Cross-Linker
Although cross-linking of micelles has numerous benefits, the
ultimate degradation of a stable nanoparticle would be desirable
to enable its clearance from the body. An added benefit would
be the triggered degradation inside the micelle, which
simultaneously leads to a burst in drug release. The differences
of pH in intracellular endosomes (pH 5.0−5.5), lysosomes (pH
2,2-Azobisisobutyronitrile (AIBN; Fluka, 98%) was purified by
recrystallization from methanol. Oligo(ethylene glycol) methylether
−1
methacrylate (OEGMEMA; MW = 300 g mol ; Aldrich) was
deinhibited by passing through a column of basic aluminum oxide.
The RAFT agent 2-(2-cyanopropyl)dithiobenzoate (CPDB) was
42,43
synthesized according to literature
and recrystallized from toluene
to yield fine pink powder. Deionized (DI) water produced by a Mili-Q
water purification system has a resistivity of 17.9 mΩ/cm.
3
8
4
.0−4.5), and extracellular compartments (pH 7.4) can act as
a trigger for intracellular drug release, if an acid cleavable cross-
linker is used in the system. For instance, acid degradable
acetal-type cross-linkers such as 3,9-divinyl-2,4,8,10-
Synthesis. Synthesis of Acid-Degradable Amine Bearing Cross-
Linker. Compound X in Scheme 2 was synthesized as described
previously with some minor modifications. In a round-bottomed flask,
N-(2-hydroxyethyl)phthalimide (3.0 g, 15.7 mmol) was dissolved in
anhydrous DMF (40 mL), and p-toluenesulfonic acid (30 mg, 0.157
mmol) was added. The solution was cooled to 0 °C using an ice bath,
and 2-methoxypropene (1.5 mL, 15.7 mmol) was carefully added to
the solution. The reaction mixture was then stirred for 1 h while
keeping the temperature at 0 °C to avoid loss of the highly volatile 2-
methoxy propene. The flask was then connected to a trap, and the
reaction mixture was heated to 45 °C under high vacuum for 6 h to
remove the methanol formed during the reaction. Finally, the reaction
mixture was cooled to room temperature and triethylamine (4 mL)
was added to quench the reaction. To facilitate further purification,
acetic anhydride (0.8 mL) was added to convert any unreacted alcohol
groups into the corresponding acetate and the reaction mixture was
allowed to stir overnight. DMF was then removed under reduced
pressure, resulting in colorless crystals that were washed three times
with ethyl acetate, yielding a thin white powder. Yield: 48%.
39
tetraoxaspiro[5.5]undecance and di(2-acryloyloxyethyoxy)-4-
18
hydroxyphenylmethane have been utilized to produce cross-
linked micelles. These micelles appear to be stable at
physiological pH but quickly degrade into unimers at low pH
values. Another example of acid-labile linkage is the reaction of
a benzaldehyde and a primary amine to generate “stealth”
polycationic micelles that are stable at a high pH environment
40
but can disassociate at endosomal pH (5.0−6.0). Imine-type
cross-linker can further lead to the “burst” release of
41
doxorubicin (DOX) at endosomal pH.
Herein, we describe the synthesis of cross-linked polymeric
micelles carrying platinum drugs using an acid-cleavable ketal
diamine cross-linker. The polymeric micelles were obtained by
self-assembly of the triblock copolymer prepared via RAFT
polymerization. Incorporation of a block with an activated ester
as a pendant group between the shell and core-forming blocks
allows cross-linking at the interface between the hydrophobic
core and the hydrophilic shell (Scheme 1). In addition, a
permanent cross-linker was used for comparison to evaluate the
effect of the degradability on drug release and in vitro studies.
1
H NMR (300.17 MHz, CDCl , 25 °C): δ (ppm, from CHCl
3
3
signal at 7.26 ppm) 1.26 (6H, s, CH −C), 3.60 (4H, t, CH −O), 3.82
3
2
(
4H, t, CH N), 7.4−7.8 (10H, dt, C H ).
2
6
5
13
C NMR (75.48 MHz, CDCl3, 25 °C): δ (ppm from TMS) 170.1+,
132.2, 123.2, 113.7, 58.0.1, 42.9, 26.5. ESI-MS. Calcd [M + H]
+
(
C H N O ) m/z = 422.15; found [M + Na] = 445.3.
23 22 2 6
1
Compound Y (1 g, 2.4 mmol) was deprotected in 6 M NaOH (6
mL) by heating the reaction mixture at reflux overnight. The product
H NMR spectroscopy, dynamic light scattering (DLS),
transmission electron microscopy (TEM), and size exclusion
chromatography (SEC) were employed to characterize the
cross-linked micelles. The differences between uncross-linked
micelles and cross-linked micelles using both cross-linkers were
investigated in the light of the drug release, cytotoxicity, cellular
uptake, and colony formation.
was extracted with CHCl /iPrOH (1/1) mixture three times, and the
3
fractions were combined and dried over anhydrous MgSO . The
4
organic layer was filtered and evaporated in vacuo. Yield: 78%.
1
H NMR (300.17 MHz, CDCl , 25 °C): δ (ppm, from CHCl
3
3
signal at 7.26 ppm) 1.36 (6H, s, CH −C), 1.57 (bs, 4H, NH ), 2.84
3
2
(4H, t, CH −NH ), 3.46 (4H, t, CH −O).
2
2
2
13
C NMR (75.48 MHz, CDCl , 25 °C): δ (ppm from TMS) 113.7,
3
6
4.2, 42.9, 26.5.
EXPERIMENTAL PART
■
Synthesis of Monomers with Pendant Carboxylic Functional
Materials. Unless otherwise specified, all chemicals were reagent
grade and were used as received: Di-tert-butyl malonate (Aldrich,
8%), ethylene glycol dimethacrylate (EGDMA, Aldrich, 98%),
potassium carbonate (Univar, anhydrous), 18-crown-6 (Sigma-Adrich,
9%), tetrahydrofuran (THF, anhydrous, 98%, Aldrich), diethyl ether
Et O anhydrous, Ajax Finechem, 99%), petroleum ether (BR 40−60
C; Ajax Finechem, 90%), ethyl acetate (ETOAc, Ajax Finechemicals,
9.5%), N,N-dimethylacetamide (DMAc; Aldrich, HPLC grade),
magnesium sulfate (Ajax Finechem, 70%), toluene (Aldrich; purum),
,4-dioxane (Sigma-Aldrich, 99%), dichloromethane (DCM) (Ajax
Groups (1,1-Di-tert-butyl 3-(2-(methacryloyloxy)ethyl) Butane-
1,1,3-tricarboxylate MAETC). 1,1-Di-tert-butyl 3-(2-
(methacryloyloxy)ethyl) butane-1,1,3-tricarboxylate (MAETC) mono-
mer (Scheme 3) was synthesized according to the previous
9
1
1,13
1
9
(
°
9
publications.
The purified monomer was characterized by
and1 C NMR spectroscopy, DEPT-135, DEPT-90, and ESI-MS.
H NMR (300.17 MHz, CDCl , 25 °C): δ (ppm) = 6.12 (s,
1H,H ), 5.58 (s, 1H, H ); 4.34 (t, 4H, H and H , J = 1.08 Hz); 3.2−
3.25 (dd, 1H, H , J = 6.45 Hz, J = 8.91 Hz); 2.45−2.57 (qq, 1H, H , J
= 6.99 Hz); 2.15 (dd, 1H, H , J = 6.54 Hz); 1.9 (dd, 4H, H and H , J
H
13
2
3
1
2
4
5
9
6
1
8
8
3
Finechem, 99%), trifluoroacetic acid (Sigma-Aldrich, 99%), chloro-
= 6.12 Hz); 1.43 (s, 18H, H ); 1.18 (d, 3H, H , J = 7.02 Hz).
10 7
13
form-d (CDCl ; Cambridge Isotape Laboratories), cis-
C NMR (75.48 MHz, CDCl3, 25 °C): δ (ppm) = 175.37 (Cg),
168.41 (C ), 168.21 (C ), 166.99 (C ), 135.79 (C ), 126.01 (C ),
3
dichlorodiaminoplatinum(II) (CDDP; Sigma-Aldrich; 99.9%), N-
succinimidyl methacrylate (NHSMA, Aldrich, 99%), N-methyl-2-
pyrrolidone (NMP, Sigma-Aldrich, 98%), 2-[2-(2-aminoethoxy)-
ethoxy]ethan-1-amine (Sigma Aldrich, 97%, HPLC grade), and p-
toluenesulfonic acid (Sigma-Aldrich, 98.5%).
n
p
d
c
a
81.50 (C ), 62.33 (C ), 61.99 (C ), 51.64 (C ), 37.08 (C ), 31.96
o
e
f
m
h
(C ), 27.79 (C ), 18.18 (C ), 17.30 (C ).
l
q
b
k
ESI-MS: theoretical m/z for C H O , 414.23; experimental m/z
21
34
8
+
437.1 (Na ).
3
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Chemistry of Materials
Scheme 3. MAETC Label for H NMR (left) and 13C NMR
Article
1
1
H NMR (300.17 MHz, CDCl , 25 °C) δ (ppm) = 4.2−4.0 (2nH,
3
CH COOCH CH O), 4.0−3.8 (11nH, CH of OEGMEMA mono-
(
right)
3
2
2
2
mer), 3.6−3.4 (3nH, CH of chain end of OEGMEMA), 3.0−2.8
3
(
4aH, CH of NHS), 2.4−2.2 (3aH, CH of NHSMA), 1.4−1.2 (3nH,
2
3
CH of the main chain), 1.2−0.9 (2nH + 2aH, CH of the main chain)
3
2
(
Figure 1). n and a are the degrees of polymerization (DP ) of
n
POEGMEMA and PNHSMA, respectively.
Synthesis of Triblock Copolymer Poly(oligo(ethylene glycol)
methylether methacrylate)-block-poly(N-hyroxysuccinic methacry-
late)-block-poly(1,1-di-tert-butyl 3-(2-(methacryloyloxy)ethyl) bu-
tane-1,1,3-tricarboxylate) (POEGMEMA-b-PNHSMA-b-PMAETC).
The triblock copolymer was prepared by chain extension of the
diblock copolymer POEGMEMA-b-PNHSMA (M
= 19 934 g
n(theo)
−
1
−1
mol , M
= 19 200 g mol , PDI = 1.24) using MAETC as a
n(SEC)
−4
monomer. Briefly, in a glass vial, MAETC (0.06 g, 1.4 × 10 mol),
−
6
POEGMEMA-b-PNHS-MA (0.029 g, 1.4 × 10
mol) as a
−4
−7
macroRAFT agent, and AIBN (0.5 × 10 g, 2.89 × 10 mol)
were dissolved in 0.29 mL of dioxane to result in [MAETC]/
−1
[
macroRAFT]/[AIBN] = 100:1:0.2 and [MAETC] = 0.5 mol L .
The vial was capped with a rubber septum and copper wire. It was
then deoxygenated using nitrogen purging for 45 min and then placed
in a preheated oil bath at 70 °C. The sample was taken after 2.5 h of
polymerization. In order to quench the polymerization reaction, the
vial was placed in an ice bath for 5 min. The final copolymer was
Polymer Synthesis. RAFT Polymerization of OEGMEMA Using
1
characterized by SEC (DMAc) and H NMR.
−2
CPADB RAFT Agent. OEGMEMA (2.5 g, 0.83 × 10 mol), CPADB
1
H NMR (300.17 MHz, CDCl , 25 °C): δ (ppm) = 4.4−4.2 (4mH,
−
4
−5
3
(
0.023 g, 0.83 × 10 mol), and AIBN (0.0027 g, 1.66 × 10 mol)
OCH CH O), 4.2−4.0 (2nH, CH COOCH CH O), 4.0−3.8 (11nH,
2
2
3
2
2
were dissolved in acetonitrile (16.6 mL) in a glass bottle equipped with
CH of OEGMEMA monomer), 3.6−3.4 (3nH, CH of chain end of
2
3
a magnetic stirrer bar to give [OEGMEMA]/[CPADB]/[AIBN] =
OEGMEMA), 3.3−3.1 (mH, tert-Bu-OOCCHCH COO-Bu-tert),
−1
2
1
00:5:0.2 and [OEGMEMA] = 0.5 mol L . The bottle was then
3
2
.0−2.8 (4aH, CH of NHS), 2.8−2.6 (mH, OOCCHCH −CH2),
2
3
sealed with a rubber septum and thoroughly deoxygenated using
nitrogen purging for 30 min in an ice bath before being placed in a
preheated oil bath at 70 °C. After 5.5 h, the polymerization was
terminated by placing the sample in an ice bath for 5 min. Acetonitrile
was removed under reduced pressure, and the concentrated reaction
mixture was purified by dialysis against methanol to remove any traces
of monomer and unreacted reactants using a membrane with
molecular weight cutoff of 3500 Da. The solvent was evaporated,
. 4− 2. 2 ( 3 aH, CH o f NHS M A) , 2. 3−2. 0 (mH,
3
OOCCHCH CH CH(COO-Bu-tert) ), 2.0−1.6 (mH+3mH,
3
2
2
OOCCHCH CH CH(COO-Bu-tert) and CH3 attached to the
3
2
2
backbone of MAETC), 1.6−1.3 (18mH, OOCCHCH CH CH(COO-
3
2
(
2
CH ) ) ), 1.4−1.2 (3nH, CH of the main chain), 1.2−0.9 (2nH +
3
3
2
3
aH + 2mH, CH of the main chain) (Figure 1). n, a, and m are the
2
degrees of polymerization (DP ) of POEGMEMA, PNHSMA, and
n
1
PMAETC, respectively.
and the final polymer was characterized by H NMR and SEC
1
The conversion of 40% was observed after 2.5 h polymerization.
(
(
DMAc). H NMR (300.17 MHz, CDCl , 25 °C) δ (ppm) = 4.2−4.0
3
−1
The molecular weight (Mn(SEC)) was 37 550 g mol and PDI was 1.29
2nH, CH COOCH CH O), 4.0−3.8 (11nH, CH of OEGMEMA
3
2
2
2
measured by SEC (DMAc) while the theoretical molecular weight
monomer), 3.6−3.4 (3nH, CH of chain end of OEGMEMA), 1.4−1.2
3
−1
1
(
Mn(theo)) was 28 600 g mol using H NMR to quantify the ratios
(
(
3nH, CH of the main chain), 1.2−0.9 (2nH, CH of the main chain)
3
2
between blocks.
Figure 1). n is the degree of polymerization (DP ) of POEGMEMA.
n
Deprotection of Block Copolymers. Deprotection of the tert-butyl
The conversion of 59% was obtained after 5.5 h polymerization. The
molecular weight of the POEGMEMA macroRAFT agent was
groups of POEGMEMA-b-PNHSMA-b-PMAETC was carried out
1
1,13
−1
similar to the procedure described in previous publications.
Briefly,
measured to be 14 200 g mol (PDI = 1.16) by SEC (DMAc),
the triblock copolymer (0.2 g) was fully dissolved in dichloromethane
(DCM, 0.5 mL). Trifluoroacetic acid (3 equiv compared to tert-butyl
group) was then added in the polymer mixture, followed by stirring at
room temperature for 72 h. The final reaction mixture was
subsequently dialyzed against acetone−water (1:1) using a tubular
membrane with 3500 Da as molecular weight cutoff (MWCO).
Acetone was then removed by dialysis against DI water. Subsequently,
the remaining solution inside the bag was freeze-dried to yield a waxy
which is in good agreement with the theoretical molecular weight
Mn(theo) = 17 921 g mol .
−1
Synthesis of Poly(oligo(ethylene glycol) methylether methacry-
late)-block-poly(N-hydroxysuccinic methacrylate) (POEGMEMA-b-
PNHSMA). POEGMEMA-b-PNHSMA was prepared by the chain
−
1
extension of POEGMEMA (M
2
= 17 921 g mol , M
= 14
n(theo)
n(SEC)
−1
00 g mol , PDI = 1.16) using N-hydroxysuccinic methacrylate
(
NHS-MA) as a monomer. Briefly, in a glass vial, the POEGMEMA
−4
1
macroRAFT agent (0.5 g, 0.28 × 10 mol), NHS-MA (0.26 g, 0.14 ×
1
polymer. The deprotected copolymer was characterized by H NMR.
−
2
−5
1
0
mol), and AIBN (0.0009 g, 0.56 × 10 mol) were dissolved in
H NMR (300.17 MHz, CDCl
CH O), 4.2−4.0 (2nH, CH
of OEGMEMA monomer), 3.6−3.4 (3nH + mH, CH
end of OEGMEMA and tert-Bu-OOCCHCH COO-Bu-tert), 3.0−2.8
(4aH, CH of NHS), 2.8−2.6 (mH, OOCCHCH −CH2), 2.4−2.2
(3aH, CH of NHSMA), 2.3−2.0 (mH, OOCCHCH CH CH(COO-
Bu-tert) ), 2.0−1.6 (mH+3mH, OOCCHCH CH CH(COO-Bu-tert)
and CH attached to the backbone of MAETC), 1.4−1.2 (3nH, CH
3
, 25 °C): δ (ppm) = 4.4−4.2 (4mH,
COOCH CH O), 4.0−3.8 (11nH,
of chain
2
.78 mL of N-methyl-2-pyrrolidone (NMP) to lead to [NHS-MA]/
OCH
CH
2
2
3
2
2
−1
[
MacroRAFT]/[AIBN] = 50:1:0.2 and [NHS-MA] = 0.5 mol L .
2
3
The vial was capped with a rubber septum and copper wire. The vial
was then deoxygenated using nitrogen purging for 30 min and placed
in a preheated oil bath at 70 °C. The polymerization was terminated
by placing it in an ice bath for 5 min. The polymerization solution was
precipitated twice in cold diethyl ether to yield pink brittle polymer.
2
2
3
3
3
2
2
3
2
2
3
3
1
The final polymer was analyzed by SEC (DMAc) and H NMR. The
of the main chain), 1.2−0.9 (2nH + 2aH + 2mH, CH of the main
chain) (Figure 1). n, a, and m are the degrees of polymerization (DP )
2
conversion of 22% was observed after 2 h of polymerization. The
n
molecular weight (Mn(SEC)) of POEGMEMA-b-PNHS-MA was 19 200
of POEGMEMA, PNHSMA, and PMAETC, respectively.
Polymer−Platinum Conjugates. Conjugation of cis-dichlorodiami-
no platinum(II) to malonic acid of the deprotected triblock copolymer
−1
g mol and PDI was 1.24 measured by SEC (DMAc). This molecular
weight was in good agreement with the theoretical value (M = 19
n(theo)
−1
9
34 g mol ), which was calculated using the conversions obtained by
POEGMEMA-b-PNHSMA-b-PMAETC was prepared following the
1
11,13
H NMR.
method described in previous publications.
In a typical experi-
3
200
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Chemistry of Materials
Article
ment, CDDP (10 mg) was suspended in 10 mL of distilled water and
times with 2% aqua regia (HCl/HNO = 3:1). The solution was then
3
mixed with silver nitrate ([AgNO ]/[CDDP] = 1.955) to form the
digested at 60 °C for 3 h and then cooled to room temperature. The
concentration of Pt released from the conjugate was expressed as a
ratio of the amount of platinum in the releasing solution (the solution
outside the dialysis membrane) and that in the initial sample. The
percentage of Pt released was calculated using the equation below:
3
aqueous complex. The solution was stirred in the dark at room
temperature for 4 h. White precipitate of silver chloride was observed
indicative of the proceeding reaction. The mixture was then
centrifuged at 9000 rpm for 20 min to remove the AgCl precipitates,
and the supernatant was purified by passing it through a 0.22 μm filter.
Polymers with carboxyl functional groups (25 mg, dissolved in 2 mL of
Release = Vtotal
(t) × C + Y
%
−1
NaOH (1 mg mL ) to be deprotonated) were added to the above
prepared CDDP aqueous solution and left to react in a water bath at
Z
where Vtotal(t) is the remaining volume in the releasing container at
time t in mL; C is the concentration of platinum determined from
37 °C for 12 h with gentle shaking The prepared conjugate was
purified by ultrafiltration using Sartorius Vivaspin 6 centrifugal filter
devices with a molecular weight cutoff of 3000 Da, followed by freeze-
drying yielding a yellow powder.
−1
ICP-MS in μg mL ; Y is the amount of platinum that has already been
collected in μg; and Z is the total amount of platinum at t = 0 present
in the dialysis bag in μg.
Self-Assembly of Triblock Copolymer and Platinum Drug
Conjugates into Micellular Structure. The platinum drug conjugated
POEGMEMA-b-PNHSMA-b-PMAETC (60 mg) was dissolved in
DMF (3 mL), which is a good solvent for the hydrophobic and the
hydrophilic block. Distilled water (10 mL) was added dropwise using a
Cell Culture. The human ovarian cancer cell line OVCAR-3 was
−3
−1
grown in RPMI-1640 [2 × 10 M glutamine, 1.5 g L sodium
bicarbonate, 0.010 M 2-hydroxyethylpiperazinesulfonic acid (HEPES),
−1
−3
4
.5 g L
glucose, and 10
M sodium pyruvate] medium
supplemented with 10% fetal bovine serum (FBS). The cells were
−1
syringe pump (3 mL h ) to 3 mL of conjugate in DMF (20 mg
grown in a 5% CO atmosphere in an incubator at 37 °C.
2
−1
mL ) under moderate stirring at room temperature. The mixture was
Cytotoxicity Assay. The sulforhodamine B (SRB) assay established
by the U.S. National Cancer Institute for rapid, sensitive, and
inexpensive screening of antitumor drugs in microplates was employed
to screen the cytotoxicity and antitumor activities of polymers and
then dialyzed against DI water for 24 h using a dialysis membrane
(
MWCO 3500 Da) to remove DMF. The targeted final polymer
−1
concentration was 5 mg mL . Care needs to be taken to avoid the
hydrolysis of the NHS ester by carefully monitoring the pH value of
the solution.
47
polymeric platinum drugs, respectively. Human ovarian cancer cells
(
OVCAR-3) diluted in 100 μL of RPMI-1640 medium (2 mM L-
Synthesis of Cross-Linked Micelles. In a typical experiment, a
−1
−1
glutamine, 1.5 g L sodium bicarbonate, 10 mM HEPES, 4.5 g L
−1
polymer solution (5 mg mL ; 4 mL) in distilled water was employed
to perform the cross-linking process. The synthesized acid degradable
amine-bearing cross-linker (compound Y or 2-[2-(2-aminoethoxy)-
glucose, 1 mM sodium pyruvate) were seeded into the wells at a
concentration of 3000 cells/well. The microtiter plates were incubated
for 24 h at 37 °C and then exposed to various doses of polymers and
micelles for 24 and 72 h before being assayed for cell growth
inhibition. Cell cultures were fixed with TCA (10%, w/v) and
incubated at 4 °C for 1 h. The wells were then washed five times with
tap water to remove TCA, growth medium, and low molecular weight
metabolites. Plates were air-dried and then stored until use. TCA-fixed
cells were stained for 30 min with 0.4% (w/v) SRB dissolved in 1% (v/
v) acetic acid. At the end of the staining period, SRB was removed and
cultures were quickly rinsed five times with 1% (v/v) acetic acid to
remove unbound dye. Subsequently, the cultured plates were air-dried
until no conspicuous moisture was visible before bound dye was
shaken in 100 μL of 10 mM Tris base for 5 min. The absorbance at
−
3
ethoxy]ethan-1-amine, 0.5 mg, 3.3 × 10 mmol) was added, and the
mixture was then stirred at an ambient temperature overnight. The
cross-linked nanoparticles were purified using membrane dialysis
(
MWCO 3500 Da) for 48 h against distilled water to remove the
unreacted cross-linker. A part of the solution was freeze-dried and
characterized by SEC (DMAc) and DLS to confirm the cross-linking
process. In this study, two types of cross-linkers including 2-[2-(2-
aminoethoxy)ethoxy]ethan-1-amine and acid degradable amine-
bearing cross-linker were used.
Critical Micelle Concentration. The critical micelle concentration
(
CMC) was measured by fluorescence spectroscopy using pyrene as a
44
fluorescent probe following an established procedure. Briefly, a stock
solution of pyrene was made by dissolving pyrene (1 mg, 5 μmol) in
5
70 nm of each well was measured using microtiter plate reader
scanning spectrophotometer (BioTek’s PowerWave HT Microplate
Reader and KC4 Software). Each sample was replicated three times.
Cellular Uptake. Cellular uptake experiments were performed
according to a previously described method with some modifica-
−5
acetone (200 mL) to form a 2.5 × 10 M solution. The pyrene
solution (48 μL) was dropped into empty vials, and the acetone was
evaporated overnight under reduced pressure. A stock solution with
micelles and cross-linked micelles was serially diluted with deionized
4
8
3
tions. OVCAR-3 cells were seeded into a 12-well plate at 16 × 10
−1
water starting with the concentration of 0 to 100 μg mL . Each
polymer solution (2 mL) was transferred to a vial containing pyrene
and stirred overnight. The final concentration of pyrene in the polymer
cells per well and incubated for 24 h. The cells were treated with
polymer−Pt micelles including uncross-linked micelles and cross-
linked micelles using different cross-linkers. After 24 h incubation at 37
°C, the medium was removed and rinsed with cold PBS (1 mL × 3).
−7
solutions was 6 × 10 M (which is less than the pyrene saturation
45
concentration in water). Fluorescence measurements were carried
out using an excitation wavelength of λ = 237.96 nm, using a 2.5 nm
slit width for excitation and a 2.5 nm slit width for emission. Emission
wavelengths were scanned from 350 to 450 nm. The intensities of I1
The cells were trypsinized and incubated with HNO (68%, v/v) at 65
3
°C for 20 h.
Platinum content uptake was determined using inductively coupled
plasma mass spectrometer (ICP-MS). A four-point standard curve was
plotted between intensity versus a serial dilution of a certified reference
standard ranging from 1 to 1000 ppb. The reported result of the
sample is the average of three replicates.
(
372 nm) to I (383 nm) vibronic bands were evaluated for each
3
sample, and the ratio of these were plotted against the log of the
4
6
concentration of each polymeric sample. The critical micelle
concentration (CMC) was taken as the intersection of two regression
lines calculated from the linear portions of the graphs.
The amount of polymer taken up by the cells was qualitatively and
quantitatively determined using fluorescence microscopy and fluo-
rescence reader, respectively. To label polymers, fluorescein-o-
methacrylate (2% mol of POEGMEMA) was copolymerized with
OEGMEMA before chain extending with NHS-MA and then with the
synthesized monomers such as MAETC to yield triblock copolymer.
The block copolymer was conjugated to CDDP and formed micelles
following the procedure mentioned above. OVCAR3 was seeded into
12 well plates at a concentration of 16 000 cells per well and incubated
for 24 h. Those cells were subsequently treated with the fluorescein-
labeled micelles for 2 and 15 h at 37 °C. All cells were washed 3 times
with PBS before observing under microscopy. Cell uptake pictures
Release of Cisplatin from Micelles. Micelles and cross-linked
micelles (2 mL) containing a platinum drug were dialyzed against pH
buffer solutions (pH 7.4 and 5.5) (200 mL) at 37 °C. 0.9% of NaCl
was added into these buffer solutions to trigger the platinum drug
release. Dialysis membranes with molecular weight cutoff of 3500 Da
were used to allow equilibrium of the free platinum drug from inside
and outside the dialysis membrane. Aliquots of 1 mL were taken at
different times (24 and 72 h). The amount of released Pt was
determined using inductively coupled plasma mass spectroscopy (ICP-
MS). To a 10 mL centrifuge tube, 1 mL of the dialysate was diluted 5
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1
Figure 1. H NMR (acetone-d ) of the macroRAFT agent, the diblock copolymer, the triblock copolymer, and the deprotected triblock copolymer.
6
were acquired by using a fluorescence microscope with mercury lamp
of λex 535 nm and λem 590 nm to track the fluorescence micelles.
Fluorescence intensity of treated cell solution was measured by
fluorescence reader at λ = 535 nm and λ = 590 nm.
sensitive cross-linked platinum polymeric micelles (2.3 μM Pt
−1
[correlative to 6 μg mL Pt−polymer conjugates, <CMC value]
−1
and 32.5 μM Pt [correlative to 85.2 μg mL Pt−polymer conjugates,
>CMC value]) for 72 h and then washed with phosphate buffer
solution (PBS). Single survived cells were then plated in six well plates
with fresh PRMI 1640 medium, and the medium was changed every 3
days. Following 10 days of incubation, the cells were washed twice
with cold PBS and incubated with methanol for 30 min at room
temperature to fix the cells. Methanol was evaporated, and cells were
stained with 0.1% crystal violet for 3 min. The excess crystal violet was
washed five times with tap water and air-dried overnight. The data
were calculated based on eqs 1 and 2.
ex
em
Colony Formation Assay. Unlike the cell proliferation assay, colony
formation assay measures the productive integrity of the cells following
withdrawal of drug treatment. The assays were performed as described
49
by Liebmann et al. with some modifications. Briefly, OVCAR-3 cells
−1
were exposed to triblock copolymer (100 μg mL ), uncross-linked
−1
platinum polymeric micelles (2.1 μM Pt [correlative to 5.5 μg mL
Pt−polymer conjugates, <CMC value] and 34.8 μM Pt [correlative to
−1
9
1.2 μg mL Pt−polymer conjugates, >CMC value]), and acid-
3
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Chemistry of Materials
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Table 1. Triblock Copolymer Synthesis for cis-Platinum Conjugation
Mntheo (g mol−1
)
a
MnSEC (g mol−1
)
a
PDI no. of RU, NOEGMEMA no. of RU, NMANHS no. of RU, NMAETC
polymers
POEGMEMA50
POEGMEMA -b-PMANHS
11
17921
19934
37550
14200
19200
28600
1.16
1.24
59
59
59
11
11
50
POEGMEMA -b-PMANHS -b-PMAETC
40
1.29
40
50
11
a
MnSEC determined by SEC (DMAc) using PSt calibration; Mntheo calculated by H NMR.
1
no. of colonies formed
no. of cells seeded
Inductively Coupled Plasma−Mass Spectrometer (ICP-MS). The
Perkin-Elmer ELAN 6000 inductively coupled plasma−mass spec-
trometer (Perkin-Elmer, Norwalk, CT, USA) was used for quantitative
determinations of platinum. All experiments were carried out at an
incident ratio frequency power of 1200 W. The plasma argon gas flow
Plating efficiency (PE) =
Surviving fraction (SF) =
× 100%
(1)
no. of colonies formed after treatment
−1
−1
no. of cells seeded × PE
of 12 L min with an auxiliary argon flow of 0.8 L min was used in
all cases. The nebulizer gas flow was adjusted to maximize ion intensity
(2)
−1
at 0.93 L min as indicated by the mass flow controller. The element/
Analyses. Size Exclusion Chromatography (SEC). SEC was
implemented using a Shimadzu modular system comprising a DGU-
2A degasser, a LC-10AT pump, aSIL-10AD automatic injector, a
195
193
mass detected was Pt, and the internal standard used was Ir. The
replicate time was set to 900 ms and the dwell time to 300 ms. Peak
hopping was the scanning mode employed, and the number of
sweeps/readings was set to 3. Ten replicates were measured at a
normal resolution. The samples were treated with aqua regia solution
at 90 °C for 2 h to digest platinum.
1
CTO-10A column oven, a RID-10A refractive index detector, and a
SPD-10A Shimadzu UV/vis detector. A 50 × 7.8 mm guard column
3
4
5
and four 300 × 7.8 mm linear columns (500, 10 , 10 , and 10 Å pore
size, 5 μm particle size) were used for the analysis. N,N′-
dimethylacetamide (DMAc) (HPLC grade, 0.05% w/v of 2,6-
dibutyl-4-methylphenol (BHT), 0.03% w/v of LiBr) with a flow rate
RESULTS AND DISCUSSION
■
−1
of 1 mL min and a constant temperature of 50 °C was used as the
mobile phase with an injection volume of 50 μL. The samples were
filtered through 0.45 μm filters. The unit was calibrated using
commercially available linear polystyrene standards (0.5−1000 kDa,
Polymer Laboratories). Chromatograms were processed using Cirrus
Synthesis of POEGMEMA-b-PNHSMA-b-PMAETC Tri-
block Copolymer via RAFT Polymerization. The main
focus of this work was to investigate any measurable benefits on
cell growth inhibition when using an acid degradable cross-
linker compared to a permanent cross-linker. The present work
follows on from a series of previous studies, in which novel
dicarboxylato ligands were synthesized for effective cis-platinum
2
.0 software (Polymer Laboratories).
Nuclear Magnetic Resonance (NMR) Spectroscopy. H and 13C
1
NMR spectra were recorded using a Bruker ACF300 (300 MHz)
spectrometer, using (CD ) SO, CD OD, or CDCl as solvents. All
13
3
2
3
3
drug conjugation showing that increased spacer length had a
chemical shifts are stated in ppm (δ) relative to tetramethylsilane (δ =
favorable influence on micelle stability, cellular uptake, and
0
ppm), referenced to the chemical shifts of residual solvent
1
1
1
13
1
anticancer activities. Cross-linking of the micelle was
considered therefore the next logical step. In order to prepare
cross-linked micelles, the triblock copolymer, POEGMEMA-b-
PNHSMA-b-PMAETC, was synthesized via RAFT polymer-
ization. Poly(oligoethylene glycol methacrylates) [POEGME-
MA] homopolymer was first synthesized in acetonitrile using 2-
resonances ( H and C). The number of scan of H NMR was 16
195
195
as default for all polymer samples. For Pt NMR measurement, Pt
resonances were externally referenced to Na PtCl at 0 ppm. Spectra
2
6
were obtained using a broadband observed 5 mm probe with z-axis
gradient capability. The Bruker pulse program zgmultiscan was
modified to execute a very short delay time (set at d = 2 ms)
followed by a hard 90° pulse. The experiment was run in increments of
0 000 scans (ns = 20 000) over a 130 kHz sweep width (9 ms
acquisition time). A loop counter parameter 13 = 100 was
incorporated such that the initial iteration of 20 000 scans was
repeated 90 times to give an accumulated number of 2 000 000 scans
1
(
2-cyanopropyl) dithiobenzoate (CPDB) as the chain transfer
2
agent and AIBN as an initiator at 70 °C to target the molecular
weight of 14 kDa (Scheme 1). The polymerization was
quenched at a monomer conversion of 60−70% to maintain
high end group fidelity. The conversion of the monomer was
(
FIDs from each iteration were automatically combined and Fourier
transformed to produce the frequency domain spectrum).
1
calculated using H NMR spectroscopy by comparing the
Dynamic Light Scattering (DLS). The average hydrodynamic
intensity of vinyl proton peaks (6.1 and 5.6 ppm) to the
aliphatic proton peaks (1.1−1.3 ppm).
diameters D and size distributions of the prepared micelle solution in
h
−1
an aqueous solution (1 mg mL ) were measured using a Malvern
ZetasizerNano ZS instrument equipped with a 4 mV He−Ne laser
operating at λ = 632 nm, an avalanche photodiode detector with high
quantum efficiency, and an ALV/LSE-5003 multiple tau digital
correlator electronics system. The samples were filtered to remove
dust using a microfilter (0.45 μm) prior to measurement.
The homopolymer POEGMEMA was then purified by
dialysis against methanol and followed by SEC analysis in
1
1
DMAc and H NMR spectroscopy in acetone-d . H NMR
6
analysis shows the characteristic peaks of POEGMEMA at 4.2,
3
.9−3.5, 3.3, 1.9, and 1.3−0.9 ppm attributed to CH O ester,
2
CH O ether, CH O, CH −C, and CH −C backbone,
respectively (Figure 1). The presence of the RAFT end
group was confirmed by H NMR analysis using the chemical
Transmission Electron Microscopy (TEM). Analyses were per-
formed using a JEOL 1400 TEM with a beam voltage of 100 kV and a
Gatan CCD for acquisition of digital images. Samples were prepared
2
3
3
2
1
−1
by placing a droplet of a 1 mg mL polymer solution on a formamide
and graphite coated copper grid. Excess solution was drained after 60 s
using filter paper. To negatively stain the samples, a droplet of 2% (w/
v) phosphotungstic acid solution was placed on the copper grid for 30
s before being drained with filter paper.
shift at 7.2−8.2 ppm (benzyl group) (Figure 1). The
POEGMEMA macroRAFT agent employed for the block
copolymerization was obtained upon a monomer conversion of
−1
−1
5
0% (Mn(theo) = 17 921 g mol , M
= 14 200 g mol , PDI
n(SEC)
of 1.16).
Thermogravimetric Analysis (TGA). Thermal decomposition
properties of polymers were recorded using a Perkin-Elmer Thermo-
gravimetric Analyzer (Pyris 1 TGA). Measurements were conducted
over a temperature range of 30−700 °C with a programmed
The POEGMEMA macroRAFT was then chain extended
with the activated ester monomer, N-succinimidyl methacry-
lates, at 70 °C for 2 h using N-methyl-2-pyrrolidone (NMP) as
a solvent (Scheme 1). The resulting block copolymer,
−1
temperature increment of 20 K min .
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Chemistry of Materials
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POEGMEMA-b-PMANHS, had a PDI of 1.24 and the
successful chain extension was confirmed by SEC analysis
showing the shift in the SEC traces from low molecular weight
Deprotection and Platinum Drug Conjugation. The
triblock copolymer POEGMEMA-b-PMANHS-b-PMAETC
was deprotected using TFA and purified via dialysis against a
−1
−1
1
(
14 200 g mol ) to higher molecular weight (19 200 g mol ).
mixture of acetone and water (1:1) followed by pure water. H
1
The monomer conversion was determined by H NMR based
on comparison of the intensities of vinyl proton peaks (6.1 and
NMR confirmed a successful deprotection as evidenced by the
absence of the methyl peaks at 1.5 ppm (Figure 1). In addition,
the molecular weight, measured by SEC, shifted to a higher
value, in opposition to the expected molecular weight loss
5
.6 ppm) to the characteristic peaks for methylene groups of
the succinimidyl functionality at 2.9 ppm. The theoretical
molecular weight (19 924 g mol ) corresponding to 11
repeating units was in good agreement with the molecular
weight observed by SEC in DMAc (19200 g mol ), based on
polystyrene standards (Table 1). The presence of RAFT end
groups of POEGMEMA-b-PMANHS was confirmed by H
−1
(
Figure 3). This phenomenon has been repeatedly observed
with similar polymers and can be explained by the increase in
−1
11
hydrodynamic volume, which was been investigated earlier.
1
NMR with the benzyl peaks located at 7.2−8.2 ppm (Figure 1).
Subsequent chain extension of the POEGMEMA-b-
PMANHS macroRAFT agent with the synthesized monomer
(
MAETC) was carried out in 1,4-dioxane after prior testing of
the solvent for peroxides, which can potentially harm the RAFT
50
end group (Scheme 1). After 2.5 h of polymerization, the
monomer conversion reached 40%, which was determined by
1
H NMR by comparing the vinyl proton peaks (6.1 and 5.6
ppm) to the methyl peak at 2.6 ppm (H in the Synthesis of
6
Monomers section). SEC confirmed that the chain extension
process was successfully evidenced by a shift toward higher
molecular weight (Figure 2). The deviation between the
Figure 3. SEC traces of uncross-linked polymer, cross-linked polymer,
and cross-linked polymer after treatment with acid.
The deprotected triblock copolymer, POEGMEMA-b-
PMANHS-b-PMAETC, was then conjugated to CDDP
1
1,13,14
following an established procedure.
The necessary
deprotonation of the carboxylic acid groups using NaOH led
to fully water-soluble polymers. In the next step, the polymers
were incubated with the prepared cis-diamminediaqua
platinum(II) complexes to form a macromolecular metal
complex. The excess platinum drug was eliminated via dialysis
against water for 24 h. The platinum loading efficiency onto
polymers was measured using TGA and ICP-MS. The platinum
loading was measured to be 70% based on the dicarboxylic
groups, which is comparable with the results reported in our
previous studies. Full platinum conjugation was restricted by
steric hindrance and the entropically unfavorable stretching of
Figure 2. SEC traces of the POEGMEMA macroRAFT agent, the
POEGMEMA-b-PMA-NHS diblock copolymer, and the triblock
copolymer, POEGMEMA-b-PMANHS-b-PMAETC.
1
1,13
the polymer chain.
as displayed in Scheme 1, was confirmed using Pt-NMR.
The structure of the platinum complex,
1
95
13
triblock copolymer molecular weight observed by SEC and the
1
one calculated from H NMR could be the result of the
Noticeably, the originally fully water-soluble triblock copolymer
has now amphiphilic character capable of self-assembling into
shell−core nanostructures.
different hydrodynamic volumes for the triblock copolymer and
the polystyrene used for the SEC calibration. This phenomen-
11,13
on has been repeatedly reported with similar polymers.
The
Self-Assembly in Aqueous Solution and Cross-Linking
of Micelles. The platinated copolymer was freeze-dried and
then redissolved in a good solvent for all three blocks, here
DMF. Slow addition of DI water led to the collapse of the
hydrophobic part of the block copolymer leading to aggregate
formation with POEGMEMA as the water-soluble shell. The
DMF was removed via dialysis against DI water. The self-
assembly in water was confirmed by DLS and TEM (Figure 4).
Results observed from DLS depicted an aggregate size of
around 90 nm. TEM analysis confirmed the presence of
SEC peak is not asymmetric, which is the result of an
accumulation of potential side reactions that can occur during
RAFT polymerization. These side reactions become more
pronounced with increasing numbers of blocks and cannot be
1
9,51
avoided. They are discussed in detail elsewhere.
Figure 1
depicts the H NMR spectrum of the homopolymer
POEGMEMA), the diblock copolymer (POEGMEMA-b-
1
(
PMANHS), and the triblock copolymer (POEGMEMA-b-
PMANHS-b-PMAETC) in acetone-d6.
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Figure 4. DLS and TEM of self-assembled micelles (polymer concentration of 1 mg mL−1 in water). Phosphotungstic acid was used as a negative
stain, and the scale bar = 100 nm.
spherical micelles with a slightly smaller size, which is expected
due to the dehydrated state (Figure 4).
shown in Figure 3. Although a bimodal distribution was
observed for the cleaved copolymers, the SEC traces revealed a
significant shift from high molecular weight (595 300 g mol )
−1
The micelle stability has been identified as one potential
contributing factor to allow the safe delivery of the drug loaded
of cross-linked polymer to low molecular weight (33 100 g
11,30
−1
carrier into the tumor cell.
Lengthening of the hydrophobic
mol ) polymer.
block leads to micelles with a lower CMC but also led to better
The hydrodynamic size of the micelles (both uncross-linked
and cross-linked) in aqueous solution or in DMAc was
determined using DLS analysis (Figure 5). In aqueous solution,
1
1
cellular uptake. Considering the massive dilution occurring in
vivo, cross-linking of the micelle might add further stabiliza-
3
0
tion.
In this study, the reaction between diamines and the
activated esters, N-succinimidyl methacrylates, which are
located at the nexus between the core and the shell, was
utilized to cross-link the micelles. The cross-linked micelles
were characterized by DLS and SEC in DMAc as DMAc is a
good solvent to dissolve both blocks. Two different types of
cross-linkers were employed (Scheme 1 and Table 2), which
Table 2. Summary of Platinum Polymeric Micelles Used in
This Study
a
CMC
(
μg
−
micelles
cross-linker
notes
mL 1)
uncross-linked
platinum polymeric
micelles
N/A
25
cross-linked platinum 2-[2-(2-aminoethoxy)
permanent
cross-linker
polymeric micelles 1
ethoxy]ethan-1-
amine
Figure 5. Hydrodynamic diameters measured by DLS of uncross-
linked and cross-linked micelles before and after treatment with acid:
cross-linked platinum 2,2′-(propane-2,2-
acid
degradable
cross-linker
polymeric micelles 2
diylbis(oxy))
diethanamine
(A) uncross-linked micelles in water; (B) uncross-linked micelles in
DMAc; (C) cross-linked micelles in water; (D) cross-linked micelles in
DMAc; and (E) cross-linked micelles after treatment with acid for 72 h
in DMAc. All samples were measured at the concentration of 1 mg
a
Measured using fluorescence spectroscopy with pyrene as a probe.
−
1
mL .
can either lead to permanently cross-linked micelles or result in
micelles that are degradable under acidic conditions. Successful
cross-linking with the acid degradable cross-linker 2,2′-
the hydrodynamic diameter of both uncross-linked and cross-
linked micelles was measured to be around 90 and 70 nm,
respectively. The smaller size observed for the cross-linked
micelles was attributed to the contraction of the core during the
(
propane-2,2-diylbis(oxy))diethanamine was confirmed by a
significant shift in molecular weight from the triblock
copolymer (33 600 g mol ) to the cross-linked micelles
−1
−1
39
(
595 300 g mol ) (Figure 3). Moreover, the SEC curve in
cross-linking reaction. While the cross-linked micelles
Figure 3 revealed the absence of free unimers (uncross-linker
triblock copolymer) in the cross-linked sample.
To investigate their pH-sensitive characteristics, the micelles
cross-linked with the acid degradable cross-linker were treated
with acid (pH 5.5) for 72 h. The acid treatment of cross-linked
copolymers led to the formation of free unimers in DMAc as
retained their size in DMAc, the uncross-linked micelles
dissociated to the underlying triblock copolymers with a size of
around 8 nm. After treatment with acid (pH 5.5) for 72 h, the
sizes of cross-linked micelles in water were similar to uncross-
linked micelles since the self-assembled structure was
maintained because of the amphiphilic nature of the block
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Chemistry of Materials
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copolymer. However, the formation free block copolymer
unimers was clearly observed in DMAc with a hydrodynamic
diameter of 9 nm indicating the destruction of the core−shell
nanoparticle (Figure 5).
Cross-linking with the cross-linker 2-[2-(2-aminoethoxy)-
ethoxy]ethan-1-amine led to similar results, but now the
treatment with acid does not lead to disassociation in a good
solvent such as DMAc; thus, a stable core−shell nanoparticle
was created.
will lead to ligand exchange of the platinum complex from
carboxylato ligand to chloride ligand. The drug is then cleaved
from the polymer as cis-diaminedichloro platinum(II) (CDDP),
which is the commercially available and FDA approved
anticancer drug cisplatin. CDDP is then quickly converted to1
the active species, the cis-diaminediaquo platinum(II) complex.
The differences in drug release between both cross-linked
micelles at pH = 7.4 after 24 h is negligible although there is a
pronounced delay in release after 72 h (Figure 6). Reduced
mobility of the polymer could potentially restrict the diffusion
Pt Release from Platinum Polymeric Micelles. The aim
of this work was to investigate the effect of the type of cross-
linker on drug release and the effect on cell viability. Three
types of micelles were, therefore, investigated utilizing the same
triblock copolymer (Table 2). While no drug release occurs in
phosphate buffer solution, the release of cisplatin from the
30
of the drug resulting in a slower release rate.
The primary objective of this study was a triggered platinum
drug release in an endosomal/lysosomal acidic environment
(
pH 5−5.5). Therefore, the release of the platinum drugs from
the uncross-linked polymeric micelles and cross-linked
polymeric micelles containing either permanent or acid
degradable cross-linkers (Table 2) was investigated using
equilibrium dialysis against two different pH buffer saline
solutions at 37 °C. The rate of release of the Pt species at pH
52
polymer can be initiated by the addition of NaCl. Around
0% and 20% of platinum drugs were released from uncross-
3
linked micelles and cross-linked micelles after 24 h, respectively,
using physiological concentrations of chloride at pH 7.4 (Figure
5
.5 for the acid-cleavable micelles was twice as much as that at
6). The release of platinum drugs from platinum polymer
pH 7.4, whereas the pH value did not have an effect when
employing noncleavable micelles. It should be noted here that
the release in acidic conditions is in general slightly higher since
lower pH values lead to the protonation of the carboxylic acid
after drug release, which lowers the density of negative charges
along the polymer chain, thus promoting release. It can be
concluded from these results that cross-linking using an acid
degradable cross-linkers has two advantages: Cross-linking
delays the release at pH 7.4, which enables better protection of
the drug during systemic circulation or in the extracellular fluid
of normal tissues. Furthermore, the release is accelerated once
the drug carrier is in an acidic environment such as malignant
conjugates is triggered by the presence of chloride ions, which
53
tissues where fluid pH is often lower. Moreover, numerous
studies have shown that micelles are mainly internalized into
cells via endocytosis, with the result that these micelles are
located within the acidic endosomes and lysosomes before
54
being able to enter the cytoplasm.
Cytotoxicity of Polymers. The cytotoxicity of uncross-
linked micelles and cross-linked micelles was examined before
platinum drug conjugation against human ovarian cancer cells
(
OVCAR-3), which was later used to test the activity of
platinum−polymer conjugates. The samples were sterilized
under UV for 30 min prior to incubation with the cell lines.
The sulforhodamine B (SRB) assay was employed to determine
the cytotoxicity of the polymers for a period of 72 h. Different
concentrations of the polymers ranging from 0 to 600 mg mL−
were exposed to OVCAR-3. The uncross-linked micelles
appeared to be nontoxic against OVCAR-3 (cell viability
1
−1
above 90%) in the concentration range of 0−600 μg mL
while the cross-linked micelles led to slightly higher toxicities,
but only at high concentrations (Figure 7). The toxicity was
almost negligible for either uncross-linked or cross-linked
micelles as long as the polymer concentrations remained below
−1
3
00 μg mL , which is well above the concentrations typically
employed in drug delivery systems. Again, it seems that the
55
RAFT end group does not introduce any significant toxicity.
In Vitro Activity of Polymeric Micelles. To evaluate the
cytotoxicity of uncross-linked platinum loaded polymeric
micelles and cross-linked platinum loaded polymeric micelles,
human ovarian OVCAR-3 cells were exposed to various doses
of the drug loaded carrier (equivalent to 0−110 μM Pt) for 24
and 72 h. The cytotoxicity was examined using the sulforhod-
amine B (SRB) microculture colorimetric assay, and the results
Figure 6. Release of CDDP from platinum polymeric conjugates/
micelles in different pH buffer saline solutions (0.9% NaCl). The Pt
amount was determined by ICP-MS.
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Table 3. IC50 Values of Uncross-Linked Platinum Polymeric
Micelles and Cross-Linked Platinum Polymeric Micelles
against OVCAR-3 Cells
IC , μM
5
0
compounds
24 h
72 h
uncross-linked platinum polymeric micelles
cross-linked platinum polymeric micelles 1
cross-linked platinum polymeric micelles 2
carboplatin
90
80
35
12
6.0
5.0
16.7
resembles the structure of the platinum complex on the
polymer, has been additionally investigated. An IC of 16.7 μM
5
0
for this drug was recorded (Table 3).
The cross-linked platinum loaded polymeric micelles
exhibited a higher efficiency than the uncross-linked platinum
loaded polymeric micelles against OVCAR-3 cells (Figure 8).
The difference was even more significant after 24 h. For
example, the IC value of the cross-linked platinum polymeric
Figure 7. Percentage of living OVCAR-3 cell after being exposed to
block copolymer micelle solutions. The micelles used in these
experiments were self-assembled from amphiphilic protected triblock
copolymers.
50
micelles (35 μM) was three times lower than the one of the
uncross-linked platinum polymeric micelles (90 μM) after 24 h
while the IC value after 72 h was still half the value (Table 3).
50
The superior cytotoxicity of cross-linked platinum polymeric
micelles may be attributed to higher cellular uptake (Table 4).
An earlier study revealed that, at low micelle concentration, the
micelles cannot maintain their integrity, especially when in
contact with cell growth media. The cellular uptake below the
CMC then drops significantly, preventing the uptake of the
30
drug carrier.
Another interesting result was the difference in cytotoxicity
of the cross-linked platinum polymeric micelles using various
cross-linkers (i.e., permanent vs acid cleavable). Although
similar activity of both systems was observed after 72 h
incubation with OVCAR-3 cells, the micelles with acid
degradable cross-linker were found to be twice as active
compared to the permanent cross-linked platinum polymeric
micelles (Table 3). Obviously, the advantage in acid degradable
micelles lies in an earlier onset of activity, which could be
assigned to a faster drug release in an acidic environment.
It should be noticed that the original idea to design the
platinum drug delivery carrier based on polymeric micelles is to
enhance plasma retention, to prolong circulation times, and to
have a better cellular uptake into solid tumors, in
7
,19,21,23,56−58
vivo,
compared to carboplatin or cisplatin. The
Figure 8. Cell viability (%) after exposure to uncross-linked platinum
polymeric micelles and cross-linked platinum polymeric micelles
against OVCAR-3 cells.
enhanced in vitro cytotoxicity was, hence, unexpected and
suggests that polymeric micelle delivery of platinum drugs may
significantly improve their efficiency, possibly through
enhanced cellular uptake. This is discussed in detail in the
next section.
Cellular Uptake. The cellular uptake of uncross-linked
platinum polymeric micelles and cross-linked platinum
polymeric micelles was carried out using OVCAR-3 as tumor
cells. To label the polymer, fluorescence-o-methacrylate was
first introduced into the hydrophilic block of block copolymers
by copolymerizing with OEGMEMA. The assumption was
made that a small amount of fluorescence in the block
copolymer would not change their polymer properties in terms
of behavior in solution as well as loading efficiency. ICP-MS
was utilized to determine the amount of platinum drugs taken
up by cells while the amount of polymer was quantified by
fluorescence spectroscopy.
are presented in Figure 8 and Table 3. The concentration of
block copolymer was equivalent to the amount of polymer
present in the platinum drug loaded micelles as shown in Table
S1, Supporting Information.
All three drug carriers prior to loading, uncross-linked
polymeric micelles and cross-linked polymeric micelles, did not
show any cytotoxicity at concentrations up to 300 μg mL−1
(Figure 7). The polymer concentrations used for drug loaded
micelles is well below this threshold, and any cell death
observed should be the result of the action of the platinum
drug.
All platinum loaded polymeric micelles showed significant
cytotoxicity (Figure 8). As expected, cell death increases with
increasing incubation time from 24 to 72 h. For comparison,
cell death of the FDA approved drug carboplatin, which
The cellular uptake was carried out following the procedure
11
described in our previous study. Briefly, OVCAR-3 was
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Table 4. Cellular Uptake with OVCAR-3 Human Ovarian Cancer Cells for 24 h
a
6
b
6
c
6
Pt content (ng Pt/10 cells)
[polymer]theo (ng polymer/10 cells)
[polymer] (ng polymer/10 cells)
5 μg mL−
1
60 μg mL−1
5 μg mL−1
60 μg mL−1
5 μg mL−1
60 μg mL−1
polymer −drug conjugates
micelles
4.5 ± 2.5
21.2 ± 1.6
28.6 ± 4.5
60.1 ± 4.5
121.4 ± 3.2
145.9 ± 4.0
60.5
285.0
384.7
807.9
1632
50.3 ± 7.1
290.3 ± 8.5
340.2 ± 4.7
810.7 ± 7.4
1710.5 ± 5.1
1874.8 ± 9.2
cross-linked 1
cross-linked 2
1961
b
a
6
Pt content determined by extracting 10 cells, followed by Pt content analysis via ICP-MS. Theoretical polymer concentrations were calculated
based on the Pt content determined by ICP-MS and the loading efficiency (70% as mentioned in the platinum loading section). Actual polymer
c
concentrations were measured by fluorescence spectrometry of the labeled polymer.
Figure 9. Scheme of the hypothesized mechanisms for platinum polymeric micellar uptake: (A) via endocytosis and (B) via passive diffusion.
Figure 10. Fluorescence microscopy images of cross-linked platinum polymeric micelles incubated with OVCAR-3 for 2 h at 37 °C. (A) image of
OVCAR-3 taken under halogen light and (B) image of OVCAR-3 taken under fluorescence light.
seeded overnight prior to incubation with the fluorescent
polymers for 24 h. Subsequent intensive washing with PBS
allowed the complete removal of residual micelles. Thus, the
intensity of fluorescence or the amount of platinum drugs
observed was equivalent to the amount of micelles or platinum
drug taken up by the cells.
been taken up. Meanwhile, the fluorescence intensity in the cell
growth media was monitored to quantify the amount of
polymer that had been taken up by the cell. Table 4 summarizes
the amount of polymer and platinum that was found inside the
cell. Since the amount of platinum per amount of polymer is
known, the measured platinum content could be correlated to
the theoretical amount of polymer inside the tumor cell. In fact,
this theoretical amount was found to be in very good
agreement with the measured amount obtained from the
fluorescence intensity. It is remarkable that the observation that
the cross-linked platinum polymeric micelles were taken up
twice as efficiently as the uncross-linked platinum polymeric
micelles, and the deviation was even more significant with the
Two possible scenarios can occur when platinum drug
loaded polymeric micelles are incubated with tumor cells
(Figure 9): The drug may have been released from polymeric
micelles as cisplatin prior to the uptake of the drug carrier
leading to passive diffusion into the tumor cell, like any other
small molecule drug (Figure 9B). Alternatively, the platinum
polymeric micelles could be endocytosed before release occurs,
which is the preferred pathway for the acidic endosomes and
lyzosomes to take effect on the acid degradable linker (Figure
−1
micelle concentration of 5 mg mL , which is below CMC
values (Table 2). As highlighted above, the stability of the
micelle was found to have a noticeable effect on cellular uptake,
which furthermore led to an increased toxicity in an earlier
9A). In order to prove which mechanism is dominant in the
current system, both platinum content and polymer concen-
tration are determined inside the cells.
As seen by the green fluorescence of the polymer inside the
cells (Figure 10), the carrier has been internalized into the
OVCAR-3 cells. The cells were extracted and subject to ICP-
MS analysis to determine the amount of platinum that had
30
study. On the other hand, no significant difference was
observed in cellular uptake of cross-linked micelles using
permanent and acid degradable cross-linkers.
Colony Formation. Tumors in vivo do not grow as
59
monolayers but as colonies. It is, therefore, necessary to
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Chemistry of Materials
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−1
Figure 11. Colony formation of OVCAR-3 cells (A) without treatment (control), (B) empty triblock copolymers micelles at 100 μg mL , (C) Pt-
loaded uncross-linked micelles below CMC values, (D) Pt-loaded uncross-linked micelles above CMC values, (E) Pt-loaded cross-linked micelles
below CMC values, and (F) Pt-loaded cross-linked micelles above CMC values.
examine whether the platinum loaded drug carriers can inhibit
colony formation in human ovarian cancer OVCAR-3 cells in a
long-term assay. For this purpose, a clonogenic assay was used
to evaluate the effects of cell regrowth after exposing them to
platinum polymer conjugates for 72 h. Contrary to the SRB
assay, that only estimates cell growth inhibition relative to a
control, this assay is perhaps more representative of a clinical
situation, where regrowth of cancer tumors is common after
Table 5. Plating Efficiency and Surviving Fraction of
OVCAR-3 after 3 Days of Exposure to Polymer and
Polymer−Pt Conjugates Followed by 10 Days of Incubation
a
in PRMI 1640 Medium
Pt
concentration,
Pt−polymer
surviving
fraction (SF)
1
samples
μM
conjugate, μg mL−
uncross-linked
micelles
2.1
5.5
0.82
11
treatment with chemotherapy. This in vitro cell survival assay
was employed following the procedure described by Franken et
34.8
2.3
91.2
6.0
0
6
0
cross-linked
micelles 2
0.3
al. Similar to the SRB assay, OVAR-3 cells were incubated for
days with the uncross-linked platinum polymeric micelles and
3
3
2.5
0
85.2
0
0
the cross-linked platinum polymeric micelles with acid
degradable cross-linkers at two different platinum concen-
trations (<CMC and >CMC values). These two concentrations
of polymers were chosen because they led to significant
differences in cellular uptake and cytotoxicity and hence could
affect colony formation. After 72 h of drug treatment, a set of
single alive cells were isolated, and they were regrown in PRMI
blank micelles
control
1.05
1
0
0
carboplatin
8.5
0.23
0.11
2
41
a
Plating efficiency (PE) was 78.4%.
cross-linked platinum polymeric micelles. Only less than 20%
inhibition can be seen with as little as 2.1 μM of platinum
concentration from uncross-linked platinum polymeric micelles
compared to 70% in the case of cross-linked platinum
polymeric micelles. These results, thus, suggest that cross-
linked platinum polymeric micelle treatment is highly effective
in suppressing the colony-forming ability of human ovarian
cancer OVCAR-3 cells, probably due to a higher cellular uptake.
1
640 medium containing 10% FBS for 10 days with fresh
medium change every 3 days. The colonies were fixed using
methanol for 30 min before being stained with crystal violet
solution (0.1% in DI water) for 5 min. The colony formation
assay was assessed via the surviving fraction, which is the ratio
of colonies formed after treatment and the number of cells
seeded that can become colonies. The polymers without
platinum drugs appeared not to inhibit colony formation of
−1
OVCAR-3 cells at the concentration of 100 μg mL while both
uncross-linked micelles and cross-linked micelles showed
restriction of the number of colonies formed (Figure 11).
Table 5 revealed that both uncross-linked and cross-linked
platinum polymeric micelles inhibit the colony formation fully
at higher CMC values. The drug loaded micelles are taken up
by the cells and remain lodged for an extended period of time,
releasing the drug slowly over a period of several days.
Interestingly, there was a significant difference in the surviving
fraction of uncross-linked platinum polymeric micelles and
CONCLUSIONS
■
Cross-linked micelles bearing platinum drugs were successfully
synthesized via RAFT polymerization and cross-linking at the
nexus between the core and the shell using permanent and acid
degradable cross-linkers. This work shows that the stability of
the micelles increased considerably after cross-linking, leading
to an increased cellular uptake at very low concentrations. As a
result, the anticancer activity of cross-linked platinum polymeric
micelles is significantly enhanced compared to the uncross-
linked ones. Moreover, SEC and DLS experiments proved that
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Chemistry of Materials
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cross-linked platinum polymeric micelles were able to degrade
at lysosomal pH (pH = 5.5), which triggers an accelerated drug
release within the tumor cells. This effect is particularly
pronounced after 24 h while the behavior of micelles cross-
linked with degradable and nondegradable linkers is similar
after 72 h incubation time. In vitro experiments also revealed
that cross-linked platinum polymeric micelles effectively
suppressed colony formation even at very low concentrations
while the uncross-linked micelle below the CMC disassembles,
which leads to lower uptake.
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(
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ASSOCIATED CONTENT
Supporting Information
Further details are given in Table S1. This material is available
free of charge via the Internet at http://pubs.acs.org.
■
*
S
(
(
31) Read, E. S.; Armes, S. P. Chem. Commun. 2007, 29, 3021.
32) Zhang, L.; Nguyen, T. L. U.; Bernard, J.; Davis, T. P.; Barner-
Kowollik, C.; Stenzel, M. H. Biomacromolecules 2007, 8, 2890.
33) Zhang, L.; Liu, W.; Lin, L.; Chen, D.; Stenzel, M. H.
Biomacromolecules 2008, 9, 3321.
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Polym. Sci., Part A: Polym. Chem. 2006, 44, 5203.
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(
AUTHOR INFORMATION
Corresponding Author
E-mail: m.stenzel@unsw.edu.au.
■
(
*
(
Notes
The authors declare no competing financial interest.
(
J.; Jeong, S. Y.; Heo, J. S.; Lee, S. C. Biomacromolecules 2011, 12, 1224.
(37) Xu, X.; Flores, J. D.; McCormick, C. L. Macromolecules 2011, 44,
ACKNOWLEDGMENTS
■
1
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38) Wike-Hooley, J. L.; Haveman, J.; Reinhold, H. S. Radiother.
Oncol. 1984, 2, 343.
39) Zhang, L.; Bernard, J.; Davis, T. P.; Barner-Kowollik, C.; Stenzel,
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M.H.S. thanks the ARC (Australian Research Council) for
funding in the form of a Future Fellowship (FT0991273) and a
Discovery Project (DP1092694). V.T.H. thanks the Australian
government for Endeavour Postgraduate Award. The authors
would like to thank the Centre for Advanced Macromolecular
Design (CAMD) and UNSW Analytical Centre for support.
(
(
(
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