The dual-role of Pt(iv) complexes as active drug and crosslinker for micelles based on β-cyclodextrin grafted polymer

Article abstract of DOI:10.1039/c5tb02429c

With a combination of RAFT and click chemistry an amphiphilic block copolymer with poly(ethylene glycol) methyl ether methacrylate (POEGMEMA) as hydrophilic block and poly(propargyl methacrylate) (PMA) as hydrophobic block has been successfully synthesized. To this, 6-azide-6-deoxy-β-cyclodextrin (N3-β-CD) was clicked creating a hosting environment for a hydrophobic small molecule platinum pro-drug. Oxoplatin, the oxidized version of cisplatin, has been modified on its axial ligand introducing cholic acid groups through esterification. This modified cisplatin forms a host-guest complex with β-cyclodextrin that has been characterised via NMR spectroscopy. The host-guest interaction that the drug established with the β-cyclodextrin grafted copolymer drove the self-assembly into nanoparticles of a diameter of 266 nm able to physical encapsulate the platinum-based drug. In the presence of ascorbic acid, 70% of the pro-drug is released over a period of 24 h. Cytotoxicity assays on ovarian cancer cells show that the polymer carrier improves the cytotoxicity of the platinum pro-drug. The IC50 value decreases from 37.7 μM with the pro-drug to 20.4 μM when the pro-drug is encapsulated into the polymer carrier. This is due to the fact that the uptake of the polymer carrier is up to 6 fold higher than the significantly smaller pro-drug by itself.

Full text of DOI:10.1039/c5tb02429c

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The dual-role of Pt(IV) complexes as active drug and crosslinker
for micelles based on β-cyclodextrin grafted polymer
Manuela Callari,1 Donald S. Thomas,2 Martina H. Stenzel1*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
With a combination of RAFT and click chemistry an amphiphilic block copolymer with poly (ethylene glycol) methyl ether
methacrylate (POEGMEMA) as hydrophilic block and poly (propargyl methacrylate) (PMA) as hydrophobic block has been
successfully synthesized. To this, 6-azide-6-deoxy-β-cyclodextrin (N3-β-CD) was clicked creating a hosting environment for
a hydrophobic small molecule platinum pro-drug. Oxoplatin, the oxidized version of cisplatin, has been modified on its
axial ligand introducing cholic acid groups through esterification. This modified cisplatin forms a host-guest complex with
β-cyclodextrin that has been characterised via NMR spectroscopy. The host-guest interaction that the drug established
with the β-cyclodextrin grafted copolymer drove the self-assembly into nanoparticles of a diameter of 266 nm able to
physical encapsulate the platinum-based drug. In the presence of ascorbic acid, 70% of the pro-drug is released over a
period of 24 h. Cytotoxicity assays on ovarian cancer cells show that the polymer carrier improves the cytotoxicity of the
platinum pro-drug. The IC50 value decreases from 37.7 μM with the pro-drug to 20.4 μM when the pro-drug is
encapsulated into the polymer carrier. This is due to the fact that the uptake of the polymer carrier is up to 6 fold higher
than the significantly smaller pro-drug by itself.
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the drug and polymer moieties can be used to increase the loading
efficiency and improve the stability of NPs. Ideal candidates to form
host-guest complexes with hydrophobic drugs are cyclodextrins. In
Introduction
Platinum based anti-cancer drugs have been used in clinic for
decades, however, to date, there are many limitations.1, 2 Platinum
drugs are generally hydrophobic making their administration
challenging and they are highly toxic causing a number of side
effects.3 In order to increase their solubility and reduce their
toxicity different types of vehicles have been created to carry and
deliver platinum drugs.4 Among these, polymer nanoparticles (NPs)
have been widely employed.5 Polymer NPs show several advantages
compared to the small drugs. They increase the circulation time and
prevent fast clearance via the kidney thus reducing the
administration dose. They can also exert an enhanced permeation
and retention (EPR) effect in solid tumours.6
fact, their water solubility combined with their hydrophobic cavity
makes them perfect holders for hydrophobic molecules.12
Cyclodextrin (CD) alone has been conjugated to platinum drugs
obtaining pro-drugs which can further encapsulate
a second
hydrophobic drug such as doxorubicin to give a dual-drug system
able to enhance the anti-tumour activity.13, 14 Up to date, there are
no examples in literature of encapsulation of platinum drugs into
cyclodextrins. However, it has been shown that chelating platinum
drugs are able to form host-guest complexes with cyclodextrins.15
A
way researchers have found to establish an interaction between
platinum drugs and cyclodextrins is to conjugate the drug to a
moiety, such as adamantine, which can form host-guest complexes
with cyclodextrins.16, 17 Although cyclodextrins can enhance water
solubility, due to their small size they cannot display the EPR effect
to the same extent NPs can.6 However, cyclodextrins can easily be
clicked onto block copolymers and strengthen the interactions
Platinum drugs can be either physically encapsulated or chemically
bound to the polymer that forms the NP.7,
8
The chemical
conjugation of the platinum drug to the polymer backbone usually
allows more efficient loading. However, the drug has to be
cleavable from the polymer to be released from the NPs into the
target.9 When using physical encapsulation the release of the drug
from the NPs is easier but stability and efficient loading can be a
challenge.10, 11 For these reasons, host-guest interactions between
19
which allow the physical encapsulation of the drug.18,
Cyclodextrin grafted block copolymers have been previously used to
physically encapsulate hydrophobic anti-cancer drugs such as
albendazole.20 Next to its role as drug carrier, CD can be employed
as a building block to generate complex polymer architectures,21, 22
hydrogels,23, 24 nanoparticles25, 26 and crosslinked micelles.27
In this work, we investigate the use of CD as an anchor point to
crosslink micelles while the Pt-containing crosslinker acts
simultaneously as anti-cancer drug. Crosslinking of micelles has
been shown to have a range of benefits including enhanced cellular
30
uptake28 and prolonged circulation in the blood stream.29,
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However, permanent crosslinking can result in the accumulation of Sigma), acetic acid (99.8%, AR), cisplatin (99.9%, Sigma), 2,2’-
nanoparticles in the organs of the reticuloendothelial system Azobisisobutyronitrile (AIBN) was recrystallized from methanol
32
(RES)31,
and it is therefore desirable to introduce functional prior to use, (Ethylene glycol methyl ether) methacrylate oligomer
groups that can be cleaved once the drug has been released. A (OEGMEMA, AR) (Mn = 300 g/mol, Aldrich) was deinhibited before
range of approaches are indeed available that allow the use by passing over basic alumina. The RAFT agent, 4-
34
degradation of crosslinking units of the micelle.33, The pathway Cyanopentanoic Acid Dithiobenzoate (CPADB) was synthesised
38
chosen here is to introduce a Pt(IV) pro-drug based on bile acid as according to literature procedures,37, p-toluenesulfonyl chloride
axial ligand. Bile acid is known to form a host-guest complex with β- (99%, Sigma), Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine
cyclodextrin,35 but it is also known to be a cholesterol metabolite (TBTA) (97%, Sigma), L-ascorbic acid (reagent grade, Sigma),
that is produced in the liver.36 Block copolymers with pendant β- tetrakis(acetonitrile)copper(I) hexafluorophosphate (97%, Sigma),
cyclodextrin groups are therefore synthesized by a combination of cholic acid (from ox or sheep bile, 98%, Sigma), 4-
RAFT polymerization and click chemistry. The resulting water- (dimethylamino)pyridine (99%, Sigma), methyl-β-cyclodextrin
soluble polymer will undergo self-assembly once the host-guest (BioReagent, Sigma), β-cyclodextrin (Chem-Impex int’l inc.), N,N′-
complexation with the hydrophobic bile acid-based Pt(IV) drug diisopropylcarbodiimide (DIC) (99%, Sigma)
takes place (Scheme 1).
Synthesis and procedures
Synthesis of POEGMEMA40. Oligo(ethylene glycol methyl ether)
methacrylate (OEGMEMA) (2 g, 6.66 mmol), 4-cyanopentanoic acid-
4-dithiobenzoate (CPADB) (30 mg, 0.11 mmol) and AIBN (1.8 mg,
0.011 mmol) were dissolved in 7 ml of toluene in a Schlenk tube to
give [OEGMEMA]/[CPADB]/[AIBN] = 60:1:0.1 ratio. The Schlenk tube
was sealed and degassed using 3 freeze-pump-thaw cycles and
leaving the reaction mixture under N2. The reaction was stirred at
60 °C for 12 h. The reaction was quenched by dropping the tube
into an ice bath and exposing the mixture to air. The polymer was
precipitated twice in petroleum spirit and dried under vacuum at 40
°C overnight. 1H NMR (300 MHz, CDCl3, 25 °C): δ (ppm) = 7.90−7.15
(aromatic ring of CPDAB), 4.08 (2nH, CH3OCH2CH2O), 3.66−3.53
(4nH, OCH2CH2O), 3.37 (3nH, CH3OCH2CH2O), 1.97−1.77 (3nH, CH3
of the main chain), 1.09−0.76 (2nH, CH2 of the main chain), n being
the degree of polymerization (DPn) of POEGMEMA. (XNMR = 67%,
Mn,theo = 12000 g mol−1, Mn,SEC = 13800 g mol−1, Ð = 1.1).
Synthesis of TMSPMA. 3-(Trimethylsilyl) propargyl alcohol (5.00 g,
5.71 ml, 39.0 mmol) was dissolved in 50 ml diethyl ether and
purged with N2 for 10 min. Dry triethylamine (4.40 ml, 49.3 mmol)
was added while stirring and the reaction mixture was purged with
N2 for further 10 min in an ice bath. Methacryloyl chloride (6.10 ml,
68.2 mmol) was added drop wise over 1 h into the reaction mixture
with stirring. The reaction was brought to room temperature and
left stirring overnight, followed by filtration and solution
concentration. The obtained residue was purified via column
chromatography taking petroleum ether: diethyl ether (50:1) as
Scheme 1. Synthesis of block copolymer and Pt-drug cross-linker
eluent and obtaining a very pale yellow oil (3.7 g, 74%). 1H NMR
(300 MHz, CDCl3, 25 °C): δ (ppm) = 6.20-5.62 (4nH, C=CH2), 4.36
(2nH, OCH2C), 1.98 (3nH, CH3), 0.15 (9nH, Si(CH3)3).
Synthesis of POEGMEMA40-b-TMSPMA60. The POEGMEMA macro-
Experimental section
RAFT (0.5 g, 5.10 x 10-2 mmol), was dissolved together with 3-
(Trimethylsilyl)-2-propyn-1-yl methacrylate (TMSPMA) (1 g, 5.10
mmol) and AIBN (0.83 mg, 5.10 x 10-3 mmol) in 4 ml of toluene in a
Materials
Schlenk tube to give [POEGMEMA]/[TMSPMA]/[AIBN] = 1:100:0.1
The following chemicals were used as received from the supplier
unless stated otherwise. Sodium azide (NaN3, Sigma-Aldrich, 99%),
3-(trimethylsilyl)-2-propyn-1-ol (99%, Alfa Aesar), methacryloyl
chloride (97%, Fluka), triethylamine (Et3N, 99.5%, Sigma-Aldrich),
tetra-n-butyl ammonium fluoride (TBAF, 1 M solution in THF,
ratio. The Schlenk tube was sealed and degassed using 3 freeze-
pump-thaw cycles and leaving the reaction mixture under N2. The
reaction was let stir at 60 °C for 12 h. The reaction was quenched by
exposing it to the air and dropping the temperature by placing the
Schlenk tube into an ice bath. The polymer was precipitated twice
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in petroleum spirit and dried under vacuum at 40 °C overnight. 1H 3.06 x 10-2 mmol) was added slowly. The reaction was stirred
NMR (300 MHz, CDCl3, 25 °C): δ (ppm) = 7.90−7.15 (aromaꢀc ring of overnight at 50 °C. The product was dialysed against DMSO first and
CPDAB), 4.59 (2mH, OCH2C), 4.08 (2nH, OCH2CH2O), 3.80−3.48 H2O after, it was then freeze dried. The solid was redissolved in
(4nH, OCH2CH2O), 3.39 (3nH, CH3OCH2CH2O), 1.97−1.77 (3n+mH, DMSO, mixed with a copper chelating resin and shaken for 24 h at
CH3 of the main chain), 1.09−0.76 (2n+mH, CH2 of the main chain), 37 °C. The resin was then filtered out; the filtrate was dialysed
0.15 (9nH, Si(CH3)3), n being the degree of polymerization (DPn) of against H2O and freeze dried (380 mg, 80%). 1H NMR (300 MHz, d6-
POEGMEMA and m being the degree of polymerization (DPn) of DMSO, 25 °C): δ (ppm) = 8.30−7.9 (triazole), 6.4-5.5, 5.3-4.7, 4.7-4.2
TMSPMA. (XNMR = 60%, Mn,theo = 11760 g mol−1, Mn,SEC = 23800 g (H3, H6, H5, H3, H4), 4.1 (2nH, OCH2CH2O), 3.80−3.48 (4nH,
mol−1, Ð = 1.20).
OCH2CH2O), 3.5-2.7 (3nH, CH3OCH2CH2O), 1.97−1.77 (3n+mH, CH3 of
the main chain), 1.09−0.76 (2n+mH, CH2 of the main chain),
Deprotection POEGMEMA40-b-TMSPMA60
.
The trimethylsilyl
protected block polymer (0.447 g, 1.85 x 10-2 mmol) was dissolved Synthesis of CA-Pt. Oxoplatin was prepared via oxidation of the
in THF (10 mL) and acetic acid (2 molar equivalent with respect to commercially available cisplatin. Briefly, cisplatin is suspended in
the alkyne-trimethylsilyl groups) in a round bottom flask. The flask hydrogen peroxide solution (30% in water) and stirred at 75 °C for
was sealed and the solution was purged with N2 for 20 min while 5h and then overnight at room temperature. The mixture is then
stirring in an ice bath. A 1 M solution of TBAF∙3H2O in THF (2 molar freeze dried giving a pale yellow powder. Oxoplatin (0.2 g, 2.99 x
equivalent with respect to the alkyne-trimethylsilyl groups) was 10-4 mol) was dissolved in DMF (3 ml) in a round bottom flask which
slowly added with a syringe. The turbid mixture was let stir at 0 °C was sealed and purged with N2. A solution of cholic acid (0.232 g,
for 30 min, then at room temperature for 24 h. The reaction was 5.68 x 10-4 mol) and DMAP (0.0365 g, 2.99 x 10-4 mol) in DMF was
passed through a short silica pad to remove the TBAF, concentrated added to the oxoplatin solution while stirring. After few minutes of
under reduced pressure and diluted in chloroform. The deprotected stirring, DIC (0.093 ml, 5.98 x 10-4 mol) was added. The reaction was
polymer was precipitated in petroleum ether (40-60 °C) and dried stirred overnight at room temperature. The yellow suspension was
1
under vacuum at 40 °C overnight. H NMR (300 MHz, CDCl3, 25 °C): filtered to remove the urea and then put under reduced pressure to
δ (ppm) = 7.90−7.15 (aromaꢀc ring of CPDAB), 4.59 (2mH, OCH2C), remove the DIC. Lastly, the product was precipitated in water,
4.08 (2nH, OCH2CH2O), 3.80−3.48 (4nH, OCH2CH2O), 3.39 (3nH, filtered and freeze dried (0.258 g, 75%). 1H NMR (300 MHz, CDCl3,
CH3OCH2CH2O), 2.55 (nH, CCH), 1.97−1.77 (3n+mH, CH3 of the main 25 °C): δ (ppm) = 3.77 (CH), 3.6 (CH), 3.17 (CH), 2.13 (CH), 2.09, 2.21
chain), 1.09−0.76 (2n+mH, CH2 of the main chain), n being the (CH2), 1.97 (CH), 1.77 (CH), 1.45, 2.21 (CH2), 1.36, 1.78 (CH2), 1.35,
degree of polymerization (DPn) of POEGMEMA and m being the 1.41 (CH2), 1.32 (CH), 1.28 (CH), 1.26, 1.41 (CH2), 1.22 (CH), 1.19,
degree of polymerization (DPn) of TMSPMA. (Mn,theo = 11760 g 1.64 (CH2), 1.15, 1.71 (CH2), 0.96, 1.63 (CH2), 0.91 (CH3), 0.83, 1.63
mol−1, Mn,SEC = 20000 g mol−1, Ð = 1.28).
Synthesis of N3-β-CD. The compound was synthesis using an Preparation of β-CD/CA-Pt complex. β-CD (99 mg, 8.72 x 10-5 mol)
established procedure.39 β-CD (60.0 g, 52.9 10-3 mol) was was dissolved in 6 ml of H2O while CA-Pt (50 mg, 4.36 x 10-5 mol)
(CH2), 0.8 (CH3), 0.58 (CH3)
x
suspended in 500 ml of water to which a solution of NaOH (6.57 g, was dissolved in 6 ml of THF. The THF solution was added drop wise
164.0 x 10-3 mol) in 20 ml of water was added drop wise over 6 min. to the H2O solution. The solution turned turbid at first but turned
The suspension became homogeneous and slightly yellow. p- clear after being stirred at high speed at 40 °C for 24 h. The THF was
Toluenesulfonyl chloride (10.08 g, 52.9 x 10-3 mol) dissolved in 30 ml then evaporated slowly at 40 °C in an open vessel. The remaining
of acetonitrile was then added causing immediate formation of a solution was freeze dried.
white precipitate. After 2 h of stirring at room temperature the
Preparation of Me-β-CD/CA-Pt complex. Me-β-CD (70 mg, 5.34 x
10-5 mol) was dissolved in 3 ml of H2O while CA-Pt (25 mg, 2.18 x 10-
precipitate was filtered and the solution concentrated. The white
5 mol) was dissolved in 3.5 ml of THF. The THF solution was added
product was filtered and dried in a vacuum oven at 40 °C. (10 g, 7.8
x 10-3 mol, 14%). The tosylated β-CD (10 g, 7.7 x 10-3 mol) was
drop wise to the H2O solution while stirring. The mixture was stirred
suspended in dry DMF (1.5 ml), the mixture becomes homogeneous
at high speed at 40 °C for 24 h. The THF was then evaporated slowly
after warming up to 60 °C. Crystalline KI (0.63 g, 3.8 x 10- 3 mol) and
at 40 °C in an open vessel. The remaining solution was freeze dried.
NaN3, (5 g, 77 x 10-3 mol) were added to the mixture and let stirring
Micelles preparation POEGMEMA40-b-PMA60-g-β-CD(80%)/CA-Pt. The
polymer (11 mg, 9.82 x 10-8 mol) and the CA-Pt (4.4 mg, 3.8 x 10-6
at 60-65 °C for 24 h. The reaction was then cooled to room
temperature, acetone (100 ml) was added to produce a white
mol) were dissolved in 4 ml of DMF and stirred together at 40 °C
precipitate that was recovered by filtration and dried in vacuum
oven at 40 °C. (3 g, 2.6 x 10-3 mol, 33%, total yield 5%). (ESI-MS
overnight. To this solution, 8 ml of H2O were added drop wise over
20 h. The final solution was dialysed against water for 3 h and
Calcd m/z for C42H69N3O34, 1159.38; experimental m/z, 1157.5) (Fig.
freeze dried.
S1).
Cell culture. The A2780 cell lines were grown in a ventilated tissue
Synthesis of POEGMEMA40-b-TMSPMA60-g-βCD. POEGMEMA40-b-
PMA60 (100 mg, 5.1 x 10-3 mmol) and N3-β-CD (355 mg, 3.06 x 10-1
culture flask T-75 using Roswell Park Memorial Institute (RPMI-
1640) media containing 10% Foetal Bovine Serum (FBS) and
mmol) (1 eq. compare to the alkyne groups) were dissolved in DMF
(2 ml). A solution of TBTA (17.8 mg, 3.36 x 10-2 mmol) and L-
antibiotics. The cells were incubated at 37 °C under a 5% CO2
Ascorbic acid (53.8 mg, 3.06 x 10-2 mmol) in DMF (2 ml) was added
humidified atmosphere and passaged every 2–3 days when
monolayers at around 80% confluence were formed. The cell
to the N3-β-CD solution while stirring and under a flow of N2. The N2
was purged for about 20 min and then Cu(CH3CN)4PF6 (11.4 mg,
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density was determined by counting the number of viable cells calibrated using a K2PtCl4 standard in D2O. [1H-13C] HSQC NMR, [1H-
using a trypan blue dye (Sigma-Aldrich) exclusion test.
13C] HMBC NMR, [1H-13C] COSY NMR and J resolved experiments
were recorded using Bruker 500 MHz and Bruker AVANCE III HD 600
MHz as indicated.
Cell viability. The cytotoxicity of the pro-drug, the polymer and the
Dynamic light scattering (DLS). The average hydrodynamic
diameters and size distribution of the prepared micelle solution in
an aqueous media at a concentration of 1 mg mL−1 were obtained
using the Malvern Nano-ZS as a particle size analyzer (laser, 4 mW,
λ = 632 nm; measurement angle 12.8° and 175°). Samples were
filtered to remove dust using a microfilter 0.45 μm prior to the
measurements and run at least three times at 25 °C.
micelles loaded with pro-drug was measured by
a standard
sulforhodamine B colorimetric proliferation assay (SRB assay). The
SRB assay was established by the U.S. National Cancer Institute for
rapid, sensitive, and inexpensive screening of antitumor drugs in
microtiter plates. The cells were seeded at a density of 5000 cells
per well in 96-well plates containing 200 μL of growth medium per
well and incubated for 24 h. The medium was then replaced with
fresh medium (200 μL) containing various concentrations of the Transmission electron microscopy (TEM). The TEM micrographs
material being tested.
were obtained using a JEOL 1400 transmission electron microscope.
The instrument operates at an accelerating voltage of 100 kV. The
samples were prepared by casting the micellar solution (1 mg mL−1)
onto a formvar-coated copper grid. Phosphotungstic acid (PTA)
staining was employed.
After 48 h incubation, cells were fixed with trichloroacetic acid 10%
w/v (TCA) before washing, incubated at 4 °C for 1 h, and 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 Thermogravimetric analysis (TGA). TGA studies were carried out
0.4% (w/v) SRB dissolved in 1% acetic acid. At the end of the under an air atmosphere and the heating rate was fixed at 5 °C
staining period, SRB was removed and cultures were quickly rinsed min−1 on a thermal analyser TGA 2950HR V5.4A. The temperature
five times with 1% acetic acid to remove the unbound dye. Then the profile for analysis ranged between 50 and 1000 °C with a five
cultures were air dried until no conspicuous moisture was visible. minute isothermal condition at 100 °C. The mass of the samples
The bound dye was shaken for 10 min. The absorbance at 570 nm used in this study was 10 mg.
of each well was measured using a microtiter plate reader scanning
Inductively Coupled Plasma−Mass Spectrometer (ICP-MS). The
spectrophotometer BioTek’s PowerWave™ HT Microplate Reader
Perkin-Elmer ELAN 6000 inductively coupled plasma−mass
and the KC4™ Software. All experiments were repeated three times.
spectrometer (Perkin-Elmer, Norwalk, CT, U.S.A.) was used for
Dose–response curves were plotted (values were expressed as the quantitative determinations of platinum. All experiments were
percentage of control, medium only) and IC50 inhibitory carried out at an incident ratio frequency power of 1200 W. The
concentrations were obtained using the software Graph Pad PRISM plasma argon gas flow of 12 L min-1 with an auxiliary argon flow of
6.
0.8 L min-1 was used in all cases. The nebulizer gas flow was
adjusted to maximize ion intensity at 0.93 L min-1, as indicated by
the mass flow controller. The element/mass detected was 195Pt and
the internal standard used was 193Ir. 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. A
total of 4 replicates were measured at a normal resolution. The
samples were treated with aqua regia solution at 90 °C for 2 h to
digest platinum.
Cell viability (%) = (OD490,sample – OD490,blank)/ (OD490,control – OD490,blank
)
× 100
Cellular Uptake. Cellular uptake experiments were performed
according to
a
previously described method with some
modifications40 A2780 cells were seeded into a 6-well plate at 1 ×
105 cells per well and incubated until a confluence of 70-80% is
reached. The cells were treated with CA-Pt loaded nanoparticles
and CA-Pt alone with equal Pt concentrations. After 3 h incubation
at 37 °C, the medium was removed and rinsed with cold PBS (3 mL ×
3). The cells were trypsinized and incubated with HNO3 (68%, v/v) at
65 °C for 20 h. Platinum content uptake was determined using
inductively coupled plasma mass spectrometer (ICP-MS).
Results and discussion
The block copolymer was obtained via RAFT polymerization using
CPADB as chain transfer agent (CTA) and AIBN as initiator.41 Firstly,
OEGMEMA was polymerised generating the macro-CTA agent and
the hydrophilic block. A ratio of [OEGMEMA]/[CPADB]/[AIBN] =
60:1:0.1 was used and the polymerization was carried out at 60 °C.
The conversion monomer/polymer was calculated from the 1H NMR
of the crude solution and the dispersity was determined via GPC
analysis. After 12 h polymerization 66% conversion was obtained
resulting in a POEGMEMA macro-CTA of 40 units, Mn = 13800 g mol-
1 and Ð = 1.1 (Fig. 1).
Analysis
1
Nuclear magnetic resonance (NMR) spectroscopy. H NMR spectra
were recorded using a Bruker DPX (300 MHz) spectrometer, using
D2O and d6-DMSO as the solvent. All chemical shifts are stated in
ppm (δ) relative to tetramethylsilane (δ = 0 ppm), referenced to the
chemical shifts of residual solvent resonances.195Pt NMR spectra The TMSPMA monomer was synthesised via coupling reaction
were recorded using a Bruker AVANCE III 400 MHz spectrometer, between 3-(trimethylsilyl) propargyl alcohol and methacryloyl
using d6-DMSO as solvent. The 195Pt NMR spectra have been chloride. The choice of this monomer hails from the presence of the
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triple bond which, once deprotected, can be easily clicked with an
azide functionality.42 The trimethylsilyl protecting group was
deemed necessary to avoid unnecessary side reactions during the
polymerization. POEGMEMA was then chain extended with
Table 1. Molecular weight measured by GPC and compared to the theoretical value
Polymer
Mn/ g mol-1
(GPC)
Ð
Mn/ g mol-1
(theo)
POEGMEMA40
POEGMEMA40-b-TMSPMA60
POEGMEMA40-b-PMA60
POEGMEMA40-b-PMA60-g-
βCD
13800
1.11
1.20
1.28
1.70
12000
TMSPMA using
a ratio of [POEGMEMA]/[TMSPMA]/[AIBN] =
23800
23760
1:100:0.1. A 12 h polymerization at 60 °C gave 60% conversion, 60
units of TMSPMA and a block copolymer with Mn = 23800 g mol-1
and Ð = 1.20 (Fig. 1 and Fig. 2 a). The alkyne groups of the block
copolymer were deprotected using acidic conditions. 1H NMR
confirmed that the reaction was successful and the block copolymer
was fully deprotected. The sharp peak at 0.15 ppm of the
trimethylsilyl disappears completely whilst the alkyne functionality
appears at 2.55 ppm (Fig. 2 b). During this process the RAFT
endfunctionality is lost and replaced by the hydrogen, which has
been shown in an earlier study using Electrospray Ionisation Mass
Spectrometry.43 A small fraction of RAFT functionality has been
converted to a double bond as a result of the Chugaev reaction as
evidenced by the vinyl signals in the area of 5.5 and 6 ppm.44 The
20000
19440
96000
74016
GPC analysis showed a reduction of the molecular weight to Mn
=
20000 g mol-1 and a similar dispersity to the polymer before
deprotection (Fig. 1).
β-CD was modified in 6-position with the azide group through
tosylation. Mono-6-(p-toluenesulfonyl)-β-cyclodextrin was prepared
from β-CD and p-toluenesulfonyl chloride. The toluenesulfonyl
leaving group was then replaced by the azide group by reaction
with NaN3 (Fig. 2 c). The so functionalised β-CD (N3- β-CD) was then
grafted onto the block copolymer by copper(I)-catalyzed azide–
alkyne cycloaddition (click-reaction).45, 46 The catalyst was removed
using a copper chelating resin. In fact, precipitation or dialysis
purification methods were not found sufficient to fully remove the
catalyst. The unreacted CD was removed via dialysis against DMSO
followed by dialysis against water. Analysis by 1H NMR confirmed
that 80% of the propargyl functionality was converted to CD (Fig. 1
and Fig. 2 d). The measured molecular weight was found to be in
good agreement with the expected value although the final product
has a rather broad distribution, which is in good agreement with
other reports that highlight the encountered similar difficulties.
Figure 2: 1H-NMR analysis of a) POEGMEMA-b-TMSPMA (CDCl3); b) after
deprotection POEGMEMA-b-PMA (CDCl3); c) POEGMEMA40-b-PMA60-g-βCD (d6-
DMSO)
Synthesis and NMR characterization of the
platinum pro-drug
Cholic acid (CA) is a primary bile acid insoluble in water. It has been
previously reported that cholic acid interacts with β-CD47 forming
an inclusion complex. Moreover, the carboxylic group present in the
cholic acid structure can be used as conjugation site in the reaction
with oxoplatin. Oxoplatin was prepared by oxidation of the
commercially used cisplatin and then coupled with the cholic acid
so forming the Pt(IV) pro-drug CA-Pt. Oxoplatin was used in slight
excess compared to cholic acid in respect to the two hydroxo
groups available (1:1.9 ratio oxoplatin/cholic acid) to avoid an
excess of unreacted cholic acid which would be difficult to separate
from CA-Pt. 195Pt NMR (Fig. 3) confirmed the success of the coupling
reaction. The peak at 1047 ppm corresponds to the mono-
substituted specie whilst the peak at 1227 ppm corresponds to the
di-substituted specie. Considering the 1:0.7 ratio between the areas
of the two peaks we estimate that di-substituted specie represent
the 70% of the mixture whilst the mono-substituted specie
abundance is 30%. The low solubility of the resulting product in
most solvents does not allow the separation of the products using
chromatography methods.
PEGMEMA40
PEGMEMA40-b-PTMSMA60
PEGMEMA40-b-PMA60
n
PEGMEMA40-b-PMA60/βCD80%
3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0 5.2 5.4 5.6 5.8 6.0 6.2
Log (Mn/g mol-1
)
Figure 1: GPC analysis in DMAc of POEGMEMA40 (black), POEGMEMA40-b-
PTMSPMA60 (red), after deprotection POEGMEMAA40-b-PMA60 (blue) and after
βCD grafting POEGMEMA40-b-PMA60-g-βCD.
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Figure 3. 195Pt NMR, d6-DMSO. The spectrum has been calibrated using a K2PtCl4
standard
Figure 4. 1H NMR, 500 MHz, a) CA-Pt in d6-DMSO, b) β-CD/CA-Pt in D2O, c) β-CD in D2O
The crosslinker CA-Pt was further characterized via NMR analysis. A
range of 2D spectra were recorded (COSY, NOESY, [1H-13C] HSQC,
TOCSY and ROESY NMR (ESI, Figure S2 – S6)) together with the J All observed shifts Δδ, which are the result of the interaction of CA-
1
resolved experiments to be able to identify all H-NMR signals and Pt with β-CD are summarised in table (S8). Moreover, 2D NMR
confirm the structure of the drug. The assigned signals are listed in experiments (Supporting Info (S9 – S11)) aid the identification of
the ESI is a tabulated manner (ESI, Table S7).
the interactions. Small changes in chemical shifts (Δδ ≤ 0.2 ppm)
can be justified by the different deuterated solvent used for the
measurement: D2O in the case of β-CD/CA-Pt and d-DMSO in the
case of CA-Pt. A major shift towards lower fields of the three methyl
groups (18, 19, 21) was noticed whilst the three hydroxyl groups are
no longer visible in spectrum b due to the exchange of the proton
with deuterium from D2O (Fig. 4, a and b). To further confirm that
the CA-Pt chemical shifts are not only caused by different
deuterated solvents used but by the presence of the cyclodextrin in
Host-guest interaction
The main object of this project was to study the affinity between a
host such as β-CD and a platinum based pro-drug. In fact, it is well
known that the interaction between β-cyclodextrin and
a
hydrophobic molecule will drastically increase the solubility of the
latter. To prepare the β-CD/CA-Pt complex two solutions were
prepared: β-CD is dissolved in distilled water whilst CA-Pt is
dissolved in THF. The THF solution is added slowly to the water
solution under fast stirring until the mixture turns turbid obtaining a
mixture β-CD/CA-Pt in a ratio of 2:1. The mixture turns clear when
heated at 40 °C. The slow evaporation of THF will force the
hydrophobic CA-Pt to interact with the β-CD to remain in the water
solution. After complete evaporation of THF the water solution was
observed to be clear with no CA-Pt precipitated. Subsequent freeze
drying yields a colourless powder that can be easily redissolved in
water. The complex formation was investigated using NMR. Fig. 4
1
the solution H NMR of the CA-Pt in D2O was recorded. Due to the
high hydrophobicity of the CA-Pt only a very low concentration
solution can be prepared resulting in poor resolution of most of the
peaks. However peaks 18, 19 and 21 are still sufficiently allowing to
compare the chemical shifts of peak 18, 19 and 21 in the three
different conditions (Figure 5). The resonances move to lower fields
when using D2O instead of d6-DMSO. However, a further shift
towards lower fields can be noticed in the presence of CD.
Generally, the shift towards lower fields is due to decreased
shielding of the electrons. In fact, d6-DMSO (Fig. 5, a) solvates well
the three methyl groups causing high shielding of the electrons.
Conversely, D2O does not solvate them very well leading to a lower
shielding of the electrons and so a shift of the peaks to lower fields.
Lastly, once the cholic acid moiety of CA-Pt is encapsulated by the
CD (Fig. 5, c) the solvent around it is pushed away lowering even
more the shielding thus shifting the peaks to even lower fields.
1
displays the H NMR of β-CD and CA-Pt alone (a and c respectively)
and β-CD/CA-Pt (b). A major change in chemical shift can be noticed
for the proton H5 of the CD (Fig. 4, b and c) The shift of H5 towards
higher fields (Δδ = 0.1) is typical of host-guests CD complexes.48, 49
6 | J. Name., 2012, 00, 1-3
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Table 2. Diffusion parameter and diffusion coefficient obtained from DOSY experiments
of β-CD, CA-Pt and complex β-CD/CA-Pt in D2O and d6-DMSO
.
Solvent
Diffusion
parameter
[log(m2/s)]
-9.31
Diffusion
coefficient
[m2/s]
4.89 x 10-10
2.09 x 10-10
2.51 x 10-10
4.36 x 10-10
β-CD
β-CD
D2O
d6-DMSO
d6-DMSO
D2O
-9.68
CA-Pt
-9.60
β-CD/CA-Pt
-9.25
Micelles preparation and their characterization
The block copolymer dissolves directly in water. It does not
show any obvious amphiphilic features and thus is not able to
self-assemble in aqueous media as evidenced by the absence
of typical nanoparticles as measured using DLS. The PEGMEMA
side chain could have potentially interacted with β-CD as seen
in earlier work,20 but aggregate formation was absent this
Figure 5. a) 1H NMR, 500 MHz, CA-Pt in d6-DMSO b) 1H NMR, 600 MHz, CA-Pt in D2O, c)
β-CD/CA-Pt in D2O
Moreover, Figure 6 depicts the overlap of [1H-13C] HSQC of CA-
Pt and the complex. It is evident that peaks 3, 7 and 12 shifts
towards lower fields in presence of the CD.
time. It also needs to be considered that
α-cyclodextrin is
more suitable for the inclusion complex formation with PEO.50
Within the concentration range measured, (c < 1 mg ml-1) the
scattering intensity changes linearly with conversion, indicative
of the absence of any aggregate formation in this range (Fig.
7). However, the interaction between CA-Pt and β-CD grafted
onto the polymer introduces hydrophobicity to the CD block
enabling the formation of micelles. Micelles were prepared by
dissolving both, block copolymer and pro-drug, in DMF at a
ratio of 2:1 of grafted β-CD to CA-Pt. The mixture was stirred
overnight at 40 °C. Addition of H2O to this solution pushes the
hydrophobic pro-drug towards the hydrophobic cavity of the
cyclodextrins giving the block copolymer a more pronounced
amphiphilicity triggering the formation of nanoparticles. The
resulting solution does not show the presence of any
precipitate suggesting that all water-insoluble CA-Pt is safely
encapsulated in the CD cavity. The nanoparticles were imaged
with TEM after dialysis using PTA staining revealing spherical
nanoparticles with diameters of 200 nm (Fig. 8), which is in
agreement with the DLS results (Fig.11). It should be noted
here that considering the block lengths of the block
copolymers the resulting nanoparticles are less likely to be
well-defined core-shell micelles, but rather large-compound
micelles. Possible reason may be the rate of water addition to
the polymer/drug mixture in organic solvent. Water pushes
the drug inside the CD cavity, but also enables the self-
assembly. Although the water addition was carried out over
one day here, it might have still been too fast to enable the
formation of well-defined micelles.
Figure 6. (red) [1H-13C] HSQC, 500 MHz, CA-Pt in d-DMSO; a 1H NMR, 500 MHz, CA-Pt in
d6-DMSO; (blue) [1H-13C] HSQC, 500 MHz, β-CD/CA-Pt in D2O; b 1H NMR, 500 MHz, β-
CD/CA-Pt in D2O
Further evidence of the occurring complexation was obtained
by measuring the diffusion coefficients of β-CD, CA-Pt and
complex in two different environments, D2O and d-DMSO. The
diffusion parameters have been measured using DOSY NMR
experiment. Results are summarized in Table 2. As expected,
β-CD diffuses with a slower rate in d6-DMSO than D2O due to
the higher viscosity of DMSO whilst CA-Pt diffuses in d6-DMSO
similarly to β-CD. The first indication of interaction between β-
CD and CA-Pt is given by the presence of a single chemical shift
which indicates that the two molecules diffuse at the same
rate. Moreover, the complex has a diffusion coefficient slightly
lower than the β-CD alone indicating, as expected, a slight
bigger volume of the complex compare to the β-CD alone.
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of crosslinks is the strong interaction between bile acid and
cyclodextrin,35, 52 which has been used earlier to generate self-
healing hydrogels.53
Figure 7: Scattering intensity vs the concentration of CA-Pt loaded POEGMEMA40-b-
PMA60-g-βCD NPs ( ) and POEGMEMA40-b-PMA60-g-βCD ( ) in water.
Figure 9: 1H NMR, 600 MHz, a) POEGMEMA40-b-PMA60-g-βCD in D2O; b) drug loaded
micelles in D2O
The release of the drug in deionized water was found to be
negligible as evidence by the high loading capacity after
purification via dialysis. The release of the platinum drug can
Figure 8. TEM analysis of CA-Pt loaded POEGMEMA40-b-PMA60-g-βCD
The content of platinum pro-drug into the micelles was
measured via thermogravimetric Analysis. The platinum
content of 4.5% equals to 93% of the drug being loaded into
the micelles. The high loading efficiency indicates that only a
negligible amount of drug was lost during dialysis and can be
seen as a measure of the high stability of the CD-cholic acid
complex in deionized water. The stability of the micelles upon
dilution was investigated using DLS. Determining the CAC
concentration is not only necessary to confirm the induced
amphiphilicity compared to the unloaded POEGMEMA40-b-
PMA60-g-βCD, but also to evaluate the stability in a delivery
scenario. The scattering intensity was recorded at a fixed
be triggered in
a reductive environment such as the
intracellular environment, which was simulated here by the
addition of ascorbic acid of ascorbic acid (5 mM). Under these
conditions the Pt(IV) drug is reduced to Pt(II), which will be
released from the nanoparticles (Figure 10). Since the system
is stable in water, it is reasonable to assume that the drug can
only be released via reduction. The micelle solution was
dialysed at 37 °C using a membrane (MWCO 3500). Samples
were taken from inside of the membrane at regular intervals.
ICP-MS was used to monitor the amount of platinum released.
As shown in Figure 10 about 70% of the drug is gradually
released over 24 hours. Direct analysis of the released species
is difficult as the released Pt(II) drug will form various
complexes with water and ascorbic acid. An initial burst of
drug from a delivery system is typical, in particular when the
drug is physically encapsulated. For polymer-drug conjugated
systems this can be avoided by stabilizing the system by
crosslinking. In our delivery system, the pro-drug acts as a
crosslinker stabilizing the PEGMEMA-b-PMA-g-βCD micelles.
Furthermore, we hypostasize that the release of platinum
occurs upon cleavage of the cholic acids moieties which keep
their interaction with the CDs. Thus, the slow release is due to
the fact that a reduction reaction has to take place for the
platinum to be release.
aperture, which revealed that
a
critical aggregate
concentration of 2.8 × 10-2 0.5 × 10-2 mg ml-1.
±
Proton NMR of the grafted polymer was measured before and
after the formation of micelles. With the formation of micelles
the relaxation time of the core-forming block changed which is
evident by the suppressed signal intensity.51 Using the
POEGMEMA signals as internal standard, the intensity of
cyclodextrin moieties drastically decreases when the polymer
is loaded with pro-drug and assembled into micelles.
Furthermore, the cyclodextrins peaks appear broad and poorly
resolved in the micellar state suggesting that the cyclodextrin
moieties are in the core of the micelles while simultaneously
entrapping the drug.
The host-guest interaction between the drug and CD induced
amphiphilic features to the polymer providing protection to
the otherwise in water insoluble drug. At this point, it can only
be assumed that the drug also acts as crosslinker since
pendant cholic acid would seek out the hydrophobic CD cavity,
a process which is supported by the 1:1 ratio between CD and
the cholic acid functionality. Further evidence for the presence
8 | J. Name., 2012, 00, 1-3
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line A2780 was investigated at different concentrations of the
block copolymer. Even at a high concentration of 75 mg mL-1,
the polymer was not found to inhibit cell proliferation, as
compared to cells exposed to normal growth conditions (Fig.
12, a). Therefore, the toxicity resulting from the synthetic
procedure could be excluded. Due to its high hydrophobicity,
the cytotoxicity of CA-Pt was investigated upon encapsulation
into Me-β-CD. Unlike β-CD, Me-β-CD is known to be non-toxic
on its own so it will not interfere with the toxicity of CA-Pt. β-
CD, instead, results to be toxic if administrated to cells but it
loses toxicity when part of a polymer carrier. On A2780 cells,
80
70
60
50
40
30
20
10
Me-β-CD/CA-Pt shows an IC50
= 34.7 μM (Fig. 12, b).
Subsequently, the drug loaded nanoparticle was studied. The
polymer concentrations employed in this in vitro study ranged
from 11 mg ml-1 to 4.3 x 10-2 mg ml-1 and lies therefore above
0
0
6
12
Time/ h
24
the measured CAC concentration, which is
a crucial
consideration since disassembled block copolymers were
shown to have a low cellular uptake only.54 The IC50 value of
the drug delivered with the nanoparticle nearly halves
compared to the free drug loaded into Me-β-CD (IC50 = 20.4
μM) (Fig. 12, c).55
Figure 10: Release of platinum drug as cisplatin in the presence of ascorbic acid (5 mM)
The micelles formation and disassembly after the release of
the pro-drug was investigated via DLS (Fig. 11). The polymer in
water has a diameter of Dh = 19 ± 0.33 nm. After self-assembly,
the micelles have a diameter of Dh = 266 ± 0.48 nm which is
maintained after lyophilisation and redispersion in water. After
the release of cisplatin in presence of ascorbic acid the volume
decreases again to Dh = 65 ± 0.38 nm. This indicates that bile
acid largely remains in the CD cavity after the release of the
platinum drug helping to maintain the amphiphilicity.
Furthermore, the possibility that cisplatin may have been
recaptured by empty CD cavities was entertained. However,
intensive NMR studies showed that there is no evidence that
cisplatin is a stable guest molecules in β-CD.
a)
b)
c)
Polymer D = 19 nm; PDI = 0.33
h
Micelles
D = 266 nm; PDI = 0.48
h
After release
D = 65 nm; PDI = 0.38
h
Lyophilized and redissolved micelles
D = 342 nm; PDI = 0.48
h
1.0
0.8
0.6
0.4
0.2
0.0
10
100
1000
size (d.nm)
Figure 11: DLS curves of the grafted polymer (black); micelles (red); lyophilized
and redissolved micelles (green); after release (blue).
Figure 12: Cytotoxicity assays on A2780 of a) polymer carrier, b) Me-β-CD/CA-Pt,
c) CA-Pt loaded micelles
It is hypothesized that the higher toxicity stems from the more
efficient cellular uptake when delivered in a nanocarrier. To
investigate the uptake of platinum into cells, A2780 were
Biological evaluation
The cytotoxicity of the block copolymer was assessed by SRB
assay. Proliferation of platinum resistant ovarian cancer cell
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seeded into 6-well plates and grown up to a confluence of 70-
80%. The cells were then loaded with Me-β-CD/CA-Pt or CA-Pt
loaded nanoparticles at a concentration of 50 μM of platinum
and incubated for 3 h at 37 °C. The cells were then washed
three times with PBS to remove free Me-β-CD/CA-Pt or CA-Pt
loaded NPs, trypsinised and collected for digestion. The
digested solutions were analysed via ICP-MS to determine the
platinum content. The difference in platinum content inside
the cells is depicted in Fig. 13. The significantly higher uptake
of platinum when encapsulated into the micelles may be one
of the factors contributing to the lower IC50 value of the
nanoparticles.
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With
a combination of RAFT-polymerization and chick
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encapsulate
a Pt(IV) pro-drug. We have modified the
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commercially available cisplatin firstly via oxidation and later
via coupling reaction with cholic acid. The Pt(IV) pro-drug
interacts with the pendant β-cyclodextrins driving the
formation of micelles where CA-Pt is entrapped. We have
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compared to the CA-Pt alone. Cytotoxicity assays on ovarian
cancer cell line A2780 have proved the non-toxicity of the
polymer carrier. Moreover, the encapsulation of CA-Pt into
micelles drastically improves the cytotoxicity profile. The lower
IC50 value showed by the micelles compare to the drug alone
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Acknowledgements
The authors like to thank the Australian Research Council (ARC
DP1092694) for funding.
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Notes and references
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DOI: 10.1039/C5TB02429C