Supporting Information
Organic Nitrate Functional Nanoparticles for the Glutathione-Triggered
Slow-Release of Nitric Oxide
Hien T.T. Duong,a Amy Ho,b Thomas P. Davisc,d* and Cyrille Boyera,b *
a- Australian Centre for Nanomedicine, School of Chemical Engineering, University of
New South Wales, Sydney, Australia 2052
b- Centre for Advanced Macromolecular Design (CAMD), School of Chemical
Engineering, University of New South Wales, Sydney, Australia 2052
c- ARC Centre for Convergent Bio-Nano Science & Technology, Monash Institute of
Pharmaceutical Sciences, Monash University, Parkville, Melbourne 3052;
d- Department of Chemistry, University of Warwick, UKCorrespondence to:
Thomas.p.davis@monash.edu and cboyer@unsw.edu.au
Materials
All reagents were purchased from Sigma-Aldrich with the highest purity and were used as
supplied unless otherwise noted. Oligo(ethylene glycol) methyl ether methacrylate (OEGMA;
Mn = 300 g mol-1, 99%, Sigma-Aldrich), styrene (ST), and vinylbenzyl chloride (VBC) were
de-inhibited by passing them through a column of basic alumina. RAFT agent, CPADB, 4cyanopentanoic acid dithiobenzoate, was synthesized according to a previously reported
procedure.1 De-inhibited OEGMA, ST, and VBC were both stored at -18 oC. The initiator,
2,2-azobis(isobutyronitrile) (AIBN) was purified by recrystallization twice from methanol.
Synthesis procedures
Synthesis of Homopolymer: Poly(oligoethylene glycol methacrylate) (P(OEGMA)).
OEGMA (3 g, 1.0 × 10-2 mol), CPADB RAFT agent (49.0 mg, 1.75 x 10-4 mol) and AIBN
(2.85 mg, 1.75 × 10-5 mol) were dissolved in 20 mL of toluene. The vial was then sealed with
rubber septum, placed in an ice bath and purged with nitrogen for 30 min. The reaction
mixtures were then immersed in a pre-heated oil bath to 70 °C for 9 h. The polymerisation
S1
was terminated by placing the sample in an ice bath for 5 min. NMR was obtained using
CDCl3 as solvent. OEGMA monomer conversion was determined via 1H-NMR spectroscopy
by the following equation: αOEGMA = 1 - (∫5.5 ppm/(∫4.1 ppm/2)), where ∫ is the peak integral of
monomer (vinyl proton at 5.5 ppm, 1H) and the polymer (ester proton at 4.1 ppm, 2H) and
the conversion of monomer was determined to be 71 %.
The polymer was purified three times by precipitation in anhydrous diethyl ether followed by
centrifugation (7000 rpm for 7 min). Residual solvent was removed under reduced pressure at
room temperature. The number-average molecular weight calculated by NMR Mn,
NMR
of
P(OEGMA) macro-RAFT was calculated to be 12 200 g mol-1 from 1H NMR (CDCl3,
300MHz). The theoretical molecular weight (Mn, theo. = 12 400 g/mol) was calculated using
the following equation: Mn, theo. = [M]/[RAFT] × MWM × αM + MWRAFT, with [M], [RAFT]
and α correspond to monomer and RAFT agent concentration and monomer conversion,
respectively. The number-average molecular weight Mn, GPC was determined by GPC to be 12
000 g mol-1 with PDI = 1.12.
Synthesis of Poly(oligo(ethylene glycol) methyl ether methacrylate)block-poly(styrene-co-vinyl benzyl chloride)) (P(OEGMA))-b-P(VBC-co-ST)).
P(OEGMA)-b-P(VBC-co-ST) was prepared by the chain extension of P(OEGMA) with 40
repeating units (Mn,
NMR
= 12 200 g mol-1, Mn,
GPC
= 12 000 g mol-1) using vinyl benzyl
chloride (VBC) and styrene (ST) as comonomers. The P(OEGMA) macroRAFT agent (1 g,
8.20  10-5 mol), VBC (1.9 g, 1.24  10-2 mol), and ST (4.8 g, 4.62  10-2 mol) were mixed
and then purged with nitrogen for 30 min at 0 oC to avoid the evaporation of styrene. The
polymerizations were carried out in an oil bath at 100 oC for 2.5 h. The copolymers were then
purified three times by precipitating in methanol. After centrifugation (7000 rpm for 15 min),
the polymer was dried overnight under reduced pressure at room temperature. The purified
copolymers were kept at 4 oC prior to further experiments.
The number of repeating units of VBC and ST in the prepared copolymer was determined by
1
H NMR analysis to be 56 and 158, respectively, by comparing the unchanged signal of –
OCH2- ester of P(OEGMA) at 4.1 ppm with the signal of -CH2Cl of VBC at 4.6 ppm and
aromatic protons of ST at 6.3-7.3 pm.
S2
The prepared block copolymer P(OEGMA40)-b-P(VBC56 -co-ST158) with Mn, NMR = 37 000 g
mol-1 and Mn, GPC = 36 000 g mol-1 (PDI = 1.18) was used for further modification with silver
nitrate.
Chemical modification of copolymers P(OEGMA40)-b-P(VBC56 -co-ST158) to nitrate
containing polymer as an NO donor
The pendant chloro group (introduced to the polymer chains by VBC units) was substituted
with silver nitrate (AgNO3) in acetonitrile at 60 oC. Briefly, P(OEGMA40)-b-P(VBC56 -coST158) copolymer (0.1 g, 2.70  10-6 mol) and silver nitrate (50 mg, 2.95  10-4 mol) were
dissolved in acetonitrile (3 mL). The solution was then stirred overnight at 60 oC to achieve
the complete conversion of chloro to nitrate moiety. Acetonitrile was removed under reduced
pressure to get the crude product. The unreacted AgNO3 and by-product AgCl in crude
product was then purified by precipitation in chloroform, the solution of nitrate containing
polymer was isolated from solid AgNO3 and AgCl by centrifugation. Chloroform was then
removed under reduced pressure to get the pure nitrate containing copolymer, which was kept
at 4 oC prior to further use. The resulting copolymers were analyzed using 1H NMR using
CDCl3 as solvent. The conversion was determined from 1H NMR by the comparison of the
signal of chloro- proton peak –CH2Cl (4.5 ppm) and nitrate proton peak –CH2-ONO2 (5.5
ppm). Complete conversion (100%) was achieved after 14 h. The copolymer was further
analysed using GPC, TEM, DLS and FT-IR.
Self-assembly of P(OEGMA40)-b-P(VBNO56-co-ST158) copolymer after modification
with nitrate pendant groups into micellar nanostructures.
Block copolymer before or after modification with silver nitrate (40 mg) was dissolved in
DMF (2 ml), which is a good solvent for both hydrophobic and hydrophilic blocks. Distilled
water (8 mL) was added dropwise to the solution of copolymer solution (20 mg ml-1) under
moderate stirring at room temperature. The mixture was then dialyzed against distilled water
for 2 days using membrane cut-off (3500 Da) to remove DMF. The targeted final polymer
concentration was 4 mg mL-1. The average diameters and size distributions of the prepared
micelles were measured using dynamic light scattering (DLS) and transmission electron
microscopy (TEM).
S3
Release of nitric oxide (NO) from nitrate containing micelles by chemical reaction with
glutathione (GSH).
Block copolymer after modification was subjected to glutathione (GSH) using a
[NO2]:[GSH] molar ratio of 1.0: 2.6. Glutathione (18 mg, 0.584  10-4 mol) was added the
solution of nitrate containing polymer (10.0 mg, 2.70  10-7 mol, NO2 group = 1.51  10-5
mol) in water (12 ml) and reaction mixture was divided in two portions which were then
incubated at 37 oC and 60 oC. The reaction mixture (1 ml) was taken out at different time
intervals over the period of 21 h. The copolymer after NO release was purified using
membrane dialysis for two days against distilled water to remove unreacted GSH and
oxidized GSH. The samples were then freeze-dried and dissolved in CDCl3 for 1H NMR
analysis. The release of NO was monitored from the decrease of signal of –CH2-ONO2 at 5.5
ppm and the increase of signal of –CH2-OH at 4.6 ppm over time using 1H NMR.
Analysis
Nuclear Magnetic Resonance (NMR) Spectroscopy
All 1H-NMR spectra for polymerisations and nitric oxide modifications and NO release
testing were taken using a Gyro 300 MHz NMR Spectrometer. The polymers using CDCl 3 as
solvent unless specified. The sample for silver nitrate reaction was analysed in CD3CN. The
nitrate modified polymers after reactions with reduced glutathione (GSH) were analysed in
either DMSO-d6 or CDCl3 depending on polymer solubility.
OEGMA monomer conversion was determined via 1H-NMR spectroscopy by the following
equation: αOEGMA = 1 - (∫5.5 ppm/(∫4.1 ppm/2)), where ∫ is the peak integral of monomer (vinyl
proton at 5.6 ppm, 1H) and the polymer (ester proton at 4.1 ppm, 2H)
The poly(oligo(ethylene glycol) methyl ether acrylate (P(OEGMA)) repeating unit was
determined using the signal at δ ~ 4.2 ppm (2H, CH2O) and the aromatic group of RAFT
agent at δ ~ 7.2, 7.4 and 7.6 ppm (5H). The VBC repeating unit on polymer were observed by
the signals at δ ~ 4.5 ppm (2H) and the aromatic group of RAFT agent at δ ~ 7.6 ppm (2H).
The ST repeating unit on polymer were observed by the signals at 6.3-7.3 ppm (5H) and the
aromatic group of RAFT agent at δ ~ 7.6 ppm (2H). Modification of chloro-groups on VBC
repeating unit in polymers to nitrate-groups were observed by the signals at δ ~ 5.5 ppm
S4
(2H). Conversion of -CH2ONO2 groups to –CH2OH groups after NO release was observed by
the signals at δ ~ 4.6 ppm.
Gel permeation chromatography (GPC)
The molecular weight and polydispersity (PDI) were characterised by gel permeation
chromatography. GPC analyses was performed using N,N-dimethylacetamide (DMAc) (0.03
% w/v Lithium bromide, 0.05 % 2,6-di-tert-butyl-4-methylphenol (BHT)) as mobile phases at
50 °C with a flow rate of 1 mL min-1. The injection concentration was 2.5 mg mL-1 polymer.
Calibration was performed with poly(styrene) standards with low PDI with molecular weight
ranging from 104 to 106 g mol-1. The retention time was converted into molecular weight
using the calibration curve.). The GPC results were evaluated using the Cirrus 2.0 software
package (PL).
Dynamic Light Scattering (DLS)
The average diameters and size distributions of the prepared micelles were measured by
using a Malvern Zetasizer Nano Series running DTS software (laser, 4 mW, λ = 633 nm;
angle 173o). Samples were filtered to remove dust using microfilter 0.45 µm prior
measurements. The distribution was represented using number distribution.
Transmission Electron Microscopy (TEM)
TEM micrographs were obtained using a JEOL 1400 transmission electron microscope. It
was operated at an acceleration voltage of 80 kV. The samples were prepared by casting the
micellar solution (1 mg mL-1) onto a coated copper grid. OsO4 vapour staining was applied
for 30 mins.
Fourier
Transform-Infrared
Spectroscopy.
Attenuated
Total
Reflectance-Fourier
Transform Infrared Spectroscopy (ATR-FTIR)
ATR-FTIR measurements were performed using a Bruker IFS66\S Fourier transform
spectrometer by averaging 32 scans with a resolution of 4 cm-1.
S5
Additional Figures and Table:
1.0
w log M
0.8
0.6
0.4
0.2
0.0
3.5
4.0
4.5
5.0
5.5
log (M/g mol-1)
Figure S1. GPC traces of POEGMA (black line), P(OEGMA)40-b-P(VBC56-co-ST158) (blue
line), P(OEGMA)40-b-P(VBNO56-co-ST158) after modification with nitrate moieties (red
line).
S6
A) POEGMA40-b-P(VBC56-co-STY158)
C=O
C-Cl
C-O
B) POEGMA40-b-P(VBNO56-co-ST158)
C=O
N=O
N-O
C) POEGMA40-b-P(VBNO56-co-ST158)
after reaction with GSH
-OH
4000
3500
C=O
N=O
3000
2500
2000
1500
N-O
1000
500
-1
Wavenumber (cm )
Figure S2. FTIR-ATR spectra of: (A) P(OEGMA)40-b-P(VBC56-co-ST158) copolymer, (B)
P(OEGMA)40-b-P(VBC56-co-ST158) copolymer after modification with silver nitrate, (C)
nitrate P(OEGMA)40-b-P(VBNO56-co-ST158) copolymer after reaction with GSH at 37oC
after 21 h.
S7
-CH2-ONO2
-CH2-OH
Figure S3. Stack plot of 1H NMR spectra of nitrated modified polymer after treatment with
glutathione at 60°C (recorded in CDCl3).
S8
Table S1. Summary of the copolymers prepared in this study. P(OEGMA)-b-P(VBC-co-ST) #1
was employed in this study.
Feed ratio
(mol-%)
Final
Composition
(mol-%)
Mn, theor.
Mn, GPC
(g/mol)
(g/mol)
PDI
VBC
ST
VBC
ST
-
-
-
-
12 400
12 200
1.12
21
79
26
74
38 000
36 0000
1.18
50
50
60
40
41 000
38 000
1.21
P(OEGMA)
P(OEGMA)-bP(VBC-co-ST) #1
P(OEGMA)-bP(VBC-co-ST) #2
Note: Molecular weight assessed in DMAc GPC.
Additional References
1. Le, T. P.; Moad, G.; Rizzardo, E.; Thang, S. H., 1998;
2. Kodela, R.; Chattopadhyay, M.; Kashfi, K. ACS Medicinal Chemistry Letters 2012, 3,
257-262.
S9