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Organocatalysis Paradigm Revisited: Are Metal-free Catalysts really Harmless? Amandine Nachtergael, Olivier Coulembier, Philippe Dubois, Maxime Helvenstein, Pierre Duez, Bertrand Blankert, and Laetitia Mespouille Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm5015443 • Publication Date (Web): 09 Dec 2014 Downloaded from http://pubs.acs.org on December 15, 2014
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Biomacromolecules
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Organocatalysis Paradigm Revisited: Are Metal-
2
free Catalysts really Harmless?
3
Amandine Nachtergael1, Olivier Coulembier2, Philippe Dubois2, Maxime Helvenstein3,
4
Pierre Duez1, Bertrand Blankert3*, Laetitia Mespouille2*
5
1
6
Pharmacy, University of Mons – UMONS, 23 Place du Parc, 7000 Mons (Belgium).
7
2
8
Polymeric and Composite Materials, University of Mons - UMONS, 23 Place du Parc, 7000
9
Mons (Belgium).
Laboratory of Therapeutic Chemistry and Pharmacognosy, Faculty of Medicine and
Center of Innovation and Research in Materials and Polymers (CIRMAP), Laboratory of
10
3
11
Mons - UMONS, 23 Place du Parc, 7000 Mons (Belgium).
12
All three laboratories are members of the Research Institute for Health Sciences and
13
Technology
Laboratory of Pharmaceutical Analysis, Faculty of Medicine and Pharmacy, University of
14 15
KEYWORDS: amino-based organic catalysts, amidine, metal-free ROP, polyester,
16
cytotoxicity.
17 18 19 20
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ABSTRACT
2
Catalysts are commonly used in polymer synthesis. Traditionally, catalysts used to be
3
metallic compounds but some studies have pointed out their toxicity for human health and
4
environment and the removal of metal impurities from synthetic polymer is quite expensive.
5
To address these issues, organocatalysts have been intensively synthetized and are now
6
widely used in ring-opening polymerization (ROP) reactions. However, for most of them,
7
there is not any evidence of their safety. The present study attempts to assess if well-
8
established organo-based ROP catalysts used for the preparation of FDA-approved polyesters
9
may present or not a certain level of cytotoxicity. In vitro toxicity is evaluated using a
10
methyl-thiazol-tetrazolium (MTT) cytotoxicity assay on two cell models (FHs74Int and
11
HepaRG). Among the investigated organocatalysts, only functionalized thiourea (fTU) shows
12
an important cytotoxicity on both cell models. 4-Dimethylaminopyridine (DMAP), 1,5,7-
13
triazabicyclo[4.4.0]dec-5-ene (TBD) and meta-(trimethylammonio)phenolate betaine (m-BE)
14
show cytotoxicity against HepaRG cell line only at a high concentration.
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INTRODUCTION
2
The interest in organocatalysis has spectacularly risen over those last few years as a result of
3
increased environmental awareness. Organocatalysis has many advantages in the context of
4
“green chemistry” and, consequently, classical chemical procedures are increasingly replaced
5
by metal-free alternatives 1. The world of polymeric materials does not contravene this trend
6
and a real effort is concentrated on developing new metal-free synthesis options to replace
7
well-established metal-based routes
8
ring-opening polymerization (ROP) reactions has become notably important to the field 3.
9
The most obvious advantage of organocatalysis is to dispense from the costly removal of
10
metal impurities from the synthesized polymers, which, beyond economic standpoints, could
11
hamper their application in the medical or microelectronic added-value fields or simply raise
12
environmental concerns if used for biodegradable polymer-based packaging production. For
13
that reason, many research groups, including us, have contributed to the development and
14
improvement of ROP metal-free catalysts as being critically important for designing
15
polymer-based materials for targeted biomedical applications
16
catalytic systems avoid residual metal trace issues in the polymer material, no real
17
investigation has been addressed yet to confirm or disprove their biocompatibility.
18
The present paper attempts to assess and verify if well-established organo-based ROP
19
catalysts used for the preparation of FDA-approved polyesters may present or not a certain
20
level of cytotoxicity. For this purpose, two cell models have been selected: epithelial
21
intestinal cells (FHs74Int) and differentiated human hepatocellular carcinoma cells
22
(HepaRG). As FHs74Int cells are derived from normal human fetal intestine and show mature
23
epithelial-like characteristics 8, they are widely used to study food or drug effects on
24
biological processes
2, 3
; the application of metal-free strategies to mediate
4-7
. If, conceptually, metal-free
9, 10
. HepaRG cells have become very useful to study xenobiotic
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metabolism and toxicity because they express major hepatic transporters and drug
2
metabolizing enzymes 11.
3
MATERIALS AND METHODS
4
1. Chemicals
5
The metal-free catalysts were (+)-Sparteine (CAS 90-39-1), a home-prepared functionalized
6
thiourea
7
Dimethylaminopyridine (DMAP) (CAS 1122-58-3), meta-(trimethylammonio)phenolate
8
betaine (m-BE), 1,8-diazabicycloundec-7-ene (DBU) (CAS 6674-22-2) and an imidazolium
9
salt (I-TFA). (+)-Sparteine (Aldrich, 99%), DBU (Acros, 98%) and TBD (Aldrich, 96%)
10
were dried over BaO for 48h, distilled under reduced pressure and stored under N2. fTU was
11
prepared according to a well-established protocol
12
N2. DMAP was dried under vacuum at 60 °C for 12 hours and stored under N2. The m-BE
13
and imidazolium salt were prepared as described in the literature
14
Metal-based catalysts were tin(IV) bromide (CAS 7789-67-5, Aldrich) and dibutyltin bis(2-
15
ethylhexanoate) (CAS 2781-10-4, Aldrich). (+)-Sparteine/fTU, DMAP and dibutyltin bis(2-
16
ethylhexanoate) were dissolved in DMSO (100 mM), whereas TBD, DBU, m-BE,
17
imidazolium salt and tin (IV) bromide were prepared in ultrapure water (100 mM) without
18
any preliminary purification.
19
2. Cell culture
20
The FHs74Int fetal human intestinal epithelial cell line (ATCC number CCL-241) was
21
obtained from the American Type Culture Collection (St Cloud, USA). Cells were cultured in
22
Dulbecco's Modified Eagle Medium high glucose (Lonza, Belgium) supplemented with 10 %
23
fetal bovine serum Gold, 10 mM HEPES, 2 mM L-glutamine, 1 % non-essential amino acids,
24
100 U/mL penicillin, 100 µg/mL streptomycin, 30 ng/µL epidermal growth factor and 10
25
µg/µL insulin, and maintained at 37 °C in a humidified atmosphere of 5 % CO2 in air. The
(fTU),
1,5,7-triazabicyclo[4.4.0]dec-5-ene
(TBD)
(CAS
5807-14-7),
4-
12
, dried under vacuum and stored under
13, 14
and stored under N2.
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HepaRG human hepatocellular carcinoma cell line was obtained from Biopredic International
2
(Saint-Grégoire, France). Cells were cultured in William’s E medium supplemented with 10
3
% of fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin, 100 µg/mL
4
streptomycin, 5 µg/mL insulin and 5 x 10-5 M hydrocortisone hemisuccinate. After 2 weeks,
5
the medium was changed to a differentiation medium provided by the manufacturer
6
corresponding to "supplemented" William’s E medium with 2 % DMSO for 2 more weeks.
7
This proprietary medium allows differentiating cells into biliary-like and hepatocyte-like
8
cells.
9
3. MTT cytotoxicity assay
10
Both FHs74Int and HepaRG cells were seeded in 96-well plates (2 x 104 cells per well) and
11
grown at 37 °C for 22-26 h before a 50 µL aliquot of a base-2 logarithmic dilution of tested
12
compound was added (concentrations 0.8 to 200 µM). After 24 h, the medium was replaced
13
by 200 µL of MTT [3(-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] solution
14
in PBS (0.5 mg/mL) and the plate was incubated at 37 °C. After 4 h, the supernatant was
15
replaced by 100 µL of DMSO to dissolve reduced formazan crystals and the absorbance was
16
measured with a Thermo Labsystems Multiskan Ascent (Thermo Fisher Scientific Inc.,
17
Waltham, USA) at 570 nm versus a 690 nm reference. Percentages of cell viability were
18
expressed compared to control. The fitted curves “% of cell viability” versus “log (catalyst
19
concentration)” allowed the determination of IC10, IC50 and IC90, the catalyst concentrations
20
that inactivate cellular growth by 10, 50 and 90 percent, respectively.
21
RESULTS AND DISCUSSION
22
The field of organic catalysis for ROP of cyclic esters and cyclic carbonates has rapidly
23
expanded and a wide range of catalysts are now available for standardized synthesis protocols
24
15
25
ROP activator
. Following the report of Nederberg et al. who used 4-dimethylaminopyridine (DMAP) as a 16
, other organic molecules have demonstrated their high activity for the
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preparation of aliphatic polyesters and polycarbonates with highly competitive kinetic
2
parameters and control levels as compared with their metallic counterparts. Among them are
3
currently
4
triazabicyclo[4.4.0]dec-5-ene (TBD) 17, 21, (+)-Spartein/functionalized thiourea (fTU) 12, 22, 23,
5
an imidazolium salt (I-TFA)
6
(Figure 1). Beyond favorable kinetic and control feature aspects, this widespread
7
development is motivated by the possibility to lift restrictions on the use of metal-free
8
polymeric materials in fields where high levels of quality are a prerequisite, e.g. for in vivo
9
implants, drug formulation, tissue engineering or biodegradable plastics. Therefore, the
10
reported
:
1,8-diazabicyclo[5.4.0]undec-7-ene
14
(DBU)
17-20
,
1,5,7-
and meta-(trimethylammonio)phenolate betaine (m-BE)
24
cytotoxicity of the above listed catalysts has to be evaluated.
Functionalized thiourea
11 12
Figure 1. Typical organocatalysts used in ring-opening polymerizations of lactones and
13
cyclic carbonates.
14
The MTT cytotoxicity assay is based on the measurement of cells metabolic activity. A
15
tetrazolium salt is reduced by metabolically active cells in purple formazan crystals that are
16
dissolved in DMSO for colorimetric measurement. Assuming that the amount of generated
17
formazan is directly proportional to the amount of metabolically active cells, the proportions
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1
of viable cells can be inferred from spectrophotometric measurements
. This rapid,
2
sensitive, reproducible and inexpensive MTT assay is considered as a good preliminary
3
indicator for the toxicity and biocompatibility of polymers and their degradation products
4
28
5
concentrations (from 0.8 to 200 µM), presumably extending way over their likely residual
6
levels in polymers. Table 1 presents the IC10, IC50 and IC90 values determined from viability
7
curves fitted from the HepaRG and FHs74Int data (Figures 2 and 3, respectively). As positive
8
control, 5-fluorouracil, a well-known cytotoxic compound used in treatment of cancer, caused
9
40 % of cells death at 193.0 µM on FHs74Int normal cells (full data not shown).
26-
. Catalysts were tested on HepaRG and FHs74Int cell lines over a wide range of
10
Table 1. IC10, IC50 and IC90 determined for organic and metallic catalysts from fitted curves
11
“% of cell viability” versus “log (catalysts concentration)”. Organocatalysts IC10
IC50
IC90
HepaRG
FHs74Int
HepaRG
FHs74Int
HepaRG
FHs74Int
DMAP
138.7 µM
n.a.
n.a.
n.a.
n.a.
n.a.
(+)-Sparteine
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
fTU
4.0 µM
n.a.
11.4 µM
27.5 µM
31.6 µM
119. 5 µM
(+)-Sparteine/fTU
2.6 µM
2.5 µM
6.1 µM
32.4 µM
16.6 µM
114.1 µM
DBU
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
TBD
130.6 µM
n.a.
n.a.
n.a.
n.a.
n.a.
m-BE
75.4 µM
n.a.
n.a.
n.a.
n.a.
n.a.
I-TFA
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
Metallic catalysts IC10
Tin(IV)bromide
IC50
IC90
HepaRG
FHs74Int
HepaRG
FHs74Int
HepaRG
FHs74Int
n.t.
n.a.
n.t.
n.a.
n.t.
n.a.
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2.5 µM n.t. 3.1 µM n.t. 4.2 µM Dibutyltinbis(2-ethylhexanoate) n.t. Legend : n.a.: not applicable (inhibitory concentration not reached in the tested concentration range). n.t.: not
13
tested in present work.
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(+)-Sparteine
200 150 100 50 0 0.1
1
10
100
1000
% Cell viability versus control
% Cell viability versus control
DMAP 150
100
50
0 0.1
Catalysts concentration [µM]
50
10
100
1000
% Cell viability versus control
% Cell viability versus control
100
1
100
50
0 0.1
1
100
50
100
1000
100
50
0 0.1
1
10
100
1000
I-TFA
100
50
100
1000
% Cell viability versus control
m-BE
10
1000
Catalysts concentration [µM]
150
1
100
150
Catalysts concentration [µM]
0 0.1
10
TBD % Cell viability versus control
% Cell viability versus control
DBU
10
1000
Catalysts concentration [µM]
150
1
100
150
Catalysts concentration [µM]
0 0.1
10
(+)-Sparteine/fTU
150
0 0.1
1
Catalysts concentration [µM]
fTU
% Cell viability versus control
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200 150 100 50 0 0.1
Catalysts concentration [µM]
1
10
100
1000
Catalysts concentration [µM]
1 2
Figure 2. Cell viability determined by the MTT assay for organocatalysts on HepaRG human
3
hepatocellular carcinoma cells. Results are expressed as average +/- standard deviation (SD) ;
4
data from 2 separate experiments in triplicate.
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(+)-Sparteine
150
100
50
0 0.1
1
10
100
1000
% Cell viability versus control
% Cell viability versus control
DMAP 150
100
50
0 0.1
Catalysts concentration [µM]
50
10
100
1000
% Cell viability versus control
% Cell viability versus control
100
1
100
50
0 0.1
1
100
50
100
1000
100
50
0 0.1
1
10
100
1000
I-TFA
100
50
100
1000
Concentration (µM)
% Cell viability versus control
m-BE
10
1000
Catalysts concentration [µM]
150
1
100
150
Catalysts concentration [µM]
0 0.1
10
TBD % Cell viability versus control
% Cell viability versus control
DBU
10
1000
Catalysts concentration [µM]
150
1
100
150
Catalysts concentration [µM]
0 0.1
10
(+)-Sparteine/fTU
150
0 0.1
1
Catalysts concentration [µM]
fTU
% Cell viability versus control
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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150
100
50
0 0.1
1
10
100
1000
Catalysts concentration [µM]
1 2
Figure 3. Cell viability curves determined by the MTT assay for organocatalysts on FHs74Int
3
fetal human intestinal epithelial cells. Results are expressed as average +/- SD ; data from 3
4
separate experiments in triplicate.
5
The association (+)-Sparteine/fTU displays an important cytotoxicity on both cell models. As
6
sparteine alone doesn’t show any toxicity against both cell models, the functionalized
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thiourea appears entirely responsible for the observed cytotoxicities. A comparative study of
2
the cytotoxicity of a series of mono- and di-substituted thiourea has shown that substituted
3
thiourea cytotoxicity is structure dependent
4
compound. The first step in the bioactivation of thiourea is the oxidation of the thionosulfur
5
by a flavin-monooxygenase (FMO) which results in the formation of sulfenic acid. The
6
oxidation of glutathione by sulfenic acid leads to oxidative stress that may result in cell death
7
or oxidation of biomolecules, proteins, lipids and nucleic acids. According to Onderwater et
8
al. 29 , the toxicity of N-phenylthiourea would not be the result of a FMO-based aromatic ring
9
bioactivation. However, a possible metabolic oxidation of another part of the molecule by
10
cytochrome P450 isoenzymes could explain a structure dependency of substituted thiourea
11
cytotoxicity
12
affirm whether the cytotoxicity is due to the thiourea or the fluorinated aromatic ring
13
moieties. This issue needs further investigation notably on the in vitro and in vivo metabolism
14
of the compound. The IC values of functionalized thiourea and (+)-Sparteine/fTU association
15
were compared across the two cell lines using a two-way ANOVA with post-hoc test
16
(Bonferroni correction). Statistically significant difference (p 20 µM). Interestingly the metallic catalyst tin(IV)bromide
23
does not show any cytotoxicity against FHs74Int cells contrary to dibutyltinbis(2-
24
ethylhexanoate) that proved to be very toxic to the intestinal cell model (Figure 4 and Table
25
1).
30
29
but not correlated with the lipophilicity of the
As this is the first time that fTU is assessed for cytotoxicity, we are unable to
31, 32
. This
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dibutyltinbis(2-ethylhexanoate)
200 150 100 50 0 0
1
10
100
1000
Catalysts concentration [µM]
% Cell viability versus control
Tin(IV)bromide % Cell viability versus control
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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150
100
50
0 0
1
10
100
1000
Catalysts concentration [µM]
1 2
Figure 4. Cell viability curves determined by the MTT assay for metallic catalysts on
3
FHs74Int fetal human intestinal epithelial cells. Results are expressed as average +/- SD; data
4
from 3 separate experiments in triplicate.
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Table 2. In vitro and in vivo toxicity assay performed for catalysts studied in the present paper. Catalyst
In vitro toxicity assay
In vivo toxicity assay
Reference
Tin(IV) bromide CAS : 7789-67-5
n.a.
n.a.
Dibutyltinbis(2-ethyl hexanoate) CAS : 2781-10-4
n.a.
Deer mice : approximate lethal dose (acute oral toxicity) : 1075 mg/kg LDfr1 : 62 mg/kg/day
33
(+)-Sparteine CAS : 90-39-1
n.a.
Wistar rats : LD50 (acute toxicity) of 30 mg/kg
34
Functionalized Thiourea (fTU)
n.a.
n.a. 3
1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) CAS : 5807-14-7
The cytotoxicity of the P(EEP-co-EMEP) copolymers synthetized by copolymerization with TBD. The product was purified by repeated precipitation into cold diethyl ether. Hela cell line: no cytotoxicity for concentrations from 1 to 600 mg/mL(resazurin assay)
n.a.
35
4-dimethylaminopyridine (DMAP) CAS : 1122-58-3
Vibrio fischeri : EC504 of 0.315 mg/mL
LD50 - Deer mice : oral, 450 mg/kg - Mice : oral, 350 mg/kg/day - Rat : oral, 250 mg/mL - Fly : oral, 0.15 mg/mL
33, 36, 37
meta-(trimethylammonio)phenolate betaine (m-BE)
n.a.
n.a.
1,8-diazabicycloundec-7-ene (DBU) CAS : 6674-22-2
Lymphocytes : no significant effect at 0.5 mg/mL Chondrocytes : no effect on cell proliferation up to 1 mg/mL and significant effect (28 % viability) with 2 mg/mL (membrane integrity assay)
n.a.
38
Imidazolium salt n.a. n.a. (I-TFA) Legend : 1LD50, dose that causes death of 50 % animals of the tested group; 2IC50-IC25, concentration which inhibits 50 % and 25 % of cell proliferation, respectively; 3EEP, 2ethoxy-2-oxo-1,3,2-dioxaphospholane and EMEP, 2-ethoxy-4-methyl-2-oxo-1,3,2-dioxaphospholane; 4EC50, effective DMAP concentration at which V. fischeri bioluminescence was reduced by 50 %; 5NR50, concentration of surfactant required to induce a 50 % inhibition in neutral red uptake; n.a., not applicable
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These data indicate that cytotoxicity is certainly compound-dependent and does not correlate
2
with the metallic or non-metallic nature of the catalyst. As polymer usages used to be mostly
3
industrial, there is only a few in vitro and in vivo toxicity assays published for the catalysts
4
studied herein (Table 2).
5
With the development of recyclable, biodegradable and biocompatible polymer, the toxicity
6
(both environmental and human) has become an important parameter that has to be taken into
7
account. Catalysts are the most susceptible elements promoting reactions with toxicological
8
impact and have to be carefully tested for toxicity before they can be used in polymer in such
9
application fields. The assumption that organocatalysts are less toxic than metallic catalysts is
10
not supported by objective data in the state-of-the art and we can even conclude from our results
11
that it is not necessarily the case. By using normal intestinal and metabolically active hepatic cell
12
lines we increase the range of toxicity that can be observed because the results reflect also the
13
metabolization aspect but it will be very interesting to further testing the influence of such a
14
metabolization on catalysts cytotoxicity. To link our results with ROP-process, we started from
15
the hypothesis that 100 % of the catalyst is released in vivo from the biodegradable polymer and
16
extrapolated the cell viability for estimated catalyst concentration in 100 µg of poly(lactide)
17
polymer of 20 000 g/mol (Figure 5). By using 100 µg of 20 000 g/mol polymer, we are
18
consistent with in vivo conditions on the one hand, and on the other hand we fit in the range of
19
catalysts concentrations tested for all catalysts whatever their quantity in polymer after ROP-
20
process (Figure 5 A). Except for fTU, our results demonstrate the relatively low toxicity of
21
organocatalysts against normal intestinal and metabolically active hepatic cell models.
22 23
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A. 1
Catalyst
Equivalent(a)
Quantity per kg of polymer(b)
DMAP
1 to 5 eq.
6 to 30 g
3
(+)-Sparteine
5 eq.
60 g
4
fTU
5 eq.
92.5 g
DBU
1 eq.
8g
TBD
0.01 to 0.1 eq.
0.07 to 0.7 g
m-BE
3 to 5 eq.
24 to 40 g
7
I-TFA
5 eq.
45 g
8
Tin(IV)bromide
1 eq.
22 g
9
Dibutyltin bis(2-ethylhexanoate)
1 eq.
26 g
2
5 6
10
Legend: (a) Equivalent of catalyst relative to the initiator alcohol. (b) Required amount of catalyst to produce 1 kg of poly(lactide) polymer of 20 000 g/mol.
11 B.
50
0
M
D
150
100
50
0
M
100
D
150
A D P( M 1 A e (+ P ( q.) )-S 5 e pa q. rt ) ei ne f TU TB D (0 DB TB .01 U D eq ( m 0.1 .) -B eq m E(3 .) -B e E q. (5 ) eq . I-T ) FA
% Cell viability versus control
HepaRG
A P M (1 A (+ P eq )-S (5 .) p a eq rt .) ei ne TB fT U D D TB (0.0 BU D 1 m (0. eq. D -B 1 ) ib ut m E ( eq. yl -B 3 ) Ti E eq nb (5 .) is T in eq (2 ( - e iv I- .) th )b TF yl ro A he m x a id e no at )
% Cell viability versus control
FHs74Int
D
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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12 13
Figure 5. A. Required amount of catalyst to produce 1 kg of poly(lactide) polymer of 20 000
14
g/mol. B. Percentage of cell viability for estimated catalyst concentration in 100 µg of
15
poly(lactide) polymer of 20 000 g/mol using FHs74Int and HepaRG cell models.
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1
Although the MTT assay is a very good preliminary indicator of a compound cellular toxicity,
2
the results should be interpreted with caution. Indeed, although a correlation can often be derived
3
between viable cell number and MTT-reducing activity
4
number of viable cells but also on their metabolic activities and on the capacity of tested
5
compounds to eventually chemically reduce tetrazolium salts
6
compounds could affect cell integrity by targeting specific cell compartments or functions and be
7
missed by MTT measurements, a complete assessment of toxicity should be obtained only by
8
combining different quantitative methods 25, 27.
9
CONCLUSION
26
, MTT data depend not only on the
39
. Furthermore, as some
10
This study demonstrated the relatively low toxicity of organocatalysts over concentrations
11
presumably extending way over their likely residual levels in polymers, which will contribute to
12
promote their interest in the field of green chemistry for all applications where toxicity and
13
environmental concerns are a key issue. While many papers have hypothesized the absence of
14
toxicity of metal-free catalysts, herein we clearly stated that most of them are indeed
15
biocompatible for the range of concentration investigated at the exception of functionalized
16
thiourea that was found to be very toxic against both cell models. This one, at the equivalency of
17
Tin IV-based structures, should be subjected of thorough toxicity studies before validating its
18
usage in medical device or in green chemistry applications. Given the probable long-term use of
19
polymeric implantable devices, further research is warranted on the biological consequences of
20
residual catalysts and initiators possibly slowly leeching from the polymer.
21 22 23
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1
AUTHOR INFORMATION
2
Corresponding Author
3
*Professor Bertrand Blankert. Laboratory of Pharmaceutical Analysis, Faculty of Medicine and
4
Pharmacy, University of Mons (UMONS) 23, Place du Parc, 7000 Mons, Belgium.
5
Tel : 0032 65 37 34 92 ; Fax : 0032 65 37 34 26 ; email : [email protected]
6
*Dr Laetitia Mespouille. Center of Innovation and Research in Materials and Polymers
7
(CIRMAP). Laboratory of Polymeric and Composite Materials (LPCM). University of Mons
8
(UMONS) 23, Place du Parc, 7000 Mons, Belgium
9
Tel : 0032 65 37 34 82 ; Fax : 0032 65 37 34 84 ; email : [email protected]
10
Author Contributions
11
The manuscript was written through contributions of all authors. All authors have given approval
12
to the final version of the manuscript.
13
ACKNOWLEDGMENT
14
Amandine Nachtergael gratefully acknowledges the fellowship granted to her by UMONS.
15
HepaRG cells (patented by Inserm Transfert) are supplied by Drs. Christiane Guguen-Guillouzo,
16
Philippe Gripon & Christian Trepo (INSERM’s laboratories U522 & U271) and used under a
17
Material Transfer Agreement between INSERM U522 & U271 and the University of Mons,
18
Belgium. CIRMAP is grateful to the “Belgian Federal Government Office Policy of Science
19
(SSTC)” for general support in the frame of the PAI-7/05, the European Commission and the
20
Wallonia Region (FEDER Program) and OPTI2MAT program of excellence. The laboratory of
21
Pharmaceutical Analysis expresses its gratitude to FNRS (Fonds National de la Recherche) for
22
support via the FRSM Grant 3.4614.11. O.C. is Research Fellow of the F.R.S.-FNRS.
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Biomacromolecules
ABBREVIATIONS
2 3
ROP, ring-opening polymerization; FDA, food and drug administration; MTT, methyl-thiazol-
4
tetrazolium; fTU, home-prepared functionalized thiourea; TBD 1,5,7-triazabicyclo[4.4.0]dec-5-
5
ene; DMAP, 4-dimethylaminopyridine; m-BE, meta-(trimethylammonio)phenolate betaine;
6
DBU, 1,8-diazabicycloundec-7-ene; I-TFA, imidazolium salt; DMSO, dimethysulfoxide; PBS,
7
phosphate buffered saline; IC, inhibitory concentration; SD, standard deviation; ANOVA,
8
analysis of variance.
9
Insert Table of Contents Graphic and Synopsis Here
10 11
REFERENCES
12 13 14 15 16 17 18 19 20 21 22 23 24
1. Sheldon, R. A.; Arends, I. W. C. E.; Hanefeld, U., Green Chemistry and Catalysis. 2007; p 1-433. 2. Tsarevsky, N. V.; Matyjaszewski, K., "Green:atom transfer radical polymerization: From process design to preparation of well-defined environmentally friendly polymeric materials. Chemical Reviews 2007, 107 (6), 2270-2299. 3. Dubois, P.; Coulembier, O.; Raquez, J. M., Handbook of Ring-Opening Polymerization. 2009; p 1-408. 4. Bourissou, D.; Moebs-Sanchez, S.; Martin-Vaca, B., Recent advances in the controlled preparation of poly(alpha-hydroxy acids): Metal-free catalysts and new monomers. C. R. Chim. 2007, 10 (9), 775-794. 5. Kamber, N. E.; Jeong, W.; Waymouth, R. M.; Pratt, R. C.; Lohmeijer, B. G. G.; Hedrick, J. L., Organocatalytic ring-opening polymerization. Chemical Reviews 2007, 107 (12), 58135840.
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6. Kiesewetter, M. K.; Shin, E. J.; Hedrick, J. L.; Waymouth, R. M., Organocatalysis: Opportunities and challenges for polymer synthesis. Macromolecules 2010, 43 (5), 2093-2107. 7. Mespouille, L.; Coulembier, O.; Kawalec, M.; Dove, A. P.; Dubois, P., Implementation of metal-free ring-opening polymerization in the preparation of aliphatic polycarbonate materials. Progress in Polymer Science 2014, 39 (6), 1144-1164. 8. Kanwar, J.; Kanwar, R., Gut health immunomodulatory and anti-inflammatory functions of gut enzyme digested high protein micro-nutrient dietary supplement-Enprocal. BMC Immunology 2009, 10 (1), 7. 9. Purup, S.; Larsen, E.; Christensen, L. P., Differential effects of falcarinol and related aliphatic C17-polyacetylenes on intestinal cell proliferation. Journal of Agricultural and Food Chemistry 2009, 57 (18), 8290-8296. 10. Bonfili, L.; Amici, M.; Cecarini, V.; Cuccioloni, M.; Tacconi, R.; Angeletti, M.; Fioretti, E.; Keller, J. N.; Eleuteri, A. M., Wheat sprout extract-induced apoptosis in human cancer cells by proteasomes modulation. Biochimie 2009, 91 (9), 1131-1144. 11. Guillouzo, A.; Corlu, A.; Aninat, C.; Glaise, D.; Morel, F.; Guguen-Guillouzo, C., The human hepatoma HepaRG cells: A highly differentiated model for studies of liver metabolism and toxicity of xenobiotics. Chemico-Biological Interactions 2007, 168 (1), 66-73. 12. Dove, A. P.; Pratt, R. C.; Lohmeijer, B. G. G.; Waymouth, R. M.; Hedrick, J. L., Thiourea-based bifunctional organocatalysis: Supramolecular recognition for living polymerization. J. Am. Chem. Soc. 2005, 127 (40), 13798-13799. 13. Tsutsumi, Y.; Yamakawa, K.; Yoshida, M.; Ema, T.; Sakai, T., Bifunctional organocatalyst for activation of carbon dioxide and epoxide to produce cyclic carbonate: betaine as a new catalytic motif. Org Lett 2010, 12 (24), 5728-5731. 14. Coulembier, O.; Josse, T.; Guillerm, B.; Gerbaux, P.; Dubois, P., An imidazole-based organocatalyst designed for bulk polymerization of lactide isomers: Inspiration from Nature. Chemical Communications 2012, 48 (95), 11695-11697. 15. Dove, A. P., Organic Catalysis for Ring-Opening Polymerization. ACS Macro Letters 2012, 1 (12), 1409-1412. 16. Nederberg, F.; Connor, E. F.; Möller, M.; Glauser, T.; Hedrick, J. L., New paradigms for organic catalysts: The first organocatalytic living polymerization. Angewandte Chemie International Edition 2001, 40 (14), 2712-2715. 17. Lohmeijer, B. G. G.; Pratt, R. C.; Leibfarth, F.; Logan, J. W.; Long, D. A.; Dove, A. P.; Nederberg, F.; Choi, J.; Wade, C.; Waymouth, R. M.; Hedrick, J. L., Guanidine and amidine organocatalysts for ring-opening polymerization of cyclic esters. Macromolecules 2006, 39 (25), 8574-8583. 18. Nederberg, F.; Lohmeijer, B. G. G.; Leibfarth, F.; Pratt, R. C.; Choi, J.; Dove, A. P.; Waymouth, R. M.; Hedrick, J. L., Organocatalytic ring opening polymerization of trimethylene carbonate. Biomacromolecules 2007, 8 (1), 153-160. 19. Pratt, R. C.; Nederberg, F.; Waymouth, R. M.; Hedrick, J. L., Tagging alcohols with cyclic carbonate: a versatile equivalent of (meth)acrylate for ring-opening polymerization. Chemical Communications 2008, (1), 114-116. 20. Coulembier, O.; Lemaur, V.; Josse, T.; Minoia, A.; Cornil, J.; Dubois, P., Synthesis of poly(l-lactide) and gradient copolymers from a l-lactide/trimethylene carbonate eutectic melt. Chemical Science 2012, 3 (3), 723-726.
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21. Pratt, R. C.; Lohmeijer, B. G. G.; Long, D. A.; Waymouth, R. M.; Hedrick, J. L., Triazabicyclodecene: A simple bifunctional organocatalyst for acyl transfer and ring-opening polymerization of cyclic esters. J. Am. Chem. Soc. 2006, 128 (14), 4556-4557. 22. Pratt, R. C.; Lohmeijer, B. G. G.; Long, D. A.; Lundberg, P. N. P.; Dove, A. P.; Li, H. B.; Wade, C. G.; Waymouth, R. M.; Hedrick, J. L., Exploration, optimization, and application of supramolecular thiourea-amine catalysts for the synthesis of lactide (co)polymers. Macromolecules 2006, 39 (23), 7863-7871. 23. Coulembier, O.; De Winter, J.; Josse, T.; Mespouille, L.; Gerbaux, P.; Dubois, P., Onestep synthesis of polylactide macrocycles from sparteine-initiated ROP. Polymer Chemistry 2014, 5 (6), 2103-2108. 24. Guillerm, B.; Lemaur, V.; Cornil, J.; Lazzaroni, R.; Dubois, P.; Coulembier, O., Ammonium betaines: efficient ionic nucleophilic catalysts for the ring-opening polymerization of l-lactide and cyclic carbonates. Chemical Communications 2014, 50 (70), 10098-10101. 25. Bunel, V.; Ouedraogo, M.; Nguyen, A. T.; Stévigny, C.; Duez, P., Methods Applied to the In Vitro Primary Toxicology Testing of Natural Products: State of the Art, Strengths, and Limits. Planta Med 2014, 80 (14), 1210-1226. 26. Sgouras, D.; Duncan, R., Methods for the evaluation of biocompatibility of soluble synthetic polymers which have potential for biomedical use: 1 — Use of the tetrazolium-based colorimetric assay (MTT) as a preliminary screen for evaluation ofin vitro cytotoxicity. J Mater Sci: Mater Med 1990, 1 (2), 61-68. 27. Ciapetti, G.; Cenni, E.; Pratelli, L.; Pizzoferrato, A., In vitro evaluation of cell/biomaterial interaction by MTT assay. Biomaterials 1993, 14 (5), 359-364. 28. Ignatius, A. A.; Claes, L. E., In vitro biocompatibility of bioresorbable polymers: poly(L, DL-lactide) and poly(L-lactide-co-glycolide). Biomaterials 1996, 17 (8), 831-839. 29. Onderwater, R. C. A.; Commandeur, J. N. M.; Groot, E. J.; Sitters, A.; Menge, W. M. P. B.; Vermeulen, N. P. E., Cytotoxicity of a series of mono- and di-substituted thiourea in freshly isolated rat hepatocytes: a preliminary structure-toxicity relationship study. Toxicology 1998, 125 (2–3), 117-129. 30. Decker, C. J.; Doerge, D. R., Rat hepatic microsomal metabolism of ethylenethiourea. Contributions of the flavin-containing monooxygenase and cytochrome P-450 isozymes. Chemical Research in Toxicology 1991, 4 (4), 482-489. 31. Itani, W.; Geara, F.; Haykal, J.; Haddadin, M.; Gali-Muhtasib, H., Radiosensitization by 2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4-dioxide under oxia and hypoxia in human colon cancer cells. Radiation Oncology 2007, 2 (1), 1. 32. El-Najjar, N.; Saliba, N.; Talhouk, S.; Gali-Muhtasib, H., Onopordum cynarocephalum induces apoptosis and protects against 1, 2 dimethylhydrazine-induced colon cancer. Oncology reports 2007, 17 (6), 1517-1523. 33. Schafer, E. W., Jr.; Bowles, W. A., Jr., Acute oral toxicity and repellency of 933 chemicals to house and deer mice. Arch. Environ. Contam. Toxicol. 1985, 14 (1), 111-129. 34. Flores-Soto, M. E.; Bañuelos-Pineda, J.; Orozco-Suárez, S.; Schliebs, R.; Beas-Zárate, C., Neuronal damage and changes in the expression of muscarinic acetylcholine receptor subtypes in the neonatal rat cerebral cortical upon exposure to sparteine, a quinolizidine alkaloid. International Journal of Developmental Neuroscience 2006, 24 (6), 401-410. 35. Steinbach, T.; Schroder, R.; Ritz, S.; Wurm, F. R., Microstructure analysis of biocompatible phosphoester copolymers. Polymer Chemistry 2013, 4 (16), 4469-4479. 36. Fogarty, P., In vivo high throughput toxicology screening method. Google Patents: 2005.
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37. Couling, D. J.; Bernot, R. J.; Docherty, K. M.; Dixon, J. K.; Maginn, E. J., Assessing the factors responsible for ionic liquid toxicity to aquatic organisms via quantitative structureproperty relationship modeling. Green Chemistry 2006, 8 (1), 82-90. 38. Havla, J. B.; Lotz, A. S.; Richter, E.; Froelich, K.; Hagen, R.; Staudenmaier, R.; Kleinsasser, N. H., Cartilage tissue engineering for auricular reconstruction: In vitro evaluation of potential genotoxic and cytotoxic effects of scaffold materials. Toxicology in Vitro 2010, 24 (3), 849-853. 39. Belyanskaya, L.; Manser, P.; Spohn, P.; Bruinink, A.; Wick, P., The reliability and limits of the MTT reduction assay for carbon nanotubes–cell interaction. Carbon 2007, 45 (13), 26432648.
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229x128mm (96 x 96 DPI)
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Organocatalysis Paradigm Revisited: Are Metal-free Catalysts really Harmless? Amandine Nachtergael, Olivier Coulembier, Philippe Dubois, Maxime Helvenstein, Pierre Duez, Bertrand Blankert, and Laetitia Mespouille Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/bm5015443 • Publication Date (Web): 09 Dec 2014 Downloaded from http://pubs.acs.org on December 15, 2014
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Biomacromolecules
1
Organocatalysis Paradigm Revisited: Are Metal-
2
free Catalysts really Harmless?
3
Amandine Nachtergael1, Olivier Coulembier2, Philippe Dubois2, Maxime Helvenstein3,
4
Pierre Duez1, Bertrand Blankert3*, Laetitia Mespouille2*
5
1
6
Pharmacy, University of Mons – UMONS, 23 Place du Parc, 7000 Mons (Belgium).
7
2
8
Polymeric and Composite Materials, University of Mons - UMONS, 23 Place du Parc, 7000
9
Mons (Belgium).
Laboratory of Therapeutic Chemistry and Pharmacognosy, Faculty of Medicine and
Center of Innovation and Research in Materials and Polymers (CIRMAP), Laboratory of
10
3
11
Mons - UMONS, 23 Place du Parc, 7000 Mons (Belgium).
12
All three laboratories are members of the Research Institute for Health Sciences and
13
Technology
Laboratory of Pharmaceutical Analysis, Faculty of Medicine and Pharmacy, University of
14 15
KEYWORDS: amino-based organic catalysts, amidine, metal-free ROP, polyester,
16
cytotoxicity.
17 18 19 20
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1
ABSTRACT
2
Catalysts are commonly used in polymer synthesis. Traditionally, catalysts used to be
3
metallic compounds but some studies have pointed out their toxicity for human health and
4
environment and the removal of metal impurities from synthetic polymer is quite expensive.
5
To address these issues, organocatalysts have been intensively synthetized and are now
6
widely used in ring-opening polymerization (ROP) reactions. However, for most of them,
7
there is not any evidence of their safety. The present study attempts to assess if well-
8
established organo-based ROP catalysts used for the preparation of FDA-approved polyesters
9
may present or not a certain level of cytotoxicity. In vitro toxicity is evaluated using a
10
methyl-thiazol-tetrazolium (MTT) cytotoxicity assay on two cell models (FHs74Int and
11
HepaRG). Among the investigated organocatalysts, only functionalized thiourea (fTU) shows
12
an important cytotoxicity on both cell models. 4-Dimethylaminopyridine (DMAP), 1,5,7-
13
triazabicyclo[4.4.0]dec-5-ene (TBD) and meta-(trimethylammonio)phenolate betaine (m-BE)
14
show cytotoxicity against HepaRG cell line only at a high concentration.
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1
INTRODUCTION
2
The interest in organocatalysis has spectacularly risen over those last few years as a result of
3
increased environmental awareness. Organocatalysis has many advantages in the context of
4
“green chemistry” and, consequently, classical chemical procedures are increasingly replaced
5
by metal-free alternatives 1. The world of polymeric materials does not contravene this trend
6
and a real effort is concentrated on developing new metal-free synthesis options to replace
7
well-established metal-based routes
8
ring-opening polymerization (ROP) reactions has become notably important to the field 3.
9
The most obvious advantage of organocatalysis is to dispense from the costly removal of
10
metal impurities from the synthesized polymers, which, beyond economic standpoints, could
11
hamper their application in the medical or microelectronic added-value fields or simply raise
12
environmental concerns if used for biodegradable polymer-based packaging production. For
13
that reason, many research groups, including us, have contributed to the development and
14
improvement of ROP metal-free catalysts as being critically important for designing
15
polymer-based materials for targeted biomedical applications
16
catalytic systems avoid residual metal trace issues in the polymer material, no real
17
investigation has been addressed yet to confirm or disprove their biocompatibility.
18
The present paper attempts to assess and verify if well-established organo-based ROP
19
catalysts used for the preparation of FDA-approved polyesters may present or not a certain
20
level of cytotoxicity. For this purpose, two cell models have been selected: epithelial
21
intestinal cells (FHs74Int) and differentiated human hepatocellular carcinoma cells
22
(HepaRG). As FHs74Int cells are derived from normal human fetal intestine and show mature
23
epithelial-like characteristics 8, they are widely used to study food or drug effects on
24
biological processes
2, 3
; the application of metal-free strategies to mediate
4-7
. If, conceptually, metal-free
9, 10
. HepaRG cells have become very useful to study xenobiotic
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1
metabolism and toxicity because they express major hepatic transporters and drug
2
metabolizing enzymes 11.
3
MATERIALS AND METHODS
4
1. Chemicals
5
The metal-free catalysts were (+)-Sparteine (CAS 90-39-1), a home-prepared functionalized
6
thiourea
7
Dimethylaminopyridine (DMAP) (CAS 1122-58-3), meta-(trimethylammonio)phenolate
8
betaine (m-BE), 1,8-diazabicycloundec-7-ene (DBU) (CAS 6674-22-2) and an imidazolium
9
salt (I-TFA). (+)-Sparteine (Aldrich, 99%), DBU (Acros, 98%) and TBD (Aldrich, 96%)
10
were dried over BaO for 48h, distilled under reduced pressure and stored under N2. fTU was
11
prepared according to a well-established protocol
12
N2. DMAP was dried under vacuum at 60 °C for 12 hours and stored under N2. The m-BE
13
and imidazolium salt were prepared as described in the literature
14
Metal-based catalysts were tin(IV) bromide (CAS 7789-67-5, Aldrich) and dibutyltin bis(2-
15
ethylhexanoate) (CAS 2781-10-4, Aldrich). (+)-Sparteine/fTU, DMAP and dibutyltin bis(2-
16
ethylhexanoate) were dissolved in DMSO (100 mM), whereas TBD, DBU, m-BE,
17
imidazolium salt and tin (IV) bromide were prepared in ultrapure water (100 mM) without
18
any preliminary purification.
19
2. Cell culture
20
The FHs74Int fetal human intestinal epithelial cell line (ATCC number CCL-241) was
21
obtained from the American Type Culture Collection (St Cloud, USA). Cells were cultured in
22
Dulbecco's Modified Eagle Medium high glucose (Lonza, Belgium) supplemented with 10 %
23
fetal bovine serum Gold, 10 mM HEPES, 2 mM L-glutamine, 1 % non-essential amino acids,
24
100 U/mL penicillin, 100 µg/mL streptomycin, 30 ng/µL epidermal growth factor and 10
25
µg/µL insulin, and maintained at 37 °C in a humidified atmosphere of 5 % CO2 in air. The
(fTU),
1,5,7-triazabicyclo[4.4.0]dec-5-ene
(TBD)
(CAS
5807-14-7),
4-
12
, dried under vacuum and stored under
13, 14
and stored under N2.
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HepaRG human hepatocellular carcinoma cell line was obtained from Biopredic International
2
(Saint-Grégoire, France). Cells were cultured in William’s E medium supplemented with 10
3
% of fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin, 100 µg/mL
4
streptomycin, 5 µg/mL insulin and 5 x 10-5 M hydrocortisone hemisuccinate. After 2 weeks,
5
the medium was changed to a differentiation medium provided by the manufacturer
6
corresponding to "supplemented" William’s E medium with 2 % DMSO for 2 more weeks.
7
This proprietary medium allows differentiating cells into biliary-like and hepatocyte-like
8
cells.
9
3. MTT cytotoxicity assay
10
Both FHs74Int and HepaRG cells were seeded in 96-well plates (2 x 104 cells per well) and
11
grown at 37 °C for 22-26 h before a 50 µL aliquot of a base-2 logarithmic dilution of tested
12
compound was added (concentrations 0.8 to 200 µM). After 24 h, the medium was replaced
13
by 200 µL of MTT [3(-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] solution
14
in PBS (0.5 mg/mL) and the plate was incubated at 37 °C. After 4 h, the supernatant was
15
replaced by 100 µL of DMSO to dissolve reduced formazan crystals and the absorbance was
16
measured with a Thermo Labsystems Multiskan Ascent (Thermo Fisher Scientific Inc.,
17
Waltham, USA) at 570 nm versus a 690 nm reference. Percentages of cell viability were
18
expressed compared to control. The fitted curves “% of cell viability” versus “log (catalyst
19
concentration)” allowed the determination of IC10, IC50 and IC90, the catalyst concentrations
20
that inactivate cellular growth by 10, 50 and 90 percent, respectively.
21
RESULTS AND DISCUSSION
22
The field of organic catalysis for ROP of cyclic esters and cyclic carbonates has rapidly
23
expanded and a wide range of catalysts are now available for standardized synthesis protocols
24
15
25
ROP activator
. Following the report of Nederberg et al. who used 4-dimethylaminopyridine (DMAP) as a 16
, other organic molecules have demonstrated their high activity for the
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1
preparation of aliphatic polyesters and polycarbonates with highly competitive kinetic
2
parameters and control levels as compared with their metallic counterparts. Among them are
3
currently
4
triazabicyclo[4.4.0]dec-5-ene (TBD) 17, 21, (+)-Spartein/functionalized thiourea (fTU) 12, 22, 23,
5
an imidazolium salt (I-TFA)
6
(Figure 1). Beyond favorable kinetic and control feature aspects, this widespread
7
development is motivated by the possibility to lift restrictions on the use of metal-free
8
polymeric materials in fields where high levels of quality are a prerequisite, e.g. for in vivo
9
implants, drug formulation, tissue engineering or biodegradable plastics. Therefore, the
10
reported
:
1,8-diazabicyclo[5.4.0]undec-7-ene
14
(DBU)
17-20
,
1,5,7-
and meta-(trimethylammonio)phenolate betaine (m-BE)
24
cytotoxicity of the above listed catalysts has to be evaluated.
Functionalized thiourea
11 12
Figure 1. Typical organocatalysts used in ring-opening polymerizations of lactones and
13
cyclic carbonates.
14
The MTT cytotoxicity assay is based on the measurement of cells metabolic activity. A
15
tetrazolium salt is reduced by metabolically active cells in purple formazan crystals that are
16
dissolved in DMSO for colorimetric measurement. Assuming that the amount of generated
17
formazan is directly proportional to the amount of metabolically active cells, the proportions
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Biomacromolecules
25
1
of viable cells can be inferred from spectrophotometric measurements
. This rapid,
2
sensitive, reproducible and inexpensive MTT assay is considered as a good preliminary
3
indicator for the toxicity and biocompatibility of polymers and their degradation products
4
28
5
concentrations (from 0.8 to 200 µM), presumably extending way over their likely residual
6
levels in polymers. Table 1 presents the IC10, IC50 and IC90 values determined from viability
7
curves fitted from the HepaRG and FHs74Int data (Figures 2 and 3, respectively). As positive
8
control, 5-fluorouracil, a well-known cytotoxic compound used in treatment of cancer, caused
9
40 % of cells death at 193.0 µM on FHs74Int normal cells (full data not shown).
26-
. Catalysts were tested on HepaRG and FHs74Int cell lines over a wide range of
10
Table 1. IC10, IC50 and IC90 determined for organic and metallic catalysts from fitted curves
11
“% of cell viability” versus “log (catalysts concentration)”. Organocatalysts IC10
IC50
IC90
HepaRG
FHs74Int
HepaRG
FHs74Int
HepaRG
FHs74Int
DMAP
138.7 µM
n.a.
n.a.
n.a.
n.a.
n.a.
(+)-Sparteine
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
fTU
4.0 µM
n.a.
11.4 µM
27.5 µM
31.6 µM
119. 5 µM
(+)-Sparteine/fTU
2.6 µM
2.5 µM
6.1 µM
32.4 µM
16.6 µM
114.1 µM
DBU
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
TBD
130.6 µM
n.a.
n.a.
n.a.
n.a.
n.a.
m-BE
75.4 µM
n.a.
n.a.
n.a.
n.a.
n.a.
I-TFA
n.a.
n.a.
n.a.
n.a.
n.a.
n.a.
Metallic catalysts IC10
Tin(IV)bromide
IC50
IC90
HepaRG
FHs74Int
HepaRG
FHs74Int
HepaRG
FHs74Int
n.t.
n.a.
n.t.
n.a.
n.t.
n.a.
12
2.5 µM n.t. 3.1 µM n.t. 4.2 µM Dibutyltinbis(2-ethylhexanoate) n.t. Legend : n.a.: not applicable (inhibitory concentration not reached in the tested concentration range). n.t.: not
13
tested in present work.
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(+)-Sparteine
200 150 100 50 0 0.1
1
10
100
1000
% Cell viability versus control
% Cell viability versus control
DMAP 150
100
50
0 0.1
Catalysts concentration [µM]
50
10
100
1000
% Cell viability versus control
% Cell viability versus control
100
1
100
50
0 0.1
1
100
50
100
1000
100
50
0 0.1
1
10
100
1000
I-TFA
100
50
100
1000
% Cell viability versus control
m-BE
10
1000
Catalysts concentration [µM]
150
1
100
150
Catalysts concentration [µM]
0 0.1
10
TBD % Cell viability versus control
% Cell viability versus control
DBU
10
1000
Catalysts concentration [µM]
150
1
100
150
Catalysts concentration [µM]
0 0.1
10
(+)-Sparteine/fTU
150
0 0.1
1
Catalysts concentration [µM]
fTU
% Cell viability versus control
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 8 of 21
200 150 100 50 0 0.1
Catalysts concentration [µM]
1
10
100
1000
Catalysts concentration [µM]
1 2
Figure 2. Cell viability determined by the MTT assay for organocatalysts on HepaRG human
3
hepatocellular carcinoma cells. Results are expressed as average +/- standard deviation (SD) ;
4
data from 2 separate experiments in triplicate.
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(+)-Sparteine
150
100
50
0 0.1
1
10
100
1000
% Cell viability versus control
% Cell viability versus control
DMAP 150
100
50
0 0.1
Catalysts concentration [µM]
50
10
100
1000
% Cell viability versus control
% Cell viability versus control
100
1
100
50
0 0.1
1
100
50
100
1000
100
50
0 0.1
1
10
100
1000
I-TFA
100
50
100
1000
Concentration (µM)
% Cell viability versus control
m-BE
10
1000
Catalysts concentration [µM]
150
1
100
150
Catalysts concentration [µM]
0 0.1
10
TBD % Cell viability versus control
% Cell viability versus control
DBU
10
1000
Catalysts concentration [µM]
150
1
100
150
Catalysts concentration [µM]
0 0.1
10
(+)-Sparteine/fTU
150
0 0.1
1
Catalysts concentration [µM]
fTU
% Cell viability versus control
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
150
100
50
0 0.1
1
10
100
1000
Catalysts concentration [µM]
1 2
Figure 3. Cell viability curves determined by the MTT assay for organocatalysts on FHs74Int
3
fetal human intestinal epithelial cells. Results are expressed as average +/- SD ; data from 3
4
separate experiments in triplicate.
5
The association (+)-Sparteine/fTU displays an important cytotoxicity on both cell models. As
6
sparteine alone doesn’t show any toxicity against both cell models, the functionalized
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1
thiourea appears entirely responsible for the observed cytotoxicities. A comparative study of
2
the cytotoxicity of a series of mono- and di-substituted thiourea has shown that substituted
3
thiourea cytotoxicity is structure dependent
4
compound. The first step in the bioactivation of thiourea is the oxidation of the thionosulfur
5
by a flavin-monooxygenase (FMO) which results in the formation of sulfenic acid. The
6
oxidation of glutathione by sulfenic acid leads to oxidative stress that may result in cell death
7
or oxidation of biomolecules, proteins, lipids and nucleic acids. According to Onderwater et
8
al. 29 , the toxicity of N-phenylthiourea would not be the result of a FMO-based aromatic ring
9
bioactivation. However, a possible metabolic oxidation of another part of the molecule by
10
cytochrome P450 isoenzymes could explain a structure dependency of substituted thiourea
11
cytotoxicity
12
affirm whether the cytotoxicity is due to the thiourea or the fluorinated aromatic ring
13
moieties. This issue needs further investigation notably on the in vitro and in vivo metabolism
14
of the compound. The IC values of functionalized thiourea and (+)-Sparteine/fTU association
15
were compared across the two cell lines using a two-way ANOVA with post-hoc test
16
(Bonferroni correction). Statistically significant difference (p 20 µM). Interestingly the metallic catalyst tin(IV)bromide
23
does not show any cytotoxicity against FHs74Int cells contrary to dibutyltinbis(2-
24
ethylhexanoate) that proved to be very toxic to the intestinal cell model (Figure 4 and Table
25
1).
30
29
but not correlated with the lipophilicity of the
As this is the first time that fTU is assessed for cytotoxicity, we are unable to
31, 32
. This
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dibutyltinbis(2-ethylhexanoate)
200 150 100 50 0 0
1
10
100
1000
Catalysts concentration [µM]
% Cell viability versus control
Tin(IV)bromide % Cell viability versus control
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
150
100
50
0 0
1
10
100
1000
Catalysts concentration [µM]
1 2
Figure 4. Cell viability curves determined by the MTT assay for metallic catalysts on
3
FHs74Int fetal human intestinal epithelial cells. Results are expressed as average +/- SD; data
4
from 3 separate experiments in triplicate.
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Page 12 of 21
Table 2. In vitro and in vivo toxicity assay performed for catalysts studied in the present paper. Catalyst
In vitro toxicity assay
In vivo toxicity assay
Reference
Tin(IV) bromide CAS : 7789-67-5
n.a.
n.a.
Dibutyltinbis(2-ethyl hexanoate) CAS : 2781-10-4
n.a.
Deer mice : approximate lethal dose (acute oral toxicity) : 1075 mg/kg LDfr1 : 62 mg/kg/day
33
(+)-Sparteine CAS : 90-39-1
n.a.
Wistar rats : LD50 (acute toxicity) of 30 mg/kg
34
Functionalized Thiourea (fTU)
n.a.
n.a. 3
1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) CAS : 5807-14-7
The cytotoxicity of the P(EEP-co-EMEP) copolymers synthetized by copolymerization with TBD. The product was purified by repeated precipitation into cold diethyl ether. Hela cell line: no cytotoxicity for concentrations from 1 to 600 mg/mL(resazurin assay)
n.a.
35
4-dimethylaminopyridine (DMAP) CAS : 1122-58-3
Vibrio fischeri : EC504 of 0.315 mg/mL
LD50 - Deer mice : oral, 450 mg/kg - Mice : oral, 350 mg/kg/day - Rat : oral, 250 mg/mL - Fly : oral, 0.15 mg/mL
33, 36, 37
meta-(trimethylammonio)phenolate betaine (m-BE)
n.a.
n.a.
1,8-diazabicycloundec-7-ene (DBU) CAS : 6674-22-2
Lymphocytes : no significant effect at 0.5 mg/mL Chondrocytes : no effect on cell proliferation up to 1 mg/mL and significant effect (28 % viability) with 2 mg/mL (membrane integrity assay)
n.a.
38
Imidazolium salt n.a. n.a. (I-TFA) Legend : 1LD50, dose that causes death of 50 % animals of the tested group; 2IC50-IC25, concentration which inhibits 50 % and 25 % of cell proliferation, respectively; 3EEP, 2ethoxy-2-oxo-1,3,2-dioxaphospholane and EMEP, 2-ethoxy-4-methyl-2-oxo-1,3,2-dioxaphospholane; 4EC50, effective DMAP concentration at which V. fischeri bioluminescence was reduced by 50 %; 5NR50, concentration of surfactant required to induce a 50 % inhibition in neutral red uptake; n.a., not applicable
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1
These data indicate that cytotoxicity is certainly compound-dependent and does not correlate
2
with the metallic or non-metallic nature of the catalyst. As polymer usages used to be mostly
3
industrial, there is only a few in vitro and in vivo toxicity assays published for the catalysts
4
studied herein (Table 2).
5
With the development of recyclable, biodegradable and biocompatible polymer, the toxicity
6
(both environmental and human) has become an important parameter that has to be taken into
7
account. Catalysts are the most susceptible elements promoting reactions with toxicological
8
impact and have to be carefully tested for toxicity before they can be used in polymer in such
9
application fields. The assumption that organocatalysts are less toxic than metallic catalysts is
10
not supported by objective data in the state-of-the art and we can even conclude from our results
11
that it is not necessarily the case. By using normal intestinal and metabolically active hepatic cell
12
lines we increase the range of toxicity that can be observed because the results reflect also the
13
metabolization aspect but it will be very interesting to further testing the influence of such a
14
metabolization on catalysts cytotoxicity. To link our results with ROP-process, we started from
15
the hypothesis that 100 % of the catalyst is released in vivo from the biodegradable polymer and
16
extrapolated the cell viability for estimated catalyst concentration in 100 µg of poly(lactide)
17
polymer of 20 000 g/mol (Figure 5). By using 100 µg of 20 000 g/mol polymer, we are
18
consistent with in vivo conditions on the one hand, and on the other hand we fit in the range of
19
catalysts concentrations tested for all catalysts whatever their quantity in polymer after ROP-
20
process (Figure 5 A). Except for fTU, our results demonstrate the relatively low toxicity of
21
organocatalysts against normal intestinal and metabolically active hepatic cell models.
22 23
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Biomacromolecules
A. 1
Catalyst
Equivalent(a)
Quantity per kg of polymer(b)
DMAP
1 to 5 eq.
6 to 30 g
3
(+)-Sparteine
5 eq.
60 g
4
fTU
5 eq.
92.5 g
DBU
1 eq.
8g
TBD
0.01 to 0.1 eq.
0.07 to 0.7 g
m-BE
3 to 5 eq.
24 to 40 g
7
I-TFA
5 eq.
45 g
8
Tin(IV)bromide
1 eq.
22 g
9
Dibutyltin bis(2-ethylhexanoate)
1 eq.
26 g
2
5 6
10
Legend: (a) Equivalent of catalyst relative to the initiator alcohol. (b) Required amount of catalyst to produce 1 kg of poly(lactide) polymer of 20 000 g/mol.
11 B.
50
0
M
D
150
100
50
0
M
100
D
150
A D P( M 1 A e (+ P ( q.) )-S 5 e pa q. rt ) ei ne f TU TB D (0 DB TB .01 U D eq ( m 0.1 .) -B eq m E(3 .) -B e E q. (5 ) eq . I-T ) FA
% Cell viability versus control
HepaRG
A P M (1 A (+ P eq )-S (5 .) p a eq rt .) ei ne TB fT U D D TB (0.0 BU D 1 m (0. eq. D -B 1 ) ib ut m E ( eq. yl -B 3 ) Ti E eq nb (5 .) is T in eq (2 ( - e iv I- .) th )b TF yl ro A he m x a id e no at )
% Cell viability versus control
FHs74Int
D
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 14 of 21
12 13
Figure 5. A. Required amount of catalyst to produce 1 kg of poly(lactide) polymer of 20 000
14
g/mol. B. Percentage of cell viability for estimated catalyst concentration in 100 µg of
15
poly(lactide) polymer of 20 000 g/mol using FHs74Int and HepaRG cell models.
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1
Although the MTT assay is a very good preliminary indicator of a compound cellular toxicity,
2
the results should be interpreted with caution. Indeed, although a correlation can often be derived
3
between viable cell number and MTT-reducing activity
4
number of viable cells but also on their metabolic activities and on the capacity of tested
5
compounds to eventually chemically reduce tetrazolium salts
6
compounds could affect cell integrity by targeting specific cell compartments or functions and be
7
missed by MTT measurements, a complete assessment of toxicity should be obtained only by
8
combining different quantitative methods 25, 27.
9
CONCLUSION
26
, MTT data depend not only on the
39
. Furthermore, as some
10
This study demonstrated the relatively low toxicity of organocatalysts over concentrations
11
presumably extending way over their likely residual levels in polymers, which will contribute to
12
promote their interest in the field of green chemistry for all applications where toxicity and
13
environmental concerns are a key issue. While many papers have hypothesized the absence of
14
toxicity of metal-free catalysts, herein we clearly stated that most of them are indeed
15
biocompatible for the range of concentration investigated at the exception of functionalized
16
thiourea that was found to be very toxic against both cell models. This one, at the equivalency of
17
Tin IV-based structures, should be subjected of thorough toxicity studies before validating its
18
usage in medical device or in green chemistry applications. Given the probable long-term use of
19
polymeric implantable devices, further research is warranted on the biological consequences of
20
residual catalysts and initiators possibly slowly leeching from the polymer.
21 22 23
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1
AUTHOR INFORMATION
2
Corresponding Author
3
*Professor Bertrand Blankert. Laboratory of Pharmaceutical Analysis, Faculty of Medicine and
4
Pharmacy, University of Mons (UMONS) 23, Place du Parc, 7000 Mons, Belgium.
5
Tel : 0032 65 37 34 92 ; Fax : 0032 65 37 34 26 ; email : [email protected]
6
*Dr Laetitia Mespouille. Center of Innovation and Research in Materials and Polymers
7
(CIRMAP). Laboratory of Polymeric and Composite Materials (LPCM). University of Mons
8
(UMONS) 23, Place du Parc, 7000 Mons, Belgium
9
Tel : 0032 65 37 34 82 ; Fax : 0032 65 37 34 84 ; email : [email protected]
10
Author Contributions
11
The manuscript was written through contributions of all authors. All authors have given approval
12
to the final version of the manuscript.
13
ACKNOWLEDGMENT
14
Amandine Nachtergael gratefully acknowledges the fellowship granted to her by UMONS.
15
HepaRG cells (patented by Inserm Transfert) are supplied by Drs. Christiane Guguen-Guillouzo,
16
Philippe Gripon & Christian Trepo (INSERM’s laboratories U522 & U271) and used under a
17
Material Transfer Agreement between INSERM U522 & U271 and the University of Mons,
18
Belgium. CIRMAP is grateful to the “Belgian Federal Government Office Policy of Science
19
(SSTC)” for general support in the frame of the PAI-7/05, the European Commission and the
20
Wallonia Region (FEDER Program) and OPTI2MAT program of excellence. The laboratory of
21
Pharmaceutical Analysis expresses its gratitude to FNRS (Fonds National de la Recherche) for
22
support via the FRSM Grant 3.4614.11. O.C. is Research Fellow of the F.R.S.-FNRS.
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1
Biomacromolecules
ABBREVIATIONS
2 3
ROP, ring-opening polymerization; FDA, food and drug administration; MTT, methyl-thiazol-
4
tetrazolium; fTU, home-prepared functionalized thiourea; TBD 1,5,7-triazabicyclo[4.4.0]dec-5-
5
ene; DMAP, 4-dimethylaminopyridine; m-BE, meta-(trimethylammonio)phenolate betaine;
6
DBU, 1,8-diazabicycloundec-7-ene; I-TFA, imidazolium salt; DMSO, dimethysulfoxide; PBS,
7
phosphate buffered saline; IC, inhibitory concentration; SD, standard deviation; ANOVA,
8
analysis of variance.
9
Insert Table of Contents Graphic and Synopsis Here
10 11
REFERENCES
12 13 14 15 16 17 18 19 20 21 22 23 24
1. Sheldon, R. A.; Arends, I. W. C. E.; Hanefeld, U., Green Chemistry and Catalysis. 2007; p 1-433. 2. Tsarevsky, N. V.; Matyjaszewski, K., "Green:atom transfer radical polymerization: From process design to preparation of well-defined environmentally friendly polymeric materials. Chemical Reviews 2007, 107 (6), 2270-2299. 3. Dubois, P.; Coulembier, O.; Raquez, J. M., Handbook of Ring-Opening Polymerization. 2009; p 1-408. 4. Bourissou, D.; Moebs-Sanchez, S.; Martin-Vaca, B., Recent advances in the controlled preparation of poly(alpha-hydroxy acids): Metal-free catalysts and new monomers. C. R. Chim. 2007, 10 (9), 775-794. 5. Kamber, N. E.; Jeong, W.; Waymouth, R. M.; Pratt, R. C.; Lohmeijer, B. G. G.; Hedrick, J. L., Organocatalytic ring-opening polymerization. Chemical Reviews 2007, 107 (12), 58135840.
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