Organocatalysis Paradigm Revisited: Are Metal-Free Catalysts Really

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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*,‡ Laboratory of Therapeutic Chemistry and Pharmacognosy, Faculty of Medicine and Pharmacy, ‡Center of Innovation and Research in Materials and Polymers (CIRMAP), Laboratory of Polymeric and Composite Materials, and §Laboratory of Pharmaceutical Analysis, Faculty of Medicine and Pharmacy, University of Mons − UMONS, 23 Place du Parc, 7000 Mons, Belgium

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ABSTRACT: Catalysts are commonly used in polymer synthesis. Traditionally, catalysts used to be metallic compounds but some studies have pointed out their toxicity for human health and environment, and the removal of metal impurities from synthetic polymer is quite expensive. Organocatalysts have been intensively synthesized and are now widely used in ring-opening polymerization (ROP) reactions to address these issues. However, for most of them, there is not any evidence of their safety. The present study attempts to assess whether well-established organo-based ROP catalysts used for the preparation of FDA-approved polyesters may present a certain level of cytotoxicity. In vitro toxicity is evaluated using a methyl-thiazol-tetrazolium cytotoxicity assay on two cell models (FHs74Int and HepaRG). Among the investigated organocatalysts, only functionalized thiourea shows an important cytotoxicity on both cell models. 4-Dimethylaminopyridine (DMAP), 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD), and meta-(trimethylammonio)phenolate betaine (m-BE) show cytotoxicity against HepaRG cell line only at a high concentration.

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INTRODUCTION The interest in organocatalysis has spectacularly risen over the past few years as a result of increased environmental awareness. Organocatalysis has many advantages in the context of “green chemistry”, and, consequently, classical chemical procedures are increasingly replaced by metal-free alternatives.1 The world of polymeric materials does not contravene this trend, and a real effort is concentrated on developing new metal-free synthesis options to replace well-established metal-based routes;2,3 the application of metal-free strategies to mediate ring-opening polymerization (ROP) reactions has become notably important to the field.3 The most obvious advantage of organocatalysis is to dispense from the costly removal of metal impurities from the synthesized polymers, which, beyond economic standpoints, could hamper their application in the medical or microelectronic added-value fields or simply raise environmental concerns if used for biodegradable polymer-based packaging production. For that reason, many research groups, including us, have contributed to the development and improvement of ROP metal-free catalysts as being critically important for designing polymer-based materials for targeted biomedical applications.4−7 If, conceptually, metal-free catalytic systems avoid residual metal trace issues in the polymer material, no real investigation has been addressed yet to confirm or disprove their biocompatibility. The present paper attempts to assess and verify whether well-established organo-based ROP catalysts used for the preparation of FDA-approved polyesters may present a certain © 2014 American Chemical Society

level of cytotoxicity. For this purpose, two cell models have been selected: epithelial intestinal cells (FHs74Int) and differentiated human hepatocellular carcinoma cells (HepaRG). Because FHs74Int cells are derived from normal human fetal intestine and show mature epithelial-like characteristics,8 they are widely used to study food or drug effects on biological processes.9,10 HepaRG cells have become very useful to study xenobiotic metabolism and toxicity because they express major hepatic transporters and drug-metabolizing enzymes.11

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MATERIALS AND METHODS

1. Chemicals. The metal-free catalysts were (+)-sparteine (CAS 90-39-1), a home-prepared functionalized thiourea (fTU), 1,5,7triazabicyclo[4.4.0]dec-5-ene (TBD) (CAS 5807-14-7), 4-dimethylaminopyridine (DMAP) (CAS 1122-58-3), meta-(trimethylammonio)phenolate betaine (m-BE), 1,8-diazabicycloundec-7-ene (DBU) (CAS 6674-22-2), and an imidazolium salt (I-TFA). (+)-Sparteine (Aldrich, 99%), DBU (Acros, 98%), and TBD (Aldrich, 96%) were dried over BaO for 48 h, distilled under reduced pressure, and stored under N2. fTU was prepared according to a well-established protocol,12 dried under vacuum, and stored under N2. DMAP was dried under vacuum at 60 °C for 12 h and stored under N2. The m-BE and imidazolium salt were prepared as described in the literature13,14 and stored under N2. Metal-based catalysts were tin(IV) bromide (CAS 7789-67-5, Aldrich) and dibutyltin bis(2-ethylhexanoate) (CAS 2781-10-4, Aldrich). Received: October 22, 2014 Revised: December 8, 2014 Published: December 9, 2014 507

dx.doi.org/10.1021/bm5015443 | Biomacromolecules 2015, 16, 507−514

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pyridine (DMAP) as a ROP activator,16 other organic molecules have demonstrated their high activity for the preparation of aliphatic polyesters and polycarbonates with highly competitive kinetic parameters and control levels as compared with their metallic counterparts. Among them are currently reported: 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),17−20 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD),17,21 (+)-spartein/functionalized thiourea (fTU),12,22,23 an imidazolium salt (I-TFA),14 and meta-(trimethylammonio)phenolate betaine (m-BE)24 (Figure 1). Beyond favorable kinetic

(+)-Sparteine/fTU, DMAP, and dibutyltin bis(2-ethylhexanoate) were dissolved in DMSO (100 mM), whereas TBD, DBU, m-BE, imidazolium salt, and tin(IV) bromide were prepared in ultrapure water (100 mM) without any preliminary purification. 2. Cell Culture. The FHs74Int fetal human intestinal epithelial cell line (ATCC number CCL-241) was obtained from the American Type Culture Collection (St. Cloud, MN). Cells were cultured in Dulbecco’s modified Eagle’s medium high glucose (Lonza, Belgium) supplemented with 10% fetal bovine serum gold, 10 mM HEPES, 2 mM L-glutamine, 1% nonessential amino acids, 100 U/mL penicillin, 100 μg/mL streptomycin, 30 ng/μL epidermal growth factor, and 10 μg/μL insulin and maintained at 37 °C in a humidified atmosphere of 5% CO2 in air. The HepaRG human hepatocellular carcinoma cell line was obtained from Biopredic International (Saint-Grégoire, France). Cells were cultured in William’s E medium supplemented with 10% of fetal bovine serum, 2 mM L-glutamine, 100 units/mL penicillin, 100 μg/mL streptomycin, 5 μg/mL insulin and 5 × 10−5 M hydrocortisone hemisuccinate. After 2 weeks, the medium was changed to a differentiation medium provided by the manufacturer corresponding to “supplemented” William’s E medium with 2% DMSO for 2 more weeks. This proprietary medium allows differentiating cells into biliary-like and hepatocyte-like cells. 3. MTT Cytotoxicity Assay. Both FHs74Int and HepaRG cells were seeded in 96-well plates (2 × 104 cells per well) and grown at 37 °C for 22−26 h before a 50 μL aliquot of a base-2 logarithmic dilution of tested compound was added (concentrations 0.8 to 200 μM). After 24 h, the medium was replaced by 200 μL of MTT [3(-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] solution in PBS (0.5 mg/mL), and the plate was incubated at 37 °C. After 4 h, the supernatant was replaced by 100 μL of DMSO to dissolve reduced formazan crystals and the absorbance was measured with a Thermo Labsystems Multiskan Ascent (Thermo Fisher Scientific, Waltham, MA) at 570 nm versus a 690 nm reference. Percentages of cell viability were expressed compared with control. The fitted curves “% of cell viability” versus “log (catalyst concentration)” allowed the determination of IC10, IC50, and IC90, the catalyst concentrations that inactivate cellular growth by 10, 50, and 90%, respectively.

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Figure 1. Typical organocatalysts used in ring-opening polymerizations of lactones and cyclic carbonates.

and control feature aspects, this widespread development is motivated by the possibility to lift restrictions on the use of metalfree polymeric materials in fields where high levels of quality are a prerequisite, for example, for in vivo implants, drug formulation, tissue engineering, or biodegradable plastics. Therefore, the cytotoxicity of the above listed catalysts has to be evaluated. The MTT cytotoxicity assay is based on the measurement of cells metabolic activity. A tetrazolium salt is reduced by metabolically active cells in purple formazan crystals that are dissolved in DMSO for colorimetric measurement. Assuming that the amount of generated formazan is directly proportional to the amount of metabolically active cells, the proportions of viable cells can be inferred from spectrophotometric measurements.25 This rapid, sensitive, reproducible, and inexpensive MTT assay is considered as a good preliminary indicator for the

RESULTS AND DISCUSSION

The field of organic catalysis for ROP of cyclic esters and cyclic carbonates has rapidly expanded and a wide range of catalysts are now available for standardized synthesis protocols.15 Following the report of Nederberg et al., who used 4-dimethylamino-

Table 1. IC10, IC50, and IC90 Determined for Organic and Metallic Catalysts from Fitted Curves “% of Cell Viability” versus “log (Catalysts Concentration)”a organocatalysts IC10 DMAP (+)-sparteine fTU (+)-sparteine/fTU DBU TBD m-BE I-TFA

IC50 FHs74Int

HepaRG

FHs74Int

HepaRG

FHs74Int

138.7 μM n.a. 4.0 μM 2.6 μM n.a. 130.6 μM 75.4 μM n.a.

n.a. n.a. n.a. 2.5 μM n.a. n.a. n.a. n.a.

n.a. n.a. 11.4 μM 6.1 μM n.a. n.a. n.a. n.a.

n.a. n.a. 27.5 μM 32.4 μM n.a. n.a. n.a. n.a. metallic catalysts



IC10 tin(IV)bromide dibutyltinbis(2-ethylhexanoate) a

IC90

HepaRG

IC50

IC90

HepaRG

FHs74Int

HepaRG

FHs74Int

HepaRG

FHs74Int

n.t. n.t.

n.a. 2.5 μM

n.t. n.t.

n.a. 3.1 μM

n.t. n.t.

n.a. 4.2 μM

Legend: n.a., not applicable (inhibitory concentration not reached in the tested concentration range); n.t., not tested in present work. 508


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Figure 2. Cell viability determined by the MTT assay for organocatalysts on HepaRG human hepatocellular carcinoma cells. Results are expressed as average ± standard deviation (SD); data from two separate experiments in triplicate.

in treatment of cancer, caused 40% of cells death at 193.0 μM on FHs74Int normal cells (full data not shown). The association (+)-sparteine/fTU displays an important cytotoxicity on both cell models. Because sparteine alone does not show any toxicity against both cell models, the functionalized thiourea appears entirely responsible for the observed cytotoxicities. A comparative study of the cytotoxicity of a series of mono- and disubstituted thiourea has shown that substituted

toxicity and biocompatibility of polymers and their degradation products.26−28 Catalysts were tested on HepaRG and FHs74Int cell lines over a wide range of concentrations (from 0.8 to 200 μM), presumably extending way over their likely residual levels in polymers. Table 1 presents the IC10, IC50, and IC90 values determined from viability curves fitted from the HepaRG and FHs74Int data (Figures 2 and 3, respectively). As positive control, 5-fluorouracil, a well-known cytotoxic compound used 509


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Figure 3. Cell viability curves determined by the MTT assay for organocatalysts on FHs74Int fetal human intestinal epithelial cells. Results are expressed as average ± SD; data from three separate experiments in triplicate.

thiourea cytotoxicity is structure-dependent29 but not correlated with the lipophilicity of the compound. The first step in the bioactivation of thiourea is the oxidation of the thionosulfur by a flavin-monooxygenase (FMO), which results in the formation of sulfenic acid. The oxidation of glutathione by sulfenic acid leads to oxidative stress that may result in cell death or oxidation of biomolecules, proteins, lipids, and nucleic

acids. According to Onderwater et al.,29 the toxicity of N-phenylthiourea would not be the result of a FMO-based aromatic ring bioactivation. However, a possible metabolic oxidation of another part of the molecule by cytochrome P450 isoenzymes could explain a structure dependency of substituted thiourea cytotoxicity30 Because this is the first time that fTU is assessed for cytotoxicity, we are unable to affirm whether the 510


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Figure 4. Cell viability curves determined by the MTT assay for metallic catalysts on FHs74Int fetal human intestinal epithelial cells. Results are expressed as average ± SD; data from three separate experiments in triplicate.

Table 2. In Vitro and in Vivo Toxicity Assay Performed for Catalysts Studied in the Present Paperb,e catalyst

in vitro toxicity assay

in vivo toxicity assay

reference

tin(IV) bromide CAS: 7789-67-5 dibutyltinbis(2-ethyl hexanoate) CAS: 2781-10-4

n.a.

n.a.

n.a.

33

(+)-sparteine CAS: 90-39-1

n.a.

functionalized thiourea (fTU) 1,5,7-triazabicyclo[4.4.0]dec5-ene (TBD) CAS: 5807-14-7

n.a.

deer mice: approximate lethal dose (acute oral toxicity): 1075 mg/kg LDfra: 62 mg/kg/day Wistar rats: LD50 (acute toxicity) of 30 mg/kg n.a. n.a.

35

4-dimethylaminopyridine (DMAP) CAS: 1122-58-3

meta-(trimethylammonio) phenolate betaine (m-BE) 1,8-diazabicycloundec-7-ene (DBU) CAS: 6674-22-2 imidazolium salt (I-TFA)

The cytotoxicity of the P(EEP-co-EMEP)c 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) Vibrio f ischeri: EC50d of 0.315 mg/mL

LD50

34

33, 36, 37 deer mice: oral, 450 mg/kg mice: oral, 350 mg/kg/day rat: oral, 250 mg/mL fly: oral, 0.15 mg/mL

n.a.

n.a.

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.

n.a.

38

n.a.

a

LD50, dose that causes death of 50% animals of the tested group. bIC50-IC25, concentration which inhibits 50 and 25% of cell proliferation, respectively. cEEP, 2-ethoxy-2-oxo-1,3,2-dioxaphospholane; EMEP, 2-ethoxy-4-methyl-2-oxo-1,3,2-dioxaphospholane. dEC50, effective DMAP concentration at which V. f ischeri bioluminescence was reduced by 50%. eNR50, concentration of surfactant required to induce a 50% inhibition in neutral red uptake; n.a., not applicable.

cytotoxicity is due to the thiourea or the fluorinated aromatic ring moieties. This issue needs further investigation notably on the in vitro and in vivo metabolism of the compound. The IC values of functionalized thiourea and (+)-sparteine/fTU association were compared across the two cell lines using a two-way ANOVA with posthoc test (Bonferroni correction). Statistically significant difference (p < 0.001) between the two cell models was obtained only for concentrations that inhibit cell growth by 90% (IC90). This difference could be explained by the metabolic capacity of the HepaRG compared with FHs74Int or by differences in sensitivity/resistance of the two cell models. Indeed, FHs74Int cells have been shown to be more resistant to cytotoxicity than cancer cell lines.31,32 This may also explain why DMAP, TBD, and m-BE show cytotoxicity only against the HepaRG cell line at high concentrations (>20 μM). Interestingly the metallic catalyst tin(IV)bromide does not show any cytotoxicity against FHs74Int cells contrary to dibutyltinbis(2-ethylhexanoate)

that proved to be very toxic to the intestinal cell model (Figure 4 and Table 1). These data indicate that cytotoxicity is certainly compounddependent and does not correlate with the metallic or nonmetallic nature of the catalyst. Because polymer usages used to be mostly industrial, there is only a few in vitro and in vivo toxicity assays published for the catalysts studied herein (Table 2). With the development of recyclable, biodegradable, and biocompatible polymer, the toxicity (both environmental and human) has become an important parameter that has to be taken into account. Catalysts are the most susceptible elements promoting reactions with toxicological impact and have to be carefully tested for toxicity before they can be used in polymer in such application fields. The assumption that organocatalysts are less toxic than metallic catalysts is not supported by objective data in the state-of-the art, and we can even conclude from our results that it is not necessarily the case. By using 511


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Figure 5. (A) Required amount of catalyst to produce 1 kg of poly(lactide) polymer of 20 000 g/mol. (B) Percentage of cell viability for estimated catalyst concentration in 100 μg of poly(lactide) polymer of 20 000 g/mol using FHs74Int and HepaRG cell models.

cells but also on their metabolic activities and on the capacity of tested compounds to eventually chemically reduce tetrazolium salts.39 Furthermore, as some compounds could affect cell integrity by targeting specific cell compartments or functions and be missed by MTT measurements, a complete assessment of toxicity should be obtained only by combining different quantitative methods.25,27

normal intestinal and metabolically active hepatic cell lines we increase the range of toxicity that can be observed because the results also reflect the metabolization aspect, but it will be very interesting to further testing the influence of such a metabolization on catalysts cytotoxicity. To link our results with ROP-process, we started from the hypothesis that 100% of the catalyst is released in vivo from the biodegradable polymer and extrapolated the cell viability for estimated catalyst concentration in 100 μg of poly(lactide) polymer of 20 000 g/mol (Figure 5). By using 100 μg of 20 000 g/mol polymer, we are consistent with in vivo conditions on the one hand, and on the other hand we fit in the range of catalysts concentrations tested for all catalysts whatever their quantity in polymer after ROP process (Figure 5 A). Except for fTU, our results demonstrate the relatively low toxicity of organocatalysts against normal intestinal and metabolically active hepatic cell models. Although the MTT assay is a very good preliminary indicator of a compound cellular toxicity, the results should be interpreted with caution. Indeed, although a correlation can often be derived between viable cell number and MTT-reducing activity,26 MTT data depend not only on the number of viable

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CONCLUSIONS This study demonstrated the relatively low toxicity of organocatalysts over concentrations presumably extending way over their likely residual levels in polymers, which will contribute to promote their interest in the field of green chemistry for all applications where toxicity and environmental concerns are a key issue. While many papers have hypothesized the absence of toxicity of metal-free catalysts, herein we clearly stated that most of them are indeed biocompatible for the range of concentration investigated at the exception of functionalized thiourea that was found to be very toxic against both cell models. This one, at the equivalency of tin IV-based structures, 512


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should be subjected of thorough toxicity studies before validating its usage in medical device or in green chemistry applications. Given the probable long-term use of polymeric implantable devices, further research is warranted on the biological consequences of residual catalysts and initiators possibly slowly leeching from the polymer.

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AUTHOR INFORMATION

Corresponding Authors

*B.B.: Tel: 0032 65 37 34 92. Fax: 0032 65 37 34 26. E-mail: [email protected]. *L.M.: Tel: 0032 65 37 34 82. Fax: 0032 65 37 34 84. E-mail: [email protected]. Notes

The authors declare no competing financial interest. All three laboratories are members of the Research Institute for Health Sciences and Technology.

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ACKNOWLEDGMENTS A.N. gratefully acknowledges the fellowship granted to her by UMONS. HepaRG cells (patented by Inserm Transfert) are supplied by Drs. Christiane Guguen-Guillouzo, Philippe Gripon, and Christian Trepo (INSERM’s laboratories U522 & U271) and used under a Material Transfer Agreement between INSERM U522 & U271 and the University of Mons, Belgium. CIRMAP is grateful to the “Belgian Federal Government Office Policy of Science (SSTC)” for general support in the frame of the PAI-7/05, the European Commission and the Wallonia Region (FEDER Program) and OPTI2MAT program of excellence. The laboratory of Pharmaceutical Analysis expresses its gratitude to FNRS (Fonds National de la Recherche) for support via the FRSM Grant 3.4614.11. O.C. is Research Fellow of the F.R.S.-FNRS.

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ABBREVIATIONS ROP, ring-opening polymerization; FDA, food and drug administration; MTT, methyl-thiazol-tetrazolium; fTU, homeprepared functionalized thiourea; TBD, 1,5,7-triazabicyclo[4.4.0]dec-5-ene; DMAP, 4-dimethylaminopyridine; m-BE, meta-(trimethylammonio)phenolate betaine; DBU, 1,8-diazabicycloundec-7-ene; I-TFA, imidazolium salt; DMSO, dimethysulfoxide; PBS, phosphate-buffered saline; IC, inhibitory concentration; SD, standard deviation; ANOVA, analysis of variance

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REFERENCES

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