S. Moinsa,
P. Loyerb,
J. Odent
a and
O. Coulembier
*a
aLaboratory of Polymeric and Composite Materials, Center of Innovation and Research in Materials and Polymers (CIRMAP), University of Mons (UMons), Place du Parc 23, 7000 Mons, Belgium. E-mail: olivier.coulembier@umons.ac.be
bInserm, INRA, Univ Rennes, Institut NUMECAN (Nutrition Metabolisms and Cancer) UMR-A 1341, UMR S 1241, F-35000 Rennes, France
First published on 4th December 2019
A one-pot, two-step method for the preparation of degradable PEG is here presented. The full process addresses the requirements imposed by green chemistry and involves the use of a single and nontoxic non-eutectic mixture of organocatalysts. The strategy relies on the polycondensation of PEG800 after its functionalization by bio-derived 5-membered γ-butyrolactone.
In the present contribution, we provide a complete description of PEG polycondensates synthesis in solvent-free condition respecting the workable definition of green chemistry. In substance, a one-pot/two-step process has been applied to both end-functionalize an oligoethylene glycol synthon and induce its polycondensation to obtain a mimetic and degradable PEG. Such a process involves the use of a Non-Eutectic Mixture of Organocatalysts (NEMO), is free of waste and avoid the use and production of toxic and/or hazardous reagents and solvents.
Due to its low strain energy,9 the bio-derived five-membered γ-butyrolactone (γ-BL) is referred as a “non-polymerizable” monomer. At the exception of reactions realized under cryogenic conditions,10 the ring-opening polymerization (ROP) of γ-BL is limited to low oligomerization under ambient pressure and high temperature due to its high equilibrium monomer concentration.11 Such incapability of polymerizing has been put to good use to end-functionalize an oligoethylene glycol (MW 800 g mol−1, PEG800) by few units of γ-BL. After screening different reaction conditions, we selected a non-stoichiometric complex of methanesulfonic acid (MSA) and 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) (3:
1) to catalyse the process at 130 °C. The interest of such Non-Eutectic Mixture of Organocatalysts (NEMO) relies on its ability to conduct bulk polycondensation of a variety of diols,12 offering then the possibility to both functionalize and polycondensate the PEG800 in a one-pot process (Scheme 1).
![]() | ||
Scheme 1 Expected end-functionalization of PEG800 and polycondensation one-pot/two-step process catalyzed by NEMO. |
Prior to any polycondensation reaction, the functionalization of PEG800 was studied and optimized. After screening different conditions, it appeared that the optimum conditions of functionalization in terms of time and efficiency of reaction necessitate to use a 10-fold excess of γ-BL as compared to the PEG800 and an amount of catalyst of 0.2 molar equivalent ([PEG]0/[γ-BL]0/[NEMO]0 = 1/10/0.2). Under such conditions, and as attested by 1H NMR analysis, 52% of the PEG800 hydroxyl end-groups were functionalized by ∼1.5 units of γ-BL after 4 hours. Increasing the reaction time to 6 hours does not allow to improve that conversion. To respond to the challenges addressed by the green chemistry in terms of waste, a simple vacuum treatment of the medium (prior to the polycondensation reaction) allows the excess of unused γ-BL to be recycled and reused for another reactions. Fig. 1 presents the 1H NMR analyses of the PEG800 after reaction with excess of γ-BL and treated under vacuum (Fig. 1A) as well as the product of the evaporation (Fig. 1B). As compared to the 1H NMR analysis of the γ-BL, the recovered and unreacted monomer proved chemically pure and reusable.
![]() | ||
Fig. 1 1H NMR analyses (in CDCl3, r.t.) of the PEG800 after end-functionalization and vacuum treatment (A) and of the product of evaporation (B). |
Recently some of us reported on the bulk self-condensation of aliphatic diols as a route to polyether homo- and copolymers.12 Following the same strategy, the polycondensation of the end-functionalized PEG800 was realized by taking advantage of the NEMO already present in the macromonomer. Briefly, the medium was kept under vacuum and was the object of successive thermal treatments starting at 130 °C for 24 h, 180 °C for 24 h and finishing by a final step at 200 °C for 24 h. Note here that during the first thermal treatment the excess of γ-BL was recovered and reused for another reaction. The polycondensation was monitored using 1H NMR spectroscopy by following the disappearance of the hydroxymethylene protons present between δ 3.72 ppm and δ 3.80 ppm. Fig. 2 presents the comparison between the end-functionalized PEG800 before and after the three thermal treatments. As expected, the hydroxymethylene protons disappeared to the benefit of a narrower polyether signal at δ 3.64 ppm while butyryl ester groups are still present (here represented by the methylene oxycarbonyl protons at δ 4.22 ppm).
![]() | ||
Fig. 2 1H NMR analyses (in CDCl3, r.t., zoomed between δ 3.55 ppm and δ 4.30 ppm) of the end-functionalized PEG800 before (blue spectrum) and after thermal treatments (red spectrum). |
SEC analyses of the polycondensates obtained after each thermal treatment show unimodal traces with dispersity values close to 2.0 and Mp,SEC increasing gradually in function of the thermal treatment applied (Fig. 3). In a view of future applications as micelle-forming copolymers, the molar mass of the prepared mimicking PEG was limited to 17 K g mol−1 knowing that most of the PEG hydrophilic block involved in biomedical micelles are comprised between 1 and 15 kDa.13
Lastly, the degradation of the mimicking poly(ethylene glycol) polycondensate14 (Mp,SEC = 17250 g mol−1) was performed by hydrolysis of the ester linkages in a 0.5 M NaOH solution. After 15 hours under stirring at r.t., the solution was lyophilized, and the recovered product analysed by SEC and 1H NMR analyses.
The 1H NMR analysis attests on the total degradation of the ester linkages as exemplified by the disappearance of the methylene oxycarbonyl signal initially present at δ 4.22 ppm and the reappearance at δ 3.72 ppm of the hydroxy methylene end-group protons of the PEG (Fig. 4). SEC analysis also concludes on a total degradation of the mimicking PEG by demonstrating that the as-obtained degraded product presents a similar chromatogram to the one of the PEG800 used before NEMO treatment (Fig. S1, ESI‡).
![]() | ||
Fig. 4 1H NMR analyses (in CDCl3, r.t., zoomed between δ 3.0 and δ 4.7 ppm) before (top) and after degradation of the mimicking PEG, Mp,SEC = 17![]() |
When considering toxicity, the biocompatibility of the initial polymer, the degradation products and the remaining catalyst must be taken into account. Literature data indicate that PEG does not present unquantified risk to humans15 and P(γ-BL) easily degrades to give γ-hydroxybutyric acid, a naturally occurring metabolite in the body.16 As NEMO catalyst remained in our mimicking PEG (∼5.5 mol% after one precipitation), we then found crucial to study its putative cytotoxicity in vitro (Fig. 5). The effect of NEMO catalyst on cell viability was first studied using the differentiated human hepatocyte-like HepaRG cells (Fig. 5A), which express most of the liver specific enzymes involved in xenobiotic metabolism17 allowing to assess hepatic toxicity of synthetic compounds.18 The NEMO catalyst was added to culture media at concentrations ranging from 0.065 to 13 mM and the cell viability was assayed by measuring the intracellular ATP content. The NEMO catalyst did not affect cell viability up to 1.3 mM while at 3.2 and 6.5 mM the ATP content was significantly decreased with an IC50 at 8.53 ± 0.24 mM. At the highest concentration of 13 mM, the ATP content was below the detection level demonstrating that only high concentrations of compound were cytotoxic. The toxicity of the NEMO catalyst was also evaluated with progenitor HepaRG cells (Fig. 5B) that actively proliferate but are undifferentiated. The NEMO catalyst also induced a strong cytotoxicity only at the high concentrations of 6.5 and 13 mM. To determine the mechanism of toxicity, we first studied the plasma cell membrane integrity of progenitor HepaRG cells using the trypan blue stain (ESI 1‡). Less than 15 minutes after incubating the cells with medium containing 13 mM of NEMO catalyst, the integrity of plasma cell membrane was lost correlating with a pH value at 2.91 (Fig. 5C and ESI 1‡). To prevent the pH decrease in culture medium, 100 mM Hepes buffer were added to culture medium that maintained the pH at 7 (Fig. 5C). In this condition, the NEMO catalyst did not significantly affect the ATP content even at the highest concentration of 13 mM.
Footnotes |
† Preparation of amphiphilic micelles based on our mimicking PEG is under investigation. Design, characterization and biological significance will be the subject of a forthcoming paper. |
‡ Electronic supplementary information (ESI) available. See DOI: 10.1039/c9ra09781c |
This journal is © The Royal Society of Chemistry 2019 |