Hiroko Takagi

Chiami Moyama

Keiko Taniguchi

Kota Ando

Ryohei Matsuda

Shota Ando

Hiromi Ii

Susumu Kageyama

Akihiro Kawauchi

Nora Chouha

Laurent Désaubry

Susumu Nakata

Affiliations

Fluorizoline Blocks the Interaction between Prohibitin-2 and γ -Glutamylcyclotransferase and Induces p21 Waf1/Cip1 Expression in MCF7 Breast Cancer Cells

Keywords: Prohibitin-2, fluorizoline, -glutamylcyclotransferase, p21 WAF1/CIP1, cell cycle arrest, cancer

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Abstract

Prohibitin-2 (PHB2) is a scaffold protein that has pleiotropic functions, which include interacting with -glutamylcyclotransferase (GGCT) in the cytoplasm and repressing the transcriptional activities of the p21 Waf1/Cip (p21) gene in the nucleus. The cytotoxic drug fluorizoline binds to PHB1/2 and exerts antiproliferative actions on cancer cells. However, the precise mechanism underlying the antiproliferative effects of fluorizoline is not fully elucidated. In the present study, we first show that fluorizoline induces p21 expression in several human cancer cell lines, including MCF7 breast cancer cells. Treatment of MCF7 cells with fluorizoline suppressed proliferation and prevented cells from entering into the DNA synthesis phase. Knockdown of p21 rescued the suppressed proliferation, indicating that fluorizoline inhibited MCF7 cell growth via the induction of p21. Overexpression of PHB2 in MCF7 cells prevented the induction of p21 expression by fluorizoline, and restored the antiproliferative effects and blockade of cell cycle progression. Moreover, treatment of MCF7 cells with fluorizoline inhibited the interaction between endogenous PHB2 and GGCT proteins, and reduced the level of nuclear localization of PHB2 proteins.

These results indicate that targeting PHB2 with fluorizoline induces the expression of p21 and consequently blocks proliferation of cancer cells.

Significance Statement

This study shows that fluorizoline may be a promising novel anticancer drug candidate that induces p21 expression and blocks cell-cycle progression in human cancer cell lines. In addition, we show that fluorizoline inhibits the interaction between PHB2 and GGCT and reduces the nuclear localization of PHB2 proteins.

Introduction

Prohibitin-1 (PHB1) and -2 (PHB2) are scaffold proteins that have pleiotropic functions. These functions include the regulation of many cellular processes such as growth factor signaling pathways, the immune response, mitochondrial dynamics, cell migration, and transcriptional activities of estrogen receptor [START_REF] Ande | Prohibitin: a potential therapeutic target in tyrosine kinase signaling[END_REF][START_REF] Chowdhury | PHB in Cardiovascular and other diseases: present knowledge and implications[END_REF][START_REF] Koushyar | Unveiling the potential of prohibitin in cancer[END_REF][START_REF] Peng | Multifaceted role of prohibitin in cell survival and apoptosis[END_REF][START_REF] Theiss | The role and therapeutic potential of prohibitin in disease[END_REF][START_REF] Wang | Prohibitin ligands: a growing armamentarium to tackle cancers, osteoporosis, inflammatory, cardiac and neurological diseases[END_REF]. PHB1/2 interact with various factors important for determining cell fate, such as C-RAF, Akt, IKK, MLK2, Shp1/2, and the 14-3-3 scaffold proteins. Due to the regulatory impact of PHB1/2 on cell proliferation, PHB1/2 are novel molecular targets for anticancer therapeutics [START_REF] Wang | Prohibitin ligands: a growing armamentarium to tackle cancers, osteoporosis, inflammatory, cardiac and neurological diseases[END_REF].

To facilitate the development of a new class of anticancer drugs, several small molecules targeting PHB1/2 have been synthesized [START_REF] Wang | Prohibitin ligands: a growing armamentarium to tackle cancers, osteoporosis, inflammatory, cardiac and neurological diseases[END_REF].

An example of one promising molecule is fluorizoline, which directly binds to PHB1/2 and is cytotoxic to cancer cells [START_REF] Pérez-Perarnau | A trifluorinated thiazoline scaffold leading to pro-apoptotic agents targeting prohibitins[END_REF].

Fluorizoline induces apoptosis in acute myeloid leukemia cells [START_REF] Pomares | Targeting prohibitins induces apoptosis in acute myeloid leukemia cells[END_REF] and chronic lymphocytic leukemia cells [START_REF] Cosialls | The prohibitin-binding compound fluorizoline induces apoptosis in chronic lymphocytic leukemia cells through the upregulation of NOXA and synergizes with ibrutinib, 5-aminoimidazole-4-carboxamide riboside or venetoclax[END_REF][START_REF] Wierz | The prohibitin-binding compound fluorizoline induces[END_REF], as well as in mouse embryonic fibroblasts and Hela cells, where this effect is mediated by upregulation of NOXA and BIM [START_REF] Moncunill-Massaguer | A novel prohibitin-binding compound induces the mitochondrial apoptotic pathway through NOXA and BIM upregulation[END_REF]. In Hela cells, fluorizoline prevents activation of MEK1/2 upon EGF stimulation by disrupting the interaction between CRAF and active RAS (Yurugi et al., 2017). Moreover, fluorizoline inhibits protein synthesis in cancer cells due to its effects on multiple components of the translational machinery, including promoting phosphorylation of initiation factor 2 (eIF2) and elongation factor 2 (eEF2) [START_REF] Jin | The prohibitin-binding compound fluorizoline affects multiple components of the translational machinery and inhibits protein synthesis[END_REF]. Recently, it was confirmed that the cytotoxicity of fluorizoline in nematodes and human cancer cells requires the expression of PHB1/2 [START_REF] Saura-Esteller | Fluorizoline-induced apoptosis requires prohibitins in nematodes and human cells[END_REF]. Despite these studies indicating the anticancer potential of fluorizoline, the precise mechanisms that underlie the antiproliferative effects of this compound are not fully understood.

Increasing evidence shows that -glutamylcyclotransferase (GGCT; formerly known as chromosome 7 open reading frame 24) promotes the proliferation of cancer cells. GGCT catalyzes the production of 5-oxoproline and free amino acids from gamma-glutamyl peptide to promote glutathione biosynthesis. It is highly expressed in a wide range of cancer types, including urothelial, breast, lung, brain, and ovary cancers [START_REF] Kageyama | Mechanisms of Tumor Growth Inhibition by Depletion of γ-Glutamylcyclotransferase (GGCT): A Novel Molecular Target for Anticancer Therapy[END_REF].

Knockdown or pharmaceutical inhibition of GGCT blocks proliferation of various human cancer cell lines. GGCT knockdown in MCF7 and MDA-MB-231 breast cancer cells upregulates the cyclin-dependent kinase inhibitors p21 WAF1/CIP1 (p21) and/or p16 INK4a to promote autophagy, followed by cellular senescence [START_REF] Matsumura | Depletion of γ-glutamylcyclotransferase inhibits breast cancer cell growth via cellular senescence induction mediated by CDK inhibitor upregulation[END_REF]Taniguchi et al., 2018a;[START_REF] Taniguchi | Depletion of gamma-glutamylcyclotransferase inhibits cancer cell growth by activating the AMPK-FOXO3a-p21 axis[END_REF].

Recently, we showed that PHB2 is a novel binding partner of GGCT (Taniguchi et al., 2018b). The interaction between PHB2 and GGCT promotes the nuclear translocation of PHB2, which binds to the promoter region of the p21 gene, thereby repressing transcription of the p21 gene in MCF7 breast cancer cells. The finding that PHB2 is a key regulator of the expression of p21, which is a pivotal cell cycle regulator and a downstream target of p53 [START_REF] El-Deiry | WAF1, a potential mediator of p53 tumor suppression[END_REF], led us to investigate the effects of fluorizoline on p21 expression in cancer cells. In the present study, we show that fluorizoline induces p21 expression in several human cancer cell lines, and also demonstrate that fluorizoline suppresses cell cycle progression, blocks the interaction between PHB2 and GGCT, and inhibits the nuclear localization of PHB2 in MCF7 cells.

Materials and Methods

Cell culture

MCF7 human breast cancer, A172 glioblastoma, and PC3 prostate cancer cells were obtained from RIKEN BioResource Center (Tsukuba, Japan) and cultured in DMEM supplemented with 10% fetal bovine serum (HyClone, South Logan, UT, USA) and 1% penicillin and streptomycin. Cells were maintained in 5% CO 2 at 37°C.

Reagent

The PHB ligand fluorizoline was synthesized following an established procedure [START_REF] Pérez-Perarnau | A trifluorinated thiazoline scaffold leading to pro-apoptotic agents targeting prohibitins[END_REF] and dissolved in dimethyl sulfoxide (DMSO).

Cell proliferation assay

Briefly, 1 × 10 5 MCF7, A172 and PC3 cells per well were seeded in 6-well plates and treated with fluorizoline at the indicated concentrations. A trypan blue dye exclusion test was performed using 0.4% trypan blue solution (Wako Pure Chemical Industries, Osaka, Japan) and a Countess II automated cell counter (Thermo Fisher Scientific, Waltham, MA, USA). Cell viability assays were performed using cell count reagent SF (WST-8 assay, Nacalai Tesque, Kyoto, Japan) and a CellTiter-Glo kit (Promega, Madison, WI, USA).

Antibodies

The following antibodies were used: mouse monoclonal antibodies specific for p21 (dilution 1:200, 556430, BD Biosciences, Franklin Lakes, NJ, USA), GAPDH (dilution 1:1000, 016-25523, Wako), the V5 epitope tag (V5) (dilution 1:1000, R960-25, Thermo Fisher Scientific), and PHB2 (dilution 1:200, Sc-133094, Santa Cruz Biotechnology, Dallas, TX, USA); a sheep monoclonal antibody against GGCT (dilution 1:500, AF5086, R&D systems, Minneapolis, MN, USA ); and a rabbit monoclonal antibody against Lamin B1 (dilution 1:1000, 12987-I-AP, Proteintech, Rosemont, IL, USA). Horse anti-mouse IgG-HRP conjugates were purchased from Vector Laboratories (dilution 1:2000, PI-2000, Burlingame, CA, USA). Rabbit anti-sheep IgG-HRP conjugates were purchased from Thermo Fisher Scientific (dilution 1:5000, 81-8620). The HRP-linked goat anti-rabbit IgG (dilution 1:2000, 7074) and mouse IgG1 isotype controls (5415) were purchased from Cell Signaling Technology (Danvers, MA, USA). A mouse GGCT mouse monoclonal antibody (TSS-M01) was purchased from Cosmo Bio (Tokyo, Japan).

Western blot analysis

Western blot analysis was performed as described previously (Taniguchi et al., 2018b). Briefly, cell lysates were prepared in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 0.1% SDS) supplemented with a protease inhibitor cocktail (Nacalai Tesque). The proteins were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Burlington, MA, USA). After blocking with 3% fat-free dried milk in TBS/0.05% Tween-20 (TBST), or PVDF blocking reagent for Can Get Signal (TOYOBO, Osaka, Japan), the membranes were incubated with primary antibodies in 3% BSA/TBST, Can Get Signal immunoreaction enhancer solution (TOYOBO), or signal enhancer HIKARI solution (Nacalai Tesque). Proteins were visualized using the Clarity Western ECL substrate (Bio-Rad, Hercules, CA, USA) or Chemi-Lumi One Super (Nacalai Tesque) and were detected using the ChemiDoc XRS plus system (Bio-Rad).

Transfection of siRNA targeting p21

Synthesized siRNAs were purchased from RNAi (Tokyo, Japan) or Gene Design (Osaka, Japan). The siRNA sequences were as follows: non-targeting, 5'-GUACCGCACGUCAUUCGUAUC-3' (sense) and 5'-UACGAAUGACGUGCGGUACGU-3' (anti-sense); p21, 5'-CUUCGACUUUGUCACCGAG-3' (sense) and 5'-CUCGGUGACAAAGUCGAAG-3' (anti-sense). Transient transfection of siRNA targeting p21 was performed using Lipofectamine RNAiMAX (Invitrogen)

according to the manufacturer's protocol. Briefly, MCF7 and PC3 cells were seeded in 6-well plates (5 × 10 4 cells / well), cultured for 24 h, and transfected with siRNAs at a concentration of 10 nM. At 24 h post-transfection, the cells were treated with fluorizoline 1 μM or 5 μM for 48 h, and then collected and analyzed with Western blot and cell counting as described above.

PHB2 overexpression

The procedure was carried out as described in (Taniguchi et al., 2018b).

Briefly, the pENTR221-PHB2 vector (clone no. 100011434, DNAFORM, Yokohama, Japan) was recombined into the pEF-DEST51 vector using a Gateway LR Clonase II enzyme mix (Thermo Fisher Scientific). Next, 2 × 10 6 MCF7 cells were transfected with 2 µg V5-tagged PHB2 vector or pEF-DEST51 empty vector by electroporation using an Amaxa cell line Nucleofector kit V and a Nucleofector 2b device (Lonza, Visp, Switzerland) with a P-020 program optimized for MCF7 cells. DMEM containing 10 µg/ml blasticidin was used for clone selection.

qRT-PCR analysis

Total RNA was extracted from MCF7 cells lysed with TRIzol (Thermo Fisher Scientific) and purified with an RNeasy mini kit (Qiagen, Hilden, Germany). cDNA was synthesized by reverse transcription using ReverTra Ace qPCR RT master mix (TOYOBO). Synthesized cDNA was analyzed by RT-qPCR with THUNDERBIRD SYBR qPCR mix (TOYOBO) using a LightCycler 96 system (Roche Diagnostic, Indianapolis, IN, USA). Gene expression levels were normalized against the housekeeping gene hARF1. Specific primers were purchased from Eurofins Genomics (Tokyo, Japan): hARF1, 5'-GACCACGATCCTCTACAAGC -3' (forward) and 5'-

TCCCACACAGTGAAGCTGATG -3' (reverse); p21 WAF1/CIP1
, 5'-GCAGACCAGCATGACAGATTT-3' (forward); 5'-GGATTAGGGCTTCCTCTTGGA-3' (reverse).

BrdU incorporation assay

MCF7 cells were treated with 1 µM fluorizoline for 24 h. The proportion of MCF7 cells in the DNA synthesis phase was assessed after BrdU-pulse for 1.5 h using an APC BrdU flow kit (BD Biosciences) according to the manufacturer's instructions. At least 10,000 cells per sample were analyzed.

Cell cycle assay

MCF7 cells or A172 cells were seeded at 1.5 x 10 5 cells in 6 cm dishes.

The next day, the cells were treated with 1 µM or 5 µM fluorizoline for 24 h. The cells were harvested in PBS, and stained with a Cell Cycle Assay Solution Blue (Dojindo, Kumamoto, Japan). Cell cycle ratios of the stained cells were analyzed using FACS Fortessa (BD Biosciences).

Apoptosis assay

Apoptosis induction was assessed using the MEBCYTO Apoptosis Kit (MBL, Nagoya, Japan). Annexin V-positive and propidium iodide (PI)-negative cells in the early phase of apoptosis, Annexin V-positive and (PI)-positive cells in the late phase of apoptosis were detected by flow cytometry using a BD LSRFortessa X-20 cell Analyzer (BD Biosciences). At least 10,000 cells were analyzed for each experiment.

Co-immunoprecipitation

The antibody-bead complexes were prepared by incubating 10 µg anti-GGCT (6-1E, Cosmo Bio) or isotype control antibody with 1.

Statistical analysis

All data were obtained from at least three independent experiments and expressed as the mean ± S.D. Statistical analysis was performed using a two-tailed Student's t-test or ANOVA with the Excel software. A p-value < 0.05 was deemed significant.

Results

Fluorizoline inhibits MCF7 proliferation and induces p21 expression

First, we examined the effects of fluorizoline treatment on the growth of MCF7 breast cancer cells. A trypan blue dye exclusion test demonstrated that treatment of MCF7 cells with fluorizoline for 72 h suppressed cell growth in a concentration-dependent manner (Fig. 1A). The number of viable cells was significantly reduced after treatment with 1 µM fluorizoline for 24 h (Fig. 1A). We next examined whether fluorizoline treatment induces p21 expression. Western blot analyses demonstrated that treatment of MCF7 cells with fluorizoline for 24 h significantly increased p21 protein expression in a concentration-dependent manner (Fig. 1B). Fluorizoline also suppressed proliferation (Fig. 1A) and induced p21 expression (Fig. 1B) in A172 human glioblastoma and PC3 prostate cancer cells. A time-course analysis of 1 µM fluorizoline treatment in MCF7 cells showed that expression levels of p21 protein increased 3 h after treatment (Fig. 1C). A qRT-PCR analysis demonstrated that 1 µM fluorizoline significantly induced mRNA expression levels of p21 from 1 h treatment (Fig. 1D). These results indicate that fluorizoline induces p21 expression and inhibits proliferation of MCF7 breast cancer cells.

Fluorizoline induces cell cycle arrest but not Apoptosis

Cell cycle analysis revealed significant increases of the G0/G1 phase population and decreases of the S phase population with fluoizoline in MCF7 cells or A172 cells. However, no increase of sub G1 population in the cell-cycle analyses was observed (Fig. 2A-B). Furthermore, Annexin V positive cells were not observed in the fluoriozoline-treated MCF7 and A172 cells (data not shown). These results suggest that fluorizoline does not induced apoptosis in MCF7 cells and A172 cells but mainly induced cell-cycle arrest.

Induction of p21 mediates proliferative suppression of MCF7 cells by fluorizoline

Next, we performed knockdown of p21 in MCF7, A172 and PC3 cells to test whether inhibition of cell proliferation by fluorizoline would be restored by suppression of p21. We confirmed the efficient knockdown of p21 by Western blot analysis (Fig. 3A). The p21 knockdown significantly rescued the suppressed proliferation of MCF7, A172 and PC3 cells by treatment with 1 μM or 5μM fluorizoline for 48 h (Fig. 3B). These results indicate that fluorizoline inhibits proliferation of MCF7 cells in a p21-induction dependent manner.

Overexpression of PHB2 restores growth in fluorizoline-treated MCF7 cells

To confirm that fluorizoline suppresses MCF7 cell proliferation by inhibiting PHB2 activity we prepared PHB2-overexpressing MCF7 cells and examined the effects of fluorizoline. We confirmed the overexpressed PHB2 proteins by Western blot analysis (Fig. 4A). Assessments of proliferation in empty vector-transfected (Control) and PHB2-overexpressing (PHB2-OE) MCF7 cells treated with 0.1 µM or 1 µM fluorizoline for 48h, the WST-8 cell viability assay showed PHB2 overexpression restored cell growth at 48 h (Fig. 4B). The ATP-based cell viability assay showed partially yet significantly restored cell growth in the PHB2-overexpressing MCF7 cells treated with 1 µM fluorizoline for 24 h (Fig. 4C). Consistent with these findings, Western blot analyses demonstrated that overexpression of PHB2 prevented the fluorizoline-mediated increase in p21 expression (Fig. 4D). These results suggest that fluorizoline increases p21 expression and suppresses cellular proliferation by modulating the activity PHB2.

Overexpression of PHB2 restores cell cycle progression in fluorizoline-treated MCF7 cells

Since p21 is an important regulator of the cell cycle, we next investigated the effects of fluorizoline on cell cycle progression in parental and PHB2-overexpressing MCF7 cells. A BrdU incorporation assay demonstrated that fluorizoline significantly reduced the number of MCF7 cells in S-phase, and that PHB2 overexpression partially but significantly restored cell cycle progression (Fig. 5A-B). These results indicate that fluorizoline suppresses the growth of MCF7 cells by inhibiting cell cycle progression, and this effect is associated with increases in p21 expression.

Fluorizoline blocks the interaction between PHB2 and GGCT proteins and reduces the nuclear localization of PHB2

PHB2 interacts with GGCT and the interaction promotes the nuclear localization of PHB2 proteins, which then bind to the promoter of the p21 gene and repress transcription (Taniguchi et al., 2018b). We finally investigated the effects of fluorizoline treatment on the interaction between PHB2 and GGCT proteins.

Results of a co-immunoprecipitation assay using monoclonal antibodies against GGCT revealed that fluorizoline treatment blocked the interaction between endogenous PHB2 and GGCT proteins (Fig. 6A). Consistent with the results, PHB2 protein expression in the insoluble nuclear fraction (Fig. 6B) and nuclear localization of PHB2 (Fig. 7) were reduced after fluorizoline treatment. These results suggest that fluorizoline prevents PHB2 from interacting with GGCT, which then inhibits the nuclear localization of PHB2 proteins, resulted in the induction of p21 (Fig. 8).

Discussion

Here, we first show that fluorizoline induces p21 expression in human cancer cell lines. Our time-course analysis demonstrated that fluorizoline treatment as short as 1 h significantly increased the mRNA levels of p21, suggesting that fluorizoline directly influences p21 expression. Moreover, knockdown of p21, which prevented from its induction by fluorizoline treatment, significantly cancelled the proliferative inhibition of MCF7 cells by fluorizoline.

We provide evidence from a co-immunoprecipitation assay showing that fluorizoline blocked the interaction between PHB2 and GGCT. A previous study demonstrated that the interaction between GGCT and PHB2 promotes the nuclear translocation of the PHB2 protein, which plays an important role in repressing p21 expression (Taniguchi et al., 2018b). Consistent with those findings, we show that fluorizoline treatment markedly reduces the amount of PHB2 protein in a chromatin-bound insoluble nuclear fraction; indeed, PHB2 protein could not be detected in this fraction even after long chemiluminescence exposure times in Western blot analysis. This meant it was not feasible to perform chromatin immunoprecipitation analysis following fluorizoline treatment using PHB2 antibodies because of the absence of the target PHB2 proteins.

Our findings showing that overexpression of PHB2 restored the cellular

Financial Disclosure

No author has an actual or perceived conflict of interest with the contents of this article.

Footnotes

This work was supported by the Japan Society for the Promotion of Science (grant numbers 18K09192 and 20K07623) and the Takeda Science Foundation, a Scholarship for Young/Women Researchers of the PMAC. MCF7 cells were treated with 1 µM fluorizoline for 24 h. Immunofluorescent analysis was performed using anti-PHB2 (green) antibody and DAPI (blue).

Scale bar, 50 μm.

  5 mgDynabeads protein G (Thermo Fisher Scientific). MCF7, A172 and PC3 cells were cultured in 10 cm dishes, treated with fluorizoline at the indicated concentrations for the indicated durations, and then lysed in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate-Na, and 0.1% SDS) supplemented with a protease inhibitor cocktail (Nacalai Tesque). Mixtures comprising cell lysate and each antibody-bead complex were rotated overnight at 4°C. After washing with PBS, the complexes were eluted in SDS-sample buffer, denatured at 95°C for 5 minutes, and analyzed by Western blotting.Fractionation of nuclear/cytoplasmic proteins MCF7, A172 and PC3 cells were treated with fluorizoline at the indicated concentrations for the indicated durations, and then cellular proteins were fractionated into cytoplasmic and nuclear fractions using a LysoPure nuclear and cytoplasmic extractor kit (Wako), according to the manufacturer's protocol. Subcellular localization analysis of PHB2 proteins by immunofluorescent staining.MCF7 cells were treated with fluorizoline for 24 h. The cells were fixed with 10%(v/v) formaldehyde in PBS for 30 min at room temperature. Then, permeabilization was performed using 0.5% triton-X 100 containing PBS for 15 min at 4 ℃. After blocking with 0.1% triton-X 100, 1% BSA containing PBS for 45 min at room temperature, the cells were incubated in primary antibodies, anti-PHB2 antibody (dilution, 1:50) in Can Get Signal immunostain solution A (TOYOBO) for 1h at room temperature. Then cells were incubated in secondary antibodies, polyclonal rabbit anti-mouse Immunoglobulins FITC (Dako, Carpinteria, CA, USA) (dilution, 1:800) in Can Get Signal Immunostain Solution A for 1h at room temperature. Nuclei were counterstained with 4, 6-Diamidino-2-phenylindole (DAPI) (Lonza Basel, Switzerland) in PBS for 5min at room temperature. Images were obtained with an Eclipse Ti confocal microscope (Nikon, Tokyo, Japan).

  proliferation that was inhibited by fluorizoline indicate that fluorizoline targets PHB2. In particular, PHB2 overexpression prevented fluorizoline-mediated increases in p21 expression. The fluorizoline-mediated blockade of cell cycle progression was prevented by PHB2 overexpression, as shown by the BrdU incorporation assay. These results also indicate that the effects of fluorizoline were mediated by p21 induction. However, recovery of proliferation and cell cycle arrest by PHB2 overexpression were partial, suggesting existence of other mechanisms for fluorizoline or potential involvement of PHB1 instead of PHB2. It has been reported PHB2 exerts pleiotropic functions in the nucleus, where it acts as a transcriptional regulator binding to MyoD, MEF2, HDAC1/5, EZH2, RNF2, or estrogen receptors (Thuaud et al., 2013). Further clarification of the binding partner for PHB2 on the p21 promoter would provide useful information on the mechanisms that underly the complicated functions of PHB2. Our findings show that fluorizoline induces p21 expression in several human cancer cell lines. The increase in p21 expression, antiproliferative effects, and blockade of cell cycle progression mediated by fluorizoline treatment were reversed by PHB2 overexpression, indicating that fluorizoline specifically targets PHB2. Fluorizoline inhibited the interaction between GGCT and PHB2, which decreased the nuclear localization of PHB2. Taken together, these results shed new light on the mechanism of action of fluorizoline and illustrate how this small molecule can be used to explore new aspects of PHB signaling. apoptosis in chronic lymphocytic leukemia cells ex vivo but fails to prevent leukemia development in a murine model. Haematologica 103(4):e154-e157. Yurugi H, Marini F, Weber C, David K, Zhao Q, Binder H, Désaubry L and Rajalingam K (2017) Targeting prohibitins with chemical ligands inhibits KRAS-mediated lung tumours. Oncogene 36(33):4778-4789.

Fig. 1 .

 1 Fig. 1. Fluorizoline inhibits MCF7 proliferation and induces p21 expression.

Fig. 2 .

 2 Fig. 2. Fluorizoline induces cell cycle arrest.

Fig. 3 .

 3 Fig. 3. p21 knockdown restores cell proliferation suppressed by fluorizoline.

Fig. 4 .Fig. 5 .

 45 Fig. 4. PHB2 plays a crucial role in regulating changes in p21 expression and

Fig. 6 .Fig. 7 .

 67 Fig.6. Fluorizoline blocks the interaction between PHB2 and GGCT and

Fig. 8 .

 8 Fig. 8. Fluorizoline disrupts the interaction of PHB2 with GGCT and its

Acknowledgments

We thank Momoka Endo for experimental support.

Authorship Contributions

Participated in research design: Kageyama, Kawauchi, Désaubry and Nakata.

Conducted experiments: Takagi, Moyama, Taniguchi, K. Ando, Matsuda, S.

Performed data analysis: Takagi, Moyama, Taniguchi, K. Ando, Matsuda, S.

Ando, Ii, Chouha, Désaubry and Nakata.

Wrote or contributed to the writing of the manuscript: Takagi, Moyama, Taniguchi, Désaubry and Nakata.


Noémie Gil

Dr Constance Thomas

Rana Mhanna

Jessica Mauriello

Dr Romain Maury

Dr Benjamin Leuschel

Jean-Pierre Malval

Jean-Louis Clément

Dr Didier Gigmes

Dr Catherine Lefay

Jean-Pierre Malval

Jean-Louis Clément

Olivier Soppera
email: olivier.soppera@uha.fr

Yohann Guillaneuf
email: yohann.guillaneuf@univ-amu.fr

] N Gil

Dr J.-L Clément

Dr J.-P Malval

Dr J.-P Malval

Thionolactone as a Resin Additive to Prepare (Bio)degradable 3D Objects via VAT Photopolymerization**

Keywords: 3D printing, degradable materials, radical ringopening polymerization, photopolymerization, thionolactone

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Additive manufacturing and more specially 3D printing has revolutionized the classic industrial manufacturing by creating directly a 3D object that was designed on a computer using successive layering of materials. [1] The rapid development of this technique was first made by the ease of the fused deposition modeling (FDM) technique that allows to prepare 3D objects by the computer-controlled layer by layer deposition of molten or semi-molten polymer via an extrusion nozzle. [1] Nevertheless, 3D printing has become extremely popular with the development of light-cured processes that are summarized by the term VAT photopolymerization. [1] Among these processes, Digital Light Processing (DLP) [1][2] in particular has attracted attention due to several attractive features, such as fast build, low resolution (<100 μm features), wide ink viscosity tolerance and low cost. The 3D object is thus obtained by the cross-linking of a liquid resin consisting mainly of multifunctional (meth)acrylates derivatives. The photopolymerization of acrylates occurs rapidly, with high yields, and generates almost no byproducts. This led to a cross-linked material whose mechanical properties could be tuned by the monomers structure. Moreover, such object is constituted by a C-C bond covalent network that impart a high thermal and chemical stability. This permanent cross-linked network could also be detrimental for diverse reasons. First the high stability is incompatible with facile degradability and re/upcyclability. The high volume/amount of 3D-printed thermoset will be incinerated or stored in landfills following use, making this so promising manufacturing process not environment-friendly or sustainable. [3] Secondly, this high stability is also not compatible with subtractive manufacturing, [4] that is a technique where printed materials could be removable or erasable after its formation. Such approach [5] is very interesting to easily remove templates or sacrificial parts of 3D objects, a technique that is widely used when the object is a positive print before casting and lastly decomposed such as in jewelry or to prepare ceramics. Recently very elegant approaches were proposed by Barner-Kowollik and coworkers that developed new monomers and resins for two-photon lithography based either on thiol-ene chemistry, [6] TAD-polyether ligation, [7] silane-based monomers, [8] o-nitrobenzyl ester monomers, [9] and chymotrypsin-cleavable monomers. [10] With these chemistries, stimuli and/or controlled degradation could be obtained. Specific and home-made di or multi-(meth)acrylate monomers with a core containing an acid-degradable acetal [11] or basic-degradable poly(beta amino ester) [START_REF] Muralidharan | Advanced Functional Materials[END_REF] were also prepared and presented a good degradability. In the context of biomaterials, poly(propylene fumarate) (PPF) was also used to print stents for example. [START_REF] Luo | [END_REF] To increase the degradability Bowman and Anseth [14][15][16][17] as well as Patton and coworkers [18][19] showed that the use of similar specific thiol-ene monomers containing a degradable core could be used. In that case the use of thiol-ene chemistry allowed the preparation of more homogeneous networks that impart good degradation profile. [16] Nevertheless, such thiol-ene polymers also tend to be too soft for many applications and are more devoted to biomaterials such as hydrogels. [1] The groups of Zhao [20] and Bowman [21] reported the 3D printing of linear polymers as a way to impart reprintability or recyclability to the printed object. Nevertheless, such materials have a lower chemical resistance due to the polymer solubility in organic solvent.

Another possibility to induce recycling of cross-linked 3D objects could be to use the concept of dynamic covalent networks [22] also known as vitrimers. Such approach in combination with 3D printing has been already investigated. [23][24][25] In contrary to these techniques the polymer ablation of conventional resins has received less attention, [26][27] since this technique is more difficult to achieve and seems less promising.

All the previously mentioned techniques either require to prepare specific monomers with degradable core that are the main component of the resins and thus cannot be readily used (due to the high quantity of monomers that has to be prepared for each 3D printing) on commercially-available resins/printers. Moreover, the properties of the photopolymers are impacted by such structure and it is difficult to find the optimized composition to target both degradability and desired mechanical properties.

Herein we thus introduce a new approach to impart degradability of 3D objects by adding a comonomer, here the dibenzo[c,e]oxepane-5-thione (DOT) only as an additive (2 wt%) to impart degradability to conventional acrylate resin (Figure 1.a). This approach is also based on the comonomer approach that has been developed by Johnson et al. [28][29] on thermoset made by Ring-Opening Methathesis Polymerization (ROMP), where he showed that if the labile/degradable linkage is located into the backbone instead of the knots, fewer amount of labile/degradable moieties is required (Figure 1.b). This study showed that the cleavable monomer approach with only a few wt% of additive is possible, [29] leading to materials with similar mechanical properties but that could be turned soluble after degradation.

Radical ring-opening polymerization (rROP) is a technique that has been developed in the 80s and that allows the incorporation of heteroatoms in the polymer backbone via a mechanism of addition/fragmentation. [30] This technique has been rejuvenated recently by the copolymerization of common vinyl monomers and such cyclic derivatives to impart (bio)degradation of conventional materials. [30] Two main cyclic monomer families emerged, the cyclic ketene acetals (CKAs) [30][31] and the thionolactones [32][33] . The non-favorable reactivity ratio between CKA and (meth)acrylates (rCKA close to 0.01 and r(meth)acrylate close to 4) [34] led to a low and non-homogeneous incorporation of the labile group in the materials. [35] To impart good degradability, high initial amounts of CKA are required or a continuous feed of monomers [36] could be used to counterbalance it. In the case of (meth)acrylate resin for 3D printing, both solutions cannot be implemented since high rate of polymerization is required implying using CKA only as additive and only bath resin can be used in 3D printer. As a reference, preliminary tests were performed using CKA inserted in (meth)acrylate resin and do not lead to any degradation (see Table S1 for details). Unlike CKAs, thionolactones and in particular dibenzo[c,e]oxepane-5-thione (DOT) introduced by Roth and coworkers [32] followed by Gutekunst et al. [33] presented an efficient copolymerization behavior (statistical incorporation of the two monomers) with acrylate derivatives, imparting homogeneous degradation that could occur via either hydrolysis or aminolysis (Figure 1.c). The combination of DOT/acrylate polymerization and VAT photopolymerization could be an efficient way to prepare (bio)degradable 3D objects. It has been shown that only few percents (5-10 vol%) of cleavable units are required to impart degradability of thermosets [28][29] and also that DOT has a slightly higher rate of incorporation [33] than acrylate derivatives during their copolymerization, allowing to use a very low amount of DOT as additive. ) under UV exposure for the photopolymer loaded or not with DOT (2 wt%). c) Samples prepared by 3D-printing for mechanical properties testing. d) Comparison of main mechanical properties of material with and without DOT.

Results and Discussion

The trifunctional character ensures a very fast initial polymerization speed, then the rapid increase in viscosity leads to an important gelification of the medium which stops the polymerization and prevents a higher conversion. The addition of DOT (2 wt%) decreases, as expected, the efficiency of the photopolymerization, both for the initial speed and the final conversion (50 %), but keep them still totally compatible to an 3D printing process. This effect can be easily explained by the absorption of DOT which acts as a filter effect for the BAPO, decreasing the amount of light available for photolysis of the photoinitiator. It should also be noted that a decrease of the kinetics during the copolymerization of acrylate derivatives and DOT is already observed during the thermal polymerization in solution. [32][33] Finally, it can be observed that it is possible to reduce the concentration of BAPO to 0.2 wt%, while maintaining effective polymerization, with in particular sufficient cross-linking of the polymer to confer interesting mechanical properties to the material, as observed qualitatively after irradiation. This result justifies the percentage of 0.2 wt% of BAPO used in the following.

Figure 2.c shows that it is possible to use DLP-UV to very easily fabricate polymer specimens that can be used to quantitatively evaluate the mechanical properties of the formed polymers. It can be seen that the addition of DOT only sligthly modifies the mechanical properties of the polymer by leading to a slightly softer and less brittle polymer (Figure 2.d). This property can be correlated with the reduced photopolymerization efficiency induced by the addition of DOT. Nevertheless, Young modulus, tensile strength and elongation values remain in the same order of magnitude, meaning that DOT can be added up to 2 wt% without changing too significantly the mechanical properties of the polymerized material. PETIA monomer is a reference monomer in microstructuring applications, 2D and 3D. [37][38][39] It is shown in the following that the addition of DOT does not degrade the resolution and the possibility to use it for 2D and 3D stereolithography applications, at the micro and macroscopic scale. In a first approach, a laser microstructuring configuration is used to demonstrate the ability of the material to be used for laser-induced 3D microfabrication. It is a laser microstructuring configuration at the output of optical fibers that has demonstrated its interest for applications in photonics or sensors. [40][41][42] Here, we use this configuration essentially as a microstructuring setup (Figure 3) that allows to test a new material in this microstructuring context with respect to its reactivity and resolution. The experimental procedure is described schematically in Figure 3.a. Further experimental details can be found in SI and reference [37] . Briefly, an optical fiber is cleaved and the resin is deposited at the cleaved end of the fiber. A 375 nm laser is coupled into the optical fiber and the light emerging from the fiber induces photocrosslinking in the volume located in the extension of the core of the optical fiber. After rinsing by immersion for a few seconds in ethanol, the microtip is obtained on the optical fiber. The photonic conditions (power, time), the geometrical and mechanical properties of the polymer formed allow to apprehend the reactivity of different photopolymers in this context of microfabrication. As shown in Figure 3.b, the microstructuring can be efficiently carried out using the DOT doped formulation. As expected after examination of the polymerization kinetics, the reaction is less efficient and the irradiation parameters need to be adjusted, by increasing the irradiation power and/or time (see SI for details).

From this result, we now consider the possibility of degrading the polymer after photopolymerization, as described schematically in Figure 1. To do this, we observe the behavior of microtips with or without DOT, in different solutions, a priori suitable to degrade the polymer. First, a KOH/MeOH/THF mixture is used. In this mixture, KOH acts as a base for the hydrolysis of the polymer. The MeOH/THF mixture is selected to solubilize KOH and provide a solvent that can swell the polymer and thus promote its dissolution. As can be seen in Figure 3.b, the results after immersion are very different with and without DOT in the photopolymer: without DOT, the microtip remains attached to the end of the optical fiber after 15 min, showing the chemical resistance of this highly crosslinked polymer. With DOT in the resin, it is removed after only 5 seconds. Degradation is also obtained in different mixtures, as shown in Figure 3.c. KOH/MeOH (without THF) and KOH in water also degrade the microtip, but with less efficiency, as demonstrated by longer degradation times. Degradation is in any case longer for objects with various sizes, which is consistent with a degradation process as shown in Figure 1. At this stage, however, it is difficult to exclude a mechanism that would implement a detachment of the microtip from the fiber end by degradation of the polymer at the interface. The results shown in Figure 3.d confirm the degradation of the material in the volume. Indeed, in this experiment, a macroscopic object (Dali mask) was fabricated by DLP-UV. This object was then immersed in the KOH/MeOH/THF solution.

After 17 h, the object is taken out of the solution and placed on a substrate to be photographed. One can easily notice the already started degradation of the object. After 24 h, this degradation is even more marked and finely, after 90 h, the object is no longer visible in the solution. This result therefore undoubtedly confirms the role of DOT in the degradation mechanism. Very remarkably, a DOT concentration of only 2 wt% allows this property to be obtained.

It seems quite logical that the degradation of macroscopic objects is significantly longer than that of microscopic objects. Moreover, the use of THF is important for these macroscopic objects to allow KOH access to hydrolysable functions. Based on this result, we further demonstrate the possibility of manufacturing various objects by VAT photopolymerization. Different objects are manufactured, with different geometries, as shown in Figure 4.a and Figure 4.b using both setups, which are interesting to build 2.5 and 3D objects with arbitrary shapes. As can be seen, the residual yellow coloration is visible but the parts keep their transparency. For relatively thin objects such as the CNRS logo, the coloration can be attenuated after bleaching.

The degradation property of PETIA/DOT combined with the stability of PETIA makes it possible to consider manufacturing objects with degradable and non-degradable parts using the same DLP-UV printer. This possibility is demonstrated in Figure 4.c. In this example, the strategy is as follows: in a first step, objects (ghosts and balls) are made with the non-degradable PETIA/BAPO resin. The object is then placed in the degradable resin with 2 wt% DOT and a second pattern is printed (Pacman figure). At the end, the object is a Pacman head in which objects are included. This matrix is soluble in the usual solution (5 wt% KOH in MeOH/THF) and its dissolution releases the included objects which are not degraded in the time necessary for the disappearance of the degradable matrix. This example is only intended to show the proof of concept of this new approach and the perspectives opened.

Figure 5 demonstrates that DOT-doped resin can also be used in two-photon stereolithography (TPS). To demonstrate this (Figure 5.a), micrometer grids (50x50 μm) were produced with different laser power (irradiation wavelength 800 nm). As for UV microfabrication, the resin doped with DOT was slightly less reactive but it could be used with photoreactivity in the same order of magnitude. A test of dissolution of the microgrids showed a complete degradation after 6 hours. Interestingly, it appeared that the dissolution time was increased when laser power was increased. This can be explained by a higher crosslinking of the polymer network at higher laser power. This proof of concept opens the doors towards 3D fabrication of arbitrary structures at the microscale as shown in In this last section, we further explore the degradation conditions for 3D objects. Before the 3D printing, cm-scale small tablets were prepared using a polypropylene mold previously printed by FDM (see Figure S1 for details) to find the best degradation conditions. The weight and visual aspect of these tablets were monitored during the decomposition process (Figure 6). According to the literature data, the degradation of the copolymers bearing thioesters weak bond could be done either via aminolysis using primary amines [32][33] or using strong bases such as TBD or KOH/NaOH. We thus started the degradation at room temperature using a 50 vol% of N-isopropylamine in dichloromethane, 2.5 wt% of TBD in THF and 5 wt% of KOH in MeOH/THF. We also prepared a pellet without DOT as a reference. After 3 months there is no degradation for both pellets using either N-isopropylamine or TBD (see SI for details). This could be related to the difficult diffusion of such compounds into the dense PETIA-based 3D network. Nevertheless, the use of 5 wt% of KOH in MeOH/THF led to the complete dissolution of the pellet in 1 week whereas the reference pellet was not degraded in 3 months (Figure 6a). To speed-up the degradation, we thus increase either the concentration of KOH or increase the temperature. Such kind of degradation conditions (120 °C, 10 wt% KOH in EtOH, 24 h) were already used by Mecking et al. [43] to perform the degradation of solid polyethylene objects containing weak ester linkages. Using these improved conditions, the complete dissolution of this dense cm-scale object could be obtained in 24 h (Figure 6a).

The main goal of this study was the 3D printing of a degradable objects and we thus used our formulation (PETIA + 2 wt% DOT + 0.2 wt% BAPO) and the reference PETIA resin with a commercial 3D printer (Miicraft 125, 385 nm). The complete parameters used for these printings are given in ESI and the same parameters were used for both resins. We first printed a hollow cube as a proof of concept. The printed objects are depicted in Figure 6b. It can be seen that the introduction of DOT inside the formulation do not modify the 3D printing since a similar object is obtained. Such cubes were then subjected to degradation. In this case, due to the porosity of the cube we choose to perform the degradation using 5 wt% of KOH in MeOH/THF at room temperature and without agitation.

After 8 h the hollow cube that was prepared with 2 wt% of DOT totally degraded (see the video in SI and Figure 6b) whereas the reference cube was not affected. This experiment validates the concept of degradable thermoset via the introduction of weak bond via radical ring-opening polymerization. These hollow cubes (the pristine PETIA and the DOT dopped cube) were also subjected to biodegradation using a home-made compost. Both cubes were inserted into the compost and the degradation monitored visually. The results are presented in Figure 6c. After 20 days in the compost, the DOT-dopped cube is quite totally degraded whereas the PETIA one was not affected.

To further showcase the interest of our approach, an Eiffel Tower was also printed to ensure that a 3D object with a high resolution could be obtained and degraded. The results are gathered on Figure 7a and confirmed that this approach could be used on cm-scale 3D objects with a high resolution. The degradation of this Eiffel Tower presented an inhomogeneous degradation with the thinner part being degraded before the thicker ones. This feature is in good agreement with our previous findings correlating the rate of degradation with the size of the object. This prompt use to investigate the potential use of this approach to address the issue of support structures. Indeed, printing complex geometries with VAT photopolymerization usually require support structures that have to be manually removed afterward. In some cases, such structures are not easily accessible, and their removal could be difficult to perform or could be time consuming since it has to be done manually. Moreover, the preparation of easily removable/cleavable support structure could enable the printing of stacked 3D objects in the zdimension. To illustrate this feature, we prepare two dices that are printed vertically (z-direction) using support structures (Figure 7b).

The dices were printed either using the PETIA and the DOT-dopped resins. In both cases the 3D objects were obtained and were immersed in a gentle basic solution (5 wt% KOH in MeOH, RT, without agitation). After one hour, the dices printed with the DOTdopped resin dissociated (Figure 6b) whereas the reference still possessed its support structures.

Conclusion

In conclusion, we showed in this work that the introduction of 2 wt% of a specific thionolactone, i.e. the dibenzo[c,e]-oxepane-5thione (DOT) that is known to copolymerize efficiently with acrylate monomers, into an acrylate based-3D resin confer a degradability property to the obtained 3D objects. Moreover, we showed that the mechanical properties of the final product are only slightly affected since only small amount of DOT was used as additive. This approach has then been applied to microstructuring configuration, two-photon stereolithography, DLP set-up and finally to commercial 3D printer. At all scales, the objects could be degraded using basic conditions. Such conditions are nevertheless dependent of the size of the object. In all cases the complete dissolution of the objects occurred that allow first to strongly decrease the volume of wastes that could be produced via VAT photopolymerization and secondly will make more feasible the use of substractive manufacturing since only an additive could be added to common acrylate-based resins. As an example, using the size-dependent rate of degradation of the printed materials, stacked 3D object (zdirections) could be prepared and whose separation could be done gently in solution. This approach could thus be improved by developing new monomers of radical ring-opening polymerization that do not absorb in the UV range and whose reactivity with methacrylate derivatives is efficient (i.e. reactivity ratios allowing statistical incorporation of thioesters in the polymer backbone).

Figure 1 .

 1 Figure 1. Schematic representation of the writing/erasing process. a) structure and mechanism of the radical polymerization involving the DOT/acrylate monomer pair. b) efficient cleavage of the thermoset via the comonomer approach (cleavage of 10 bonds in the case of both cleavable knots or cleavage linkage in the polymer backbone) adapted from ref. [19], c) Resin formulation, preparation of 3D objects via VAT 3D printer and cleavage mechanism.

  Resin formulations were prepared by mixing only 2 wt% of DOT with pentaerythritol triacrylate (PETIA) as model multi-acrylate monomer and 0.2-0.5 wt% of bis(2,4,6-trimethylbenzoyl)-phenyl phosphine oxide (BAPO) as photoinitiator. Figure2.a compares the absorption spectra of the resins, with and without DOT. The monomer chosen is transparent in UV (λ > 350 nm) and the photoinitiator was chosen for its absorption at the wavelength of interest in this work (365-385 nm), both for laser microstructuring and for the DLP printer. DOT, added at 2 wt%, shows significant absorption in the UV and visible (< 500 nm) range. This absorption corresponds to a yellow coloration of the resin and is due to the (C=S)-O-group. Once polymerized and due to the isomerization (Figure1.a), the resulting ring-opened (C=O)-Sthioester bond loose its color. It should thus be noted that this coloration also decreases strongly after UV irradiation (Figure2.a). Nevertheless, since the overall conversion is not complete during the printing, the 3D objects are still yellow. The absorption of the DOT slightly modifies the reactivity of the resin but does not alter too much the polymerization rate. It has to be reminded that UV resins for 3D printing usually comprise photoabsorbers to control the penetration depth of actinic light. The kinetics of the photopolymerization were studied to quantify the effect of the DOT on the photopolymerization process. The kinetics are plotted in Figure2.b. They are based on monitoring the C=C conversion of the acrylate functions in the monomer. With a BAPO concentration of 0.5 wt%, the polymerization is efficient, leading to a final conversion of more than 60 % and a conversion plateau quickly reached. This type of kinetics is classical for such a triacrylate monomer.

Figure 2 .

 2 Figure 2. a) UV-vis absorption spectra of monomer (PETIA), monomer + photoinitiator (BAPO, 0.2 wt%), monomer + photoinitiator + DOT (2 wt%) before and after UV irradiation. b) Photopolymerization kinetics followed by FTIR (conversion of C=C band at 1635 cm -1

Figure 3 .

 3 Figure 3. a) Steps of process for preparing polymer microtips at the end of optical fiber showing the photopolymer drop deposition, exposure through the optical fiber and microtip after development (loaded with 2 wt% DOT in this case). b) Dissolution experiments with KOH (5 wt%) in MeOH/THF showing the efficiency of

Figure 4 .

 4 Figure 4. a) Examples of objects made by DLP-printing, b) 3D-printing (scale bar is 1 cm) and c) Embedding a non-degradable object in the degradable resin: example of a degradable Pacman head containing ghosts and balls that are non-degradable.

Figure 5 :

 5 Figure 5: a) Two-photon stereolithography of 50x50 μm microgrid structures and their dissolution in KOH/MeOH/H2O. Grids were written using a 40X, 0.65-NA objective with increasing laser input power from 4.8 to 9.6 W. b) SEM images of a 20x20x19 μm pyramid prepared by TPS with a resist doped with DOT. c) top view of 3 pyramids prepared with DOT. d) Example of pyramid with the same formulation without DOT. e) Optical microscopy images of dissolution tests of the micropyramids prepared with (left column) and without DOT (right column). Scale bar is 20 μm.

  Figure 5.b ans 5.c. The micro-pyramid shown in the SEM image illustrates that no significant loss of resolution is visible as compared to the reference formulation not doped with DOT (Figure 5.d). Dissolution tests (Figure 5.e) cleary shows that objects doped with DOT can be dissolved, unlile objects non-doped with DOT.

Figure 6 .

 6 Figure 6. a). Degradation of the PETIA-based pellets using various degradation conditions. b) 3D printing of hollow cubes using a MiiCraft 125 UV-printer and their degradation (5 wt% of KOH in MeOH/THF at room temperature and without agitation). c) Biodegradation of the hollow cubes in a home-made compost.

Figure 7 .

 7 Figure 7. a) Preparation of the Eiffel Tower using the Miicraft 3D printer and its degradation using 5 wt% of KOH in MeOH/THF at room temperature and without agitation. b) Preparation of stack dices using the Miicraft 3D printer and its degradation using 5 wt% of KOH in MeOH/THF at room temperature and without agitation.

Acknowledgements

We thank the CNRS for support and the French National Research Agency (ANR-18-CE06-0014 and ANR-19-CE19-0012) for the PhD funding of Noémie Gil and Constance Thomas.

Entry for the Table of Contents

3D UV-printing produces cross-linked materials at the expense of degradability. The introduction of thionolactone (2 wt% of dibenzo[c,e]-oxepane-5-thione (DOT)) in acrylate-based resins confer (bio)degradability via the introduction of thioester units into the network via a radical ring-opening polymerization process. The resin was used in different set-up including commercial 3D printers and the objects were shown to degrade in basic solution.

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