Novel poly(azoamide triazole)s containing twin azobenzene units in the backbone. Synthesis, characterization, and in vitro degradation studies

Polymer Degradation and Stability 193 (2021) 109726 Contents lists available at ScienceDirect Polymer Degradation and Stability journal homepage: www.elsevier.com/locate/polymdegradstab Novel poly(azoamide triazole)s containing twin azobenzene units in the backbone. Synthesis, characterization, and in vitro degradation studies Adrián Suárez-Cruz, Inmaculada Molina-Pinilla, Khalid Hakkou, Cristian Rangel-Núñez, Manuel Bueno-Martínez ∗ Departamento de Química Orgánica y Farmacéutica. Facultad de Farmacia. Universidad de Sevilla, C/ Profesor García González 2, 41012 Sevilla, Spain a r t i c l e i n f o Article history: Received 5 June 2021 Revised 15 September 2021 Accepted 19 September 2021 Available online 28 September 2021 Keywords: Click polymerization Degradable polymer Azo polymer Azobenzene Dithionite reduction Biodegradable polymer a b s t r a c t We describe the synthesis and characterization of four new light and reduction sensitives poly(azoamide triazole)s, in which the azobenzene units are found along the main chain of the macromolecule. These polymers were prepared by the azide-alkyne cycloaddition reaction catalyzed with copper (I) (CuAAC). They were obtained in high yield and with apparent molecular weights in the range from 95 to 148 kDa. All poly(azoamide triazole)s are soluble in polar aprotic solvents, and two of them are also soluble in chloroform showing good coating and film-forming properties. They were characterized by Fourier transform infrared, nuclear magnetic resonance (NMR), ultraviolet-visible spectroscopy and gel permeation chromatography (GPC). The photoisomerization study of the synthesized polymers has been carried out by UV–Vis spectroscopy, as well as their trans - cis - trans reversibility behavior. Differential scanning calorimetry (DSC) and themogravimetric analysis (TGA) were used to investigate their thermal properties. Results show that the polymers were amorphous and stable up to 300 °C under nitrogen. The hydrolytic degradation of films of these polymers has been studied in vitro under various conditions of pH and temperature and was monitored by GPC. Furthermore, the presence of azo units along the polymer backbone as cleavable groups provides access to their degradation by reduction. In this sense, the degradation of polymers has also been studied using sodium dithionite as a mimic of the enzyme azoreductase. The results of these studies show that the polymers are stable enough under hydrolytic physiological conditions, but they degrade rapidly when sodium dithionite is used. A preliminary study of biocompatibility of polymers PAAT1 and PAAT4 has been carried out. A hemolysis study with human red blood cells (hRBC) and a cytotoxicity study with human gingival fibroblasts (HGnF) have been carried out. The results obtained suggest that these polymers could be good candidates to be used as drug coating materials. ©2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ) 1. Introduction The design and preparation of drug delivery systems that allow the release of bioactive compounds in a controlled and localized way have received a great deal of attention in recent decades. These systems, which were initially sensitive to a particular stimulus, such as light, pH, oxidizing or reducing conditions, certain enzymes, or temperature [1–9] , have been the basis to develop systems that respond to dual or multiple signals, such as redox and pH [10] , temperature and pH [11] , temperature and enzyme [12] , ∗Corresponding author. E-mail address: [email protected] (M. Bueno-Martínez). reducing conditions and light [13] , enzyme and pH [14–16] ; light and pH [17–19] ; light and enzyme [20] and others [21] . Azo derivatives have been widely used for several applications, for example, as surfactants [22] , gelators [23] , liquid crystals [24] or biocides [25] . Azobenzene derivatives have also been used for the preparation of stimulus-sensitive drug delivery systems based on their photochemical behavior [26] . As it is well known, azobenzenes are photosensitive chromophores that have an unique sensitivity to light (or heat), which causes reversible trans - cis photoisomerization [27] leading to significant changes in their molecular size and dipole moments [28] . This photoisomerization process can also have an impact on other characteristics of the azo group, such as its sensitivity to reduction. Boulègue and collaborators have observed an increase in the reduction rate of cis https://doi.org/10.1016/j.polymdegradstab.2021.109726 0141-3910/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ) A. Suárez-Cruz, I. Molina-Pinilla, K. Hakkou et al. Polymer Degradation and Stability 193 (2021) 109726 azobenzene in relation to the trans isomer [29] , which may allow a greater control of the degradation of this type of material. There are a wide variety of applications in biomedicine that use the sensitivity of azo polymers to light and reduction [ 14 , 30–38 ]. Although azobenzene derivatives can also be used in any tumor tissue that suffers from hypoxic conditions or that expresses azoreductases, as for example, in the case of liver cancer cells [39] , we are interested in their use in the design and preparation of systems that allow the vectorization of drugs to the colon through oral administration. Many compounds containing azobenzene in their structure have been studied for enabling oral administration and colonic release of specific drugs. Thus, various non-polymeric prodrugs [40–44] , as well as macromolecular prodrugs which contain the azo group in the main or side chain, have been developed [45–53] . The bioactive compounds can be protected by polymeric coatings or matrixes based on azobenzene to reach the colonic delivery. In seminal work, Saffran and coworkers first used this approach for oral administration of azo polymer-coated peptide drugs [54] . In this way, drugs are protected from the adverse environmental conditions faced by these bioactive compounds in the gastrointestinal tract, delaying the release until the combination of azo-derivative materials and drugs reaches the colon. Azo compounds degrade in the colon because microbiome excretes the enzyme azoreductase, which can reduce selectively the azobenzene group to aromatic amines [ 36 , 55–57 ]. Although azoreductase enzymes have the most suitable characteristics to carry out the reduction of azo group, managing this type of enzyme at laboratory conditions can be difficult and results heavily depend on the gene codification of the enzyme. For this reason, other non-biological compounds such as hydrazine hydrate [ 58 , 59 ] or sodium dithionite [ 30 , 31 , 35 , 60 , 61 ] can be used for the same purpose. In particular, it is common to find the use of sodium dithionite since this reducing agent acts in a gentle way, it is cheap and potentially bioorthogonal [38] . The design of azo polymers that exhibit adequate sensitivity under biological and chemical conditions is an important research objective with implications in different fields. We previously reported on the synthesis of linear copoly(azoester triazole)s containing different amounts of the prodrug olsalazine in the polymer backbone [53] . Herein, we describe the synthesis and characterization of four new light and reduction sensitive poly(azoamide triazole)s, in which the azo units are found along the main chain of the macromolecule. These polymers were prepared by the azidealkyne cycloaddition reaction catalyzed with copper (I) (CuAAC). The hydrolytic degradation of these polymers has been studied under various conditions of pH and temperature. Likewise, the degradation using sodium dithionite as a mimic of the enzyme azoreductase has also been studied. 2. Material and methods 2.1. Materials All solvents and reagents were obtained from Merck and were used without further purification. 96-wells plates, Petri dishes, bovine fetal serum, Dulbecco’s Modified Eagle’s Medium, antibiotics, phosphate buffered saline (PBS) and trypsin were obtained from Thermo Fisher Scientific (Massachusetts, USA). Human gingival fibroblasts were purchased from Innoprot (Vizcaya, Spain). 2.2. Measurements Thin-layer chromatography (TLC) was performed on Silica Gel 60 F254 (E. Merck) with detection by UV light or charring with H 2 SO 4 or phosphomolybdic acid. Flash column chromatography was performed using E. Merck Silica Gel 60 (230–400 mesh). Fourier transform infrared (FTIR) spectra were recorded on a JASCO FT/IR-4200 spectrometer in the wavenumber range from 650 to 40 0 0 cm −1 using films or KBr disks. 1 H and 13 C NMR spectra were recorded in the CITIUS of the Universidad de Sevilla using a Bruker AV300, Bruker AMX-500 or Bruker AVIII-700 spectrometers. Chemical shifts are reported as parts per million (ppm) and are referenced to the residual solvent signals as the internal standard. Two-dimensional 1 H–1 H homonuclear and 13 C1 H heteronuclear shift correlation spectra were recorded with the COSY and HETCOR pulse sequences, respectively. Elemental analyses were carried out in the Microanalysis Laboratories of the CITIUS Service at the Universidad de Sevilla. Chemical ionization (CI) and fast-atom bombardment mass spectra were performed on a Micromass Autospec spectrometer. FABMS spectra were obtained using thioglycerol-NaI as a matrix. The thermal behavior of the polymers was examined by differential scanning calorimetry (DSC) using a TA DSC Q200 Instrument, calibrated with indium. Samples of about 2–3 mg were heated at a rate of 10 °C/min under a nitrogen flow rate of 20 mL/min and cooled to −35 °C. Thermogravimetric analyses (TGA) were carried out by a SDT Q600 TA instrument at a heating rate of 10 °C/min under a nitrogen flow of 100 mL/min, and the temperature range was from room temperature to 600 °C. The size exclusion chromatography instrument consisted of a Waters apparatus equipped with a Waters 2414 refractive-index detector and two mStyragel HR columns (7.8 mm ×300 mm) linked in series, thermostatted at 60 °C. N, N -dimethylformamide containing 0.5 mg/mL LiBr was used as the eluent with a flow rate of 1.0 mL/min. Twelve polystyrene samples of narrow molecular weight distribution were used to calibrate the apparatus. Absorbances at 540 and 570 nm were measured using a Biotek Synergy HT plate reader (Vermont, USA). UV–visible spectra were recorded using a spectrophotometer UV-1280 (Shimadzu) in quartz cuvettes with 10 mm length of the optical pathway. The UV light source consisted of OSRAM Ultravitalux 300 W, set at 20 cm (2 mW / cm 2 ) from the sample. The visible light source was a Schott KL1500 LCD set at 1 cm from the sample. 2.3. Synthesis of the monomers Ethyl 4-(4-hydroxyphenyl)azobenzoate (1) [62] . A solution of NaNO 2 (4.8 g, 56.4 mmol) in water (30 mL) was added dropwise to a stirred suspension of ethyl 4-aminobenzoate (9.3 g, 56.2 mmol) in HCl 2 M (100 mL) previously cooled around 0–5 °C. Afterwards, phenol (6.34 g, 67.3 mmol) was added. The reaction mixture was kept cooled and stirred for 90 min. Then the reaction mixture was neutralized with a saturated solution of NaHCO 3 and filtered. Finally, the solid was recrystallized in methanol: water (1:1), obtaining 1 as a brownish crystalline solid. Yield: 9.4 g (63%), m. p. 162– 164 °C; IR: v max 3389 (OH), 1691 (CO), 1591 cm −1 (Ar); NMR data (300 MHz, CDCl 3 ): 1 H, δ8.17 (d, 2H, H-b), 7.95–7.85 (2d, 4H, H-c, H-f), 6.97 (d, 2H, H-g), 5.95 (bs, 1H, OH), 4.42 (q, 2H, C H 2 ), 1.43 (t, 3H, C H 3 ). Ethyl 4-(4-propynyloxyphenyl)azobenzoate (2) [33] . To a stirred suspension of 1 (16 g, 59.2 mmol) and K 2 CO 3 (24 g, 173.6 mmol) in dry acetonitrile (572 mL), propargyl bromide (240 mmol, 26 mL) was added dropwise and the reaction mixture was refluxed for 24 h. Then the reaction mixture was filtered and the solid was recrystallized in ethanol, obtaining an orange crystalline solid. Yield: 14.2 g (78%), m. p. 108–110 °C; IR: v max 3252 (HC ≡), 2126 (C ≡C), 1702 (CO), 1599 cm −1 (Ar); NMR data (300 MHz, CDCl 3 ): 1H, δ8.17 (m, 2H, H-b), 7.95 (m, 2H, H-f), 7.90 (m, 2H, H-c), 7.01 (m, 2H, H-g), 4.77 (d, 2H, J 2.0 Hz, H-i), 4.40 (q, 2H, J 7.12 Hz, C H 2 CH 3 ), 2.57 (t, 1H, ≡C H ), 1.42 (t, 3H, C H 3 ); 13 C, δ166.12 (CO), 160.40 (C-h), 155.21 (C-d), 147.49 (C-e), 131.70 (C-a), 130.55 (C-b), 125.08 (C-f), 122.40 (C-c), 115.24 (C-g), 77.92 (C ≡C –C), 76.14 ( ≡C H), 61.21 ( C H 2 CH 3 ), 56.04 (C-i), 14.36 (CH 3 ). 2 A. Suárez-Cruz, I. Molina-Pinilla, K. Hakkou et al. Polymer Degradation and Stability 193 (2021) 109726 4-(4-propynyloxyphenyl)azobenzoic acid (3) [33] . A suspension of 2 (1 g, 3.24 mmol) in 49 mL of ethanol was refluxed for 10 min. Afterwards, a solution of KOH (0.292 g, 5.2 mmol) in 25 mL of water was added, and the reaction mixture was heated at 85 °C for 2 h. The suspension was filtered and HCl 2 M was added to the resulting solution until pH 3–4. The formed precipitate was filtered and dried, obtaining an orange solid. Yield: 815 mg (90%). NMR data (300 MHz, DMSO–d 6 ): 1 H, δ8.13 (d, 2H, H-b), 7.98–7.85 (2d, 4H, H-c, H-f), 7.21 (d, 2H, H-g), 4.95 (d, 2H, H-i), 3.65 (t, 1H, ≡C H ); 13 C, δ167.23 (CO), 160.86 (C-h), 154.84 (C-d), 147.09 (C-e), 132.77 (C-a), 131.06 (C-b), 125.32 (C-f), 122.75 (C-c), 116.06 (C-g), 79.23 (C ≡C –C), 79.18 ( ≡C H), 56.38 (C-i). Succinimide 4-(4-propynyloxyphenyl)azobenzoate (4) [63] . To a mixture of 3 (1 g, 3.56 mmol) and N -hydroxysuccinimide (0.410 g, 3.56 mmol) in dry acetonitrile (15 mL) and dimethylformamide (5 mL), EDC •HCl (0.85 g, 4.44 mmol) was added. The mixture was stirred under argon atmosphere for 24 h. Afterwards, the reaction mixture was dropped in water, and the resulting precipitate was filtered and dried to obtain an orange solid. Yield: 1.2 g (87%), IR: v max 3271 (HC ≡), 2134 (C ≡C), 1765, 1729 (CO), 1597 cm −1 (Ar); NMR data (300 MHz, CDCl 3 ): 1 H, δ8.28 (d, 2H, H-b), 8.00–7.90 (2d, 4H, H-c, H-f), 7.12 (d, 2H, H-g), 4.80 (d, 2H, H-i), 2.93 (s, 4H, succ), 2.58 (t, 1H, ≡C H ). 2-(2-methoxyethoxy)ethyl methanesulfonate (5) [64] . To a stirred mixture of diethylene glycol methyl ether (2 g, 16.65 mmol), triethylamine (2.53 g, 25 mmol) and dry dichloromethane (40 mL) under argon atmosphere at 0 °C, mesyl chloride (1.5 mL, 18.7 mmol) was added dropwise. After 1.5 h, the reaction mixture was washed with water (10 mL), HCl 2 N (5 mL), saturated NaHCO 3 solution until basic pH, and water. Organic phase was dried with Na 2 SO 4 and concentrated. The residue was purified by column chromatography (ethyl acetate) obtaining a colourless syrup. Yield: 2.9 g (87%). NMR data (300 MHz, CDCl 3 ): 1 H, δ4.38 (m, 2H, MsOC H 2 CH 2 ), 3.77 (m, 2H, MsOCH 2 C H 2 ), 3.66 (m, 2H, CH 2 C H 2 OCH 3 ), 3.55 (m, 2H, C H 2 CH 2 OCH 3 ), 3.38 (s, 3H, CH 3 ), 3.07 (s, 3H, Ms); 13 C, δ71.83 ( C H 2 CH 2 OCH 3 ), 70.61 (CH 2 C H 2 OCH 3 ), 69.18 (MsO C H 2 C), 69.05 (MsOCH 2 C ), 58.98 (CH 3 ), 37.66 (Ms). 1,12-Diazido-4,9-dioxadodecan-2,11-diol (6) [65] . A mixture of 1,4-butanediol diglycidyl ether (3.3 g, 16.0 mmol), tetrabutylammonium sulfate (0.68 g, 2.0 mmol), and sodium azide (13 g, 200 mmol) in water-dioxane (1:1, 200 mL) was refluxed for 4 h. Then, the reaction mixture was concentrated to about half its volume and extracted with ethyl acetate (3 ×100 mL). The combined organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure to obtain a syrupy residue which was purified by column chromatography ( tert –butyl methyl ether:hexane, 1:1), giving after evaporation of the solvents 6 as a solid (3.2 g, 68%), m.p. 56–58 °C, IR: v max 3400 (OH), 2087 cm −1 (N 3 ); NMR data (CDCl 3 ): 1 H, δ3.99–3.88 (m, 2H, H-2), 3.55–3.30 (m, 12H, H-1, H-3, H-4), 2.72 (d, 2H, OH), 1.69–1.63 (m, 4H, H-5); 13 C, δ71.92, 71.30 (C-3, C-4), 69.65 (C-2), 53.41 (C-1), 26.27 (C-5). Diazide monomer 7. To a mixture of 6 (2.4 g, 8.32 mmol), powdered KOH (2.3 g, 41 mmol), tetrabutylammonium bromide (1.07 g, 3.32 mmol) and water (1 mL), a solution of 5 (4.0 g, 20.18 mmol) in toluene (10 mL) was added. The reaction mixture was refluxed for 24 h. Afterwards dichloromethane was added, and the resulting solution was filtered and concentrated. The residue was purified by column chromatography (hexane: acetone 3:1), obtaining a colourless syrup. Yield: 2.3 g (56%), IR: v max 2094 (N 3 ), 1106 cm −1 (C –O); NMR data (300 MHz, CDCl 3 ): 1 H, δ3.85–3.72 (m, 4H, H-6), 3.69–3.64 (m, 10H, H-2,7,8), 3.68–3.44 (m, 12H, H-3,4,9), 3.40 (s, 6H, H-10), 3.39–3.33 (m, 4H, H-1), 1.6 (m, 4H, H-5); 13 C, δ78.49 (C-2), 71.94 (C-9), 71.33 (C-4), 70.80 (C-7), 70.57 (C-8), 70.12 (C-3), 69.86 (C-6), 59.03 (C-10), 51.94 (C-1), 26.29 (C-5). Anal. Calcd for C 20 H 40 N 6 O 8 : C, 48.77; H, 8.19; N, 17.06. Found: C, 48.67; H, 8.351; N, 16.96. HRMS: m/z 515.2796 (calcd. for [M] + : 515.2800). 1,12-Diamino-4,9-dioxadodecan-2,11-diol (8). Compound 6 (0.577 g, 2 mmol), Pd/C 10% (0.286 g) and methanol (50 mL) were added to a hydrogenation flask. Hydrogenation was carried out at 40 psi for 3 h. Then, the mixture was filtered through a celite pad, and the solvent evaporated until dryness, obtaining a waxy purple solid. Yield: 0.43 g (90%). NMR data (300 MHz, DMSO–d 6 ): 1 H, δ3.53–3.40 (m, 2H, H-2  ), 3.40–3.30 (m, 4H, H-4  ), 3.30–3.24 (m, 4H, H-3  ), 2.57 (dd, 2H, J 1’a, 1  b 12.8 Hz, J 1’a, 2  4.4 Hz, H-1  a), 2.41 (dd, 2H, J 1’b, 2  6.7 Hz, H-1  b), 1.51 (m, 4H, H-5  ); 13 C, δ73.48 (C3  ), 71.37 (C-2  ), 70.75 (C-4  ), 45.63 (C-1  ), 26.45 (C-5  ). HRMS: m/z 237.1809 (calcd. for [M] + : 237.1809). 2,11-bis(2-(2-methoxyethoxy)ethoxy) −4,9-dioxadodecan-1,12diamine (9). Compound 7 (0.575 g, 1.17 mmol), Pd/C 10% (0.173 g) and methanol (30 mL) were added to a hydrogenation flask. Hydrogenation was carried out at 40 psi for 3 h. Then, the mixture was filtered through a celite pad, and the solvent evaporated until dryness, obtaining a colourless syrup. Yield: 0.460 g (89%). NMR data (300 MHz, CDCl 3 ): 1 H, δ3.89–3.67 (m, 4H, H-6  ), 3.67–3.59 (m, 8H, H-7  ,8  ), 3.59–3.50 (m, 4H, H-9  ), 3.50–3.37 (m, 10H, H-2  ,3  ,4  ), 3.38 (s, 6H, H-10  ), 2.90–2.63 (m, 4H, H-1  ), 1.68 (bs, 4H, NH), 1.61 (m, 4H, H-5  ); 13 C, δ80.67 (C-2  ), 71.95 (C-9  ), 71.38, 71.30, 70.87, 70.47 (C-3  , 4  , 7  , 8  ), 69.52 (C-6  ), 59.03 (C-10  ), 43.45 (C-1  ), 26.37 (C-5  ). HRMS: m/z 441.3161 (calcd. for [M] + : 441.3170). Dialkyne monomer 10. To a solution of 4 (0.2 g, 0.53 mmol) in dry dimethylformamide (1 mL) under argon atmosphere, 3,6dioxaoctan-1,8-diamine (40 μL, 0.266 mmol,) and triethylamine (111 μL, 0.8 mmol) were added. The resulting mixture was stirred for 24 h. Then water was added, precipitate was filtered and purified by column chromatography (dichloromethane-methanol 20:1 to 1:1), obtaining an orange solid. Yield: 0.152 g (85%), m. p. 193– 196 °C, IR: v max 3297 (HC ≡, NH), 3064 (Ar), 2134 (C ≡C), 1625 (CO), 1596 cm −1 (Ar); NMR data (300 MHz, DMSO–d 6 ): 1 H, δ 8.72 (t, 2H, NH), 8.07 (d, 2H, H-b), 7.96 (d, 2H, H-c), 7.92 (d, 2H, H-f), 7.23 (d, 2H, H-g), 4.98 (d, 4H, J 2.3 Hz, H-i), 3.69 (t, 2H, ≡C H ), 3.66–3.56 (m, 8H, H-2  , 3  ), 3.56–3.44 (m, 4H, H-1  ); 13 C, δ 166.08 (CO), 160.69 (C-h), 153.82 (C-d), 147.08 (C-e), 136.47 (C-a), 128.91 (C-b), 125.18 (C-f), 122.55 (C-c), 116.01 (C-g), 79.21 ( C ≡C ), 70.10 (C-3  ), 69.34 (C-2  ), 56.37 (C-i), 39.8 (C-1  ). Anal. Calcd for C 38 H 36 N 6 O 6 .H 2 O: C, 66.08; H, 5.55; N, 12.17. Found: C, 65.81; H, 5.55; N, 12.11. Dialkyne monomer 11. To a solution of 4 (0.438 g, 1.16 mmol) in dry dimethylformamide (2.2 mL) under argon atmosphere, 8 (0.137 g, 0.58 mmol) and triethylamine (0.24 mL, 1.75 mmol) were added. The mixture was stirred for 24 h. Then water was added, precipitate was filtered and dried to obtain an orange solid. Yield: 0.34 g (76%), m. p. 172–177 °C, IR: v max 3292 (HC ≡, OH), 2124 (C ≡C), 1625 (CO), 1600 cm −1 (Ar); NMR data (300 MHz, DMSO– d 6 ): 1 H, δ8.56 (t, 2H, NH), 8.05 (d, 2H, H-b), 7.94 (d, 2H, H-c), 7.90 (m, 2H, H-f), 7.20 (d, 2H, H-g), 4.97 (d, 2H, J 5.2 Hz, OH), 4.94 (d, 4H, J 2.3 Hz, H-i), 3.82 (m, 2H, H-2  ), 3.65 (t, 2H, ≡C H ), 3.51–3.16 (m, 12H, H-1  , 3  ,4  ), 1.56 (m, 4H, H-5  ); 13 C, δ166.22 (CO), 160.70 (C-h), 153.82 (C-d), 147.11 (C-e), 136.67 (C-a), 128.98 (C-b), 125.19 (C-f), 122.51 (C-c), 116.04 (C-g), 79.23 ( C ≡C ), 73.71 (C-3  ), 70.89 (C4  ), 68.79 (C-2  ), 56.38 (C-i), 43.92 (C-1  ), 26.47 (C-5  ). Anal. Calcd for C 42 H 44 N 6 O 8 : C, 66.30; H, 5.83; N, 11.05. Found: C, 66.04; H, 6.174; N, 11.03. Dialkyne monomer 12. To a solution of 4 (0.168 g, 0.4 4 4 mmol) in dry dimethylformamide (0.84 mL) under argon atmosphere, 9 (0.098 g, 0.222 mmol) and triethylamine (0.1 mL, 0.64 mmol) were added. The mixture was stirred for 24 h. Then water was added, the precipitate was decanted and dried to obtain an orangish syrup. Yield: 0.19 g (89%). NMR data (300 MHz, CDCl 3 ): 1 H, δ8.07–7.85 (m, 6H, H-b,c,f), 7.31 (t, 2H, NH), 7.10 (d, 2H, H-g), 3 A. Suárez-Cruz, I. Molina-Pinilla, K. Hakkou et al. Polymer Degradation and Stability 193 (2021) 109726 Table 1 GPC a data of poly(azoamide triazole)s. Polymer Yield (%) M w M w /M n PAAT1 99 148,500 1.24 PAAT2 93 138,000 1.31 PAAT3 85 131,000 1.27 PAAT4 84 95,000 1.46 a Determined by GPC analysis with polystyrene standards. Measured in DMFLiBr. Table 2 Thermal analysis data of poly(azoamide triazole)s. Polymer T g a ( °C) T 10% b ( °C) T dec b ( °C) PAAT1 35.5 318.3 319.5, 382.6 PAAT2 87.7 325.2 304.8, 371.3 PAAT3 2.1 323.6 322.9, 385.0 PAAT4 2.4 325.4 325.8, 390.8 a Determined by DSC, second heating. b Measured by TGA. Table 3 Qualitative solubilities of poly(azoamide triazole)s. Solvent PAAT1 PAAT2 PAAT3 PAAT4 TBME –––– Hexane –––– Ethyl acetate - ––± Methanol - ––± Acetone –––++ Chloroform ++ ––++ Water –––– DMF ++ ±+ ++ DMSO ++ ±++ ++ (-) insoluble, ( ±) slightly soluble, ( + ) soluble on warming, ( ++ ) soluble at room temperature. 4.78 (d, 4H, J 2.4 Hz, H-i), 3.97–3.30 (m, 30H, H-1  −4  , H-6  −9  ), 3.27 (s, 6H, H-10  ), 2.57 (t, 2H, ≡C H ), 1.66 (m, 4H, H-5  ); 13 C, δ 166.96 (CO), 160.26 (C-h), 154.25 (C-d), 147.49 (C-e), 136.07 (C-a), 128.16 (C-b), 124.96 (C-f), 122.55 (C-c), 115.23 (C-g), 77.94 (C –C ≡C), 77.78 (C-2  ), 76.08 ( ≡C H), 71.90, 71.81, 71.45, 70.79, 70.35, 69.6 (C3  ,4  ,9  ,8  ,7  ,6  ), 58.90 (C-10  ), 56,05 (C-i), 41.56 (C-1  ), 26.38 (C-5  ). Anal. Calcd for C 52 H 64 N 6 O 12 : C, 64.71; H, 6.68; N, 8.71. Found: C, 62.60; H, 7.06; N, 8.49. HRMS: m/z 987.4467 (calcd. for [ M + Na] + : 987.4474). 2.4. Synthesis of the polymers 2.4.1. General procedure for the synthesis of poly(azoamide triazole)s (PAATn) Stoichiometric amounts of diazide and dialkyne were mixed and dissolved in dimethyl sulfoxide. Water was added until a slight cloudiness appeared. Afterwards CuSO 4 pentahydrate (20%) and sodium ascorbate (40%) were added, and the reaction mixture was stirred and heated to 50 °C under argon atmosphere for 24 h. Reaction mixture was then processed in different ways depending on the polymer formed. Polymer PAAT1 was recovered by precipitation in tert –butyl methyl ether (TBME), followed by re-dissolution in dichloromethane and precipitation in acetone. Polymers PAAT2 and PAAT3 were isolated by filtration on a glass filter and washed with water and acetone. These polymers were purified by dissolution in dimethyl sulfoxide and precipitation in TBME. Polymer PAAT4 was also isolated by filtration but purified by dissolution in CH 2 Cl 2 and precipitation in TBME. Tables 1–4 show the studied characteristics of the prepared polymers. The infrared and NMR spectroscopy data are listed below. Table 4 UV Absorption data of the PAATn polymers in DMSO. Compound λ(nm) 10 429, 357, 255 11 437, 358, 255 12 449, 357, 255 PAAT1 426, 360, 255 PAAT2 432, 359, 255 PAAT3 432, 360, 255 PAAT4 442, 361, 256 Fig. 1. Synthesis of diazides and diamines. PAAT1 . IR: v max 3326 (NH), 1644 cm −1 (CO); NMR data (500 MHz, CDCl 3 ): 1 H, δ7.95 (s, 2H, H-j), 8.00–7.80 (m, 12H, Hb,c,f), 7.10 (d, 4H, H-g), 6.90 (bs, 2H, NH), 5.30 (s, 4H, H-i), 4.64 (bd, 2H, H-1a), 4.42 (dd, 2H, J 1b,2 6.94 Hz, J 1a,1b 14.0 Hz, H-1b), 3.86 (bs, 2H, H-2), 3.80–3.30 (m, 36H, H-1  ,2  ,3  ,3,4,6,7,8,9), 3.30 (s, 6H, H-10), 1.63 (bs, 4H, H-5); 13 C, δ166.96 (CO), 161.19 (Ch), 154.37 (C-d), 147.20 (C-e), 143.18(C-k), 135.67 (C-a), 127.98 (Cb), 125.08 (C-f), 124.90 (C-j), 122.63 (C-c), 115.16(C-g), 77.73 (C2), 71.87, 71.43, 70.65, 70.41, 70.31, 69.84, 69.68 (C-2  ,3  ,3,4,6,7,8,9), 62.21 (C-i), 58.95 (C-10), 51.62 (C-1), 39.88 (C-1  ), 26.30 (C-5). PAAT2 . IR: v max 3309 (OH, NH), 1630 cm −1 (CO); NMR data (500 MHz, DMSO–d 6 ): 1 H, δ8.67 (bs, 2H, NH), 8.19 (s, 2H, H-j), 4 A. Suárez-Cruz, I. Molina-Pinilla, K. Hakkou et al. Polymer Degradation and Stability 193 (2021) 109726 Fig. 2. Synthesis of dialkyne monomers. 8.02 (d, 4H, H-b), 7.91, 7.87 (2d, 8H, H-c,f), 7.25 (d, 4H, H-g), 5.32 (d, J 5.1 Hz, OH), 5.26 (s, 4H, H-i), 4.48 (bd, 2H, H-1a), 4.31 (dd, 2H, J 1b,2 7.8 Hz, J 1a,1b 13.6 Hz, H-1b), 3.99 (bs, 2H, H-2), 3.62– 3.20 (m, 20H, H-1  ,2  ,3  ,3,4), 1.56 (bs, 4H, H-5); 13 C, δ166.11 (CO), 161.65 (C-h), 153.83 (C-d), 146.80 (C-e), 142.27(C-k), 136.37 (C-a), 128.89 (C-b), 126.21 (C-j), 125.29 (C-f), 122.51 (C-c), 115.87 (C-g), 72.54, 70.94, 70.07, 69.31, 68.71 (C-2  ,3  ,3,4,2), 61.97 (C-i), 53.43 (C1), 40.84 (C-1  ), 26.32 (C-5). PAAT3 . IR: v max 3399 (OH, NH), 1644 cm −1 (CO); NMR data (500 MHz, DMSO–d 6 ): 1 H, δ8.54 (bs, 2H, NH), 8.23 (s, 2H, Hj), 8.04 (bd, 4H, H-b), 7.92, 7.89 (2bd, 8H, H-c,f), 7.25 (d, 4H, H-g), 5.28 (s, 4H, H-i), 4.96 (bs, 2H, OH), 4.58 (m, 2H, H-1a), 4.43 (m, 2H, H-1b), 3.84 (m, 4H, H-2  ,2), 3.60–3.10 (m, 36H, H1  ,3  ,4  ,3,4,6,7,8,9), 3.18 (s, 6H, H-10), 1.55 (bs, 8H, H-5  ,5); 13 C, δ 166.23 (CO), 161.60 (C-h), 154.84 (C-d), 146.84 (C-e), 142.48(C-k), 136.58 (C-a), 128.97 (C-b), 126.21 (C-j), 125.27 (C-f), 122.46 (C-c), 115.93 (C-g), 77.39 (C-2), 73.71, 71.68, 70.93, 70.67, 70.30, 69.95, 69.26, 68.81 (C-2  ,3  ,4  ,3,4,6,7,8,9), 61.99 (C-i), 58.47 (C-10), 51.09 (C-1), 43.92 (C-1  ), 26.47, 26.32 (C-5  ,5). PAAT4 . IR: v max 3336 (NH), 1644 cm −1 (CO); NMR data (500 MHz, CDCl 3 ): 1 H, δ8.05–7.85 (m, 14H, H-j,b,c,f), 7.30 (bs, 2H, NH), 7.14 (d, 4H, H-g), 5.31 (s, 4H, H-i), 4.68 (m, 2H, J 1a,2 2.9 Hz, H-1a), 4.44 (m, 2H, J 1b,2 7.3 Hz, J 1a,1b 14.3 Hz, H-1b), 4.00–3.15 (m, 56H, H-1  ,2  ,3  ,4  ,2,3,4,6  ,6,7  ,7,8  ,8,9  ,9), 3.33, 3.28 (2 s, 12H, H-10  ,10), 1.67 (m, 8H, H-5  ,5); 13 C, δ166.97 (CO), 161.15 (C-h), 154.29 (C-d), 147.25 (C-e), 143.19 (C-k), 136.02 (C-a), 128.12 (C-b), 125.01 (C-f), 124.83 (C-j), 122.52 (C-c), 115.16 (C-g), 77.81, 77.75 (C2  ,2), 71.86, 71.79, 71.43, 70.77, 70.65, 70.40, 70.33, 69.68, 69.59 (C3  ,3,4  ,4,6  ,6,7  ,7,8  ,8,9  ,9), 62,23 (C-i), 58.93, 58.86 (C-10  ,10), 51.64 (C-1), 41.57(C-1  ), 26.36, 26.29 (C-5  ,5). 2.5. Alternating irradiation of the sample with ultraviolet and visible light A solution of the polymer (0.028 mg/mL) in dimethyl sulfoxide was consecutively irradiated with ultraviolet light, for different periods of time, followed by visible light for 1 min. The solution was studied by ultraviolet-visible spectroscopy after each irradiation. 2.6. Degradation of polymers 2.6.1. Degradation in buffer solution The hydrolytic degradation study was carried out on films prepared by evaporations of polymer solutions PAAT1 and PAAT4 in dichloromethane (20 mg/mL). Films formed were dried under vacuum until no weight loss was observed. The thickness of the films obtained was approximately 70 μm. Afterwards, 10 mL of buffered solutions at different pH were added, and they were incubated at either 37 or 70 °C. After different periods of time, about 40 days, the sample was recovered by filtration, and the film was washed with distilled water and dried under vacuum. Finally, the remaining film was analyzed by Gel Permeation Chromatography. 2.6.2. Degradation with dithionite 2.6.2.1. Degradation of monomer 12 . To a solution of 12 in methanol (5 mg, 1.5 mL) water was added until turbidity (0.7 mL). Then, sodium dithionite (10 equivalents) was added and stirred at 37 °C protected from light. The reaction mixture was monitored by TLC, adding more dithionite until reduction was complete. After four days, the solvent was evaporated to dryness and dichloromethane was added to obtain a suspension. The solid formed was filtered off and the filtrate was evaporated again in vacuo . Finally, the mixture obtained was dried to constant weight, being used directly for its analysis by UV-visible spectroscopy, in order to evaluate the reduction of the azo group. 2.6.2.2. Degradation of polymer. Polymers PAAT1 and PAAT4 were dissolved in CH 2 Cl 2 (4 mg/mL), and the solvent was slowly removed by evaporation at room temperature to obtain a film that was dried under vacuum to constant weight. Afterwards, a pH 6 buffered solution of sodium dithionite (155 mM, 2.0 mL) was added, and the vials were heated at 37 °C with stirring. Samples were withdrawn at 2, 4, 8 and 11 days, replacing the sodium dithionite solution in the remaining samples. The film was washed with distilled water and dried. Finally, the samples were analyzed by size exclusion chromatography. 2.7. Biocompatibility studies 2.7.1. Sample preparation Polymers PAAT1 and PAAT4 were dissolved in DMSO for cell culture (4 mg/mL). Then, 200 μL of each solution was added to a 96-well plate, and DMSO was removed in a vacuum oven until dryness, obtaining the wells coated with thin films of the samples to be analyzed. 2.7.2. Hemolysis assay Toxicity to human red blood cells (hRBC) was tested by carefully preparing a mixture of fresh human blood and PBS. The mixture 5 A. Suárez-Cruz, I. Molina-Pinilla, K. Hakkou et al. Polymer Degradation and Stability 193 (2021) 109726 was centrifuged at 700 G for 10 min, supernatant was discarded, and the procedure was repeated three times. Then, PBS was added to prepare a 5% (v/v) suspension of hRBC. Aliquots of this suspension (150 μL) was added to each coated well. PBS (150 μL) was used as blank and hRBC suspension (150 μL) containing 1% of Triton X-100 was used as positive control (total hemolysis). The 96wells plate was incubated for 1 hour at 37 °C. Then, centrifuged for 10 min at 700 G. Supernatants were placed in a new microtiter plate and absorbance was measured at 540 nm. The experiment was performed three times in triplicate, and the equation to calculate the hemolytic activity is: 2.7.3. Cell culture DMEM was supplemented with 10% of FBS, 1% Penicillin and streptomycin, 1% l -glutamine, 1% sodium pyruvate and 1% nonessential aminoacids. A cryotube containing 0.5 million of Human Gingival Fibroblasts (HGnF) cells was heated to 37 °C, diluted with 9 mL of DMEM and centrifuged at 300 G for 5 min. Supernatant was discarded and pellet resuspended in 5 mL of supplemented DMEM. Then, plated in a cell culture flask and incubated at 37 °C with 5% CO 2 . After 48 h, the medium was removed, the culture was washed with PBS, and treated with trypsin-EDTA. HGnF were counted and diluted with DMEM to obtain a suspension of 1 ×10 5 cells per milliliter. Then, 150 μL of the cell suspension were added to the wells of a 96-wells plate to seed 1.5 10 4 HGnF per well. 96wells plate was incubated for 24 h at 37 °C with 5% CO 2 . 2.7.4. MTT assay To evaluate the toxicity produced by PAAT1 and PAAT4 , a cell viability test was carried out using the MTT method, which consists of reducing this tetrazolium dye to a water-insoluble crystal (formazan). To perform this assay, a 96 wells microtiter plate was prepared according to the Section 2.7.1 . A HGnF suspension (150 μL) containing 1 ×10 5 cells per milliliter was added to each polymer coated well. HGnF suspension (150 μL) was dispensed to a well which did not contain any polymer as positive control. Wells containing only supplemented DMEM was used as negative control. Microtiter plate was incubated at 37 °C with 5% CO 2 . After 24 h, 20 μL of a MTT solution (5 mg/mL) was added to each well and microtiter plate was incubated. After 4 h, supernatant was carefully withdrawn and 150 μL of DMSO was added to dissolve the formazan crystals and films. This solution was transferred to a new microtiter plate and absorbance of the solutions was measured at 570 nm using a microplate reader. This assay was repeated three times in triplicate. Positive control was used as 100% of viability, and results of the samples were relative to this positive control. 3. Results and discussion 3.1. Synthesis and chemical structure of the azo polymers In this work, we describe the preparation of four novel linear poly(azoamide triazole)s, referred to as PAATn , which were prepared from diazide ( 6, 7 ) and dialkyne functionalized monomers ( 10 –12 ) ( Figs. 1 and 2 , respectively), by CuAAC polyaddition reaction in solution. Diazide monomer 6 was readily prepared from commercially available 1,4-butanediol diglycidyl ether by opening of the epoxide rings with sodium azide as nucleophile [65] . Afterward, the alcohol functions obtained in the opening of the epoxides were reacted with 2-(2-methoxyethoxy)ethyl methanesulfonate ( 5 ), previously prepared according to a procedure described in the literature [64] , to obtain monomer 7 . The synthesis of dialkyne monomers 10, 11 and 12 were conveniently carried out by reaction of active ester 4 , prepared as displayed in Fig. 3 in four steps by previously described methods [63] , with three different diamines: commercial 3,6-dioxaoctan1,8-diamine and diamines 8 and 9 , respectively ( Fig. 1 ). These diamines were easily obtained in high yield by reduction of the diazide functions of the corresponding monomers 6 and 7 ( Fig. 1 ). All these compounds were conveniently characterized by infrared, 1 H and 13 C NMR spectroscopies, elemental analysis and/or highresolution mass spectrometry. Fig. 3. Synthesis of the azobenzene derivatives. 6 A. Suárez-Cruz, I. Molina-Pinilla, K. Hakkou et al. Polymer Degradation and Stability 193 (2021) 109726 Fig. 4. Synthesis of the PAATn polymers. The azo polymers PAATn were prepared by polymerization of the bis-azide and bis-alkyne monomers using copper-catalyzed azide–alkyne cycloaddition (CuAAC) reactions ( Fig. 4 ). These polymers were isolated in yields greater than 80% ( Table 1 ) and were purified as described in the experimental part. The chemical compositions anticipated for these poly(azoamide triazole)s were confirmed by FTIR and NMR spectroscopy, and the corresponding data are detailed in the experimental section. All the FTIR spectra displayed the expected absorption bands of the amide functions presents along the polymer chain. No residual bands corresponding to azide or alkyne functional groups of the starting monomers were detected in the corresponding polymeric materials. The absorption bands appearing around 3300 and 1650 cm −1 were attributed to the stretching band of NH and carbonyl group of the amide functions, respectively. As an example, the 1 H and 13 C NMR spectra ( Figs. 5 and 6 , respectively) of poly(azoamide triazole) PAAT2 , recorded in deuterated dimethyl sulfoxide, are displayed with the structural correlation of the signals that appear in both types of spectrum. The 1 H NMR spectrum of PAAT2 ( Fig. 5 ) shows five signals in the region of 8.80 to 7 ppm. The signals appearing as singlets at approximately 8.70 and 8.20 ppm were attributed to the amide function present in the bis-alkyne monomer, and to the H-j protons of the 1,4-disubstituted triazole aromatic ring obtained in the CuAAC reaction, respectively. Likewise, the hydroxyl groups present in the repeating unit of this polymer give rise to the signal that appears at 5.30 ppm, as a doublet, displaying a coupling constant of about 5 Hz. The proposed structure for the PAAT2 polymer is also confirmed by its 13 C NMR spectrum ( Fig. 6 ). According to the regioselectivity expected in the CUAAC polymerization reaction, the polymer should have 1,4-disubstituted 1,2,3triazole rings in its structure. This ring produces two signals in the 13 C NMR spectrum at 142.27 and 126.21 ppm that were attributed to the C-K and C-j carbons of the triazole, respectively. Fig. 5. 1 H NMR spectrum of PAAT2 recorded in deuterated dimethyl sulfoxide. 3.2. Gel permeation chromatography The purity of all polymers was confirmed by size exclusion chromatography, using μStyragel columns calibrated against polystyrene standards, finding that the chromatograms of the PAATn polymers were unimodal. Mass-averaged molar masses (Mw), measured using lithium bromide in dimethylformamide as mobile phase, showed values between 95,0 0 0 and 148,0 0 0 g/mol ( Table 1 ). The apparent molar masses of the azo homopolymers 7 A. Suárez-Cruz, I. Molina-Pinilla, K. Hakkou et al. Polymer Degradation and Stability 193 (2021) 109726 Fig. 6. 13 C NMR spectrum of PAAT2 recorded in dimethyl sulfoxide. PAAT1, PAAT2 and PAAT3 are quite like each other while PAAT4 has a clearly lower molar mass. However, the PAATn polymers have different chemical structures so they are expected to have different hydrodynamic volumes for the same absolute molar mass. This makes it difficult to make comparisons between the apparent molar masses measured by GPC for these polymers. 3.3. Thermal analysis The thermal study of the poly(azoamide triazole)s PAATn was carried out using a combination of DSC and TGA. The data resulting from this study are shown in Table 2 . The DSC study revealed that all the polymers exhibited one glass transition step (T g ) during the second heating trace measured with a heating rate of 10 °C/min. Annealing experiments were carried out to favor that the intermolecular interactions could lead to the crystallization of the samples. However, under the tested conditions the thermograms only showed the presence of glass transitions. The PAAT2 polymer has the highest T g value (T g 87.7 °C) while the PAAT3 and PAAT4 polymers have much lower values of T g , about 2 °C. In view of these data ( Table 2 ), it seems that the values of the glass transition could be related to the separation of the azobenzene groups, which are the most rigid segments along the macromolecules main chain. Thus, the PAAT1 and PAAT2 polymers, where the azobenzene groups are separated by segments containing 8 atoms, display higher T g values than the PAAT3 and PAAT4 polymers where these segments always contain 12 atoms. Likewise, the presence or absence of diethylene glycol side chains also affects the T g values. Thus, polymer PAAT1 have a T g value lower than PAAT2 due to the diethylene glycol side chains of monomer 7 , possibly exerting a plasticizing effect. Thermogravimetric analysis of the PAATn polymers was performed by TGA, under inert atmosphere and heating the sample from room temperature to 600 °C. Table 2 shows the values obtained from the thermogravimetric analysis of the polymers. The temperatures at 10% weight loss determined at a heating rate of 10 °C/min were higher than 315 °C ( Table 2 ), which shows its good thermal stability. All polymers thermally decompose in two stages, as it is shown in Table 2 . Although the decomposition temperature values are quite similar, it can be observed that the polymers that have free alcohol functions in the main chain, PAAT2 and PAAT3 , present somewhat lower decomposition temperature values than their analogues with diethylene glycol side chains, PAAT1 and PAAT4 . 3.4. Qualitative solubilities Table 3 shows the solubilities [66] of PAATn polymers in different solvents. None of the synthesized polymers was soluble in water, but they were dissolved in dimethyl sulfoxide or dimethylformamide. In general, they were also not soluble in common organic solvents. It can be observed that the PAAT2 and PAAT3 polymers, which contain free secondary alcohol functions along the polymer chain, have lower solubility than the other polymers in common organic solvents. Thus, for example, PAAT1 is easily soluble in chloroform or the polymer PAAT4 can be dissolved in several organic solvents. 3.5. Photophysical properties Solutions of the PAATn polymers in dimethyl sulfoxide, kept in the dark overnight, were used to obtain their ultraviolet-visible spectra. The values corresponding to the maximum absorption are shown in Table 4 . The absorption spectra of azobenzenes are known to consist of three main bands appearing at approximately 430, 320 and 230 nm, and which are assigned to the nπ∗, ππ∗transitions for trans azobenzene and ππ∗in the phenyl rings, respectively. Similar bands appear in the absorption spectra corresponding to the PAAT1 - PAAT4 polymers, which in turn are very similar to the spectra exhibited by the respective monomers 10 –12 , from which they are prepared. The polymers were irradiated with an ultraviolet lamp placed at 20 cm from the sample. Fig. 7 shows how the band that appears at 320–400 nm, corresponding to the absorption of trans -azobenzene, progressively decreases as the irradiation time with UV light increases due to its isomerization to cis - azobenzene. As can be seen, the PAAT1 polymer reached the photostationary state after 240 s ( Fig. 7 A), while the rest of the polymers needed only 180 s to reach the maximum degree of photoisomerization ( Fig. 7 B-D). The degree of photoisomerization achieved is not high, probably because the azobenzene groups are forming part of the main chain, which will hinder their movements. The reversibility of photoisomerization was studied by ultraviolet– visible spectroscopy employing two different methods: thermally and by irradiating with visible light. Thus, if previously UV irradiated samples are exposed to visible light, the absorption band at 320–400 nm increases markedly until it almost recovers its initial absorbance, which represents the isomerization of cis -azobenzene to trans -azobenzene. This process of interconversion between the cisand trans -azobenzene remains reversible when irradiation cycles with UV and visible light are carried out consecutively for all the polymers. Fig. 8 illustrated the cis - trans - cis reversibility process for polymer PAAT1 as an example. As previously mentioned, we also studied the reversibility of photoisomerization by a thermal method. Thus, for example, when a PAAT1 polymer solution in DMSO was irradiated with ultraviolet light, and subsequently kept in the dark at 60 °C, it was possible to verify how the absorbance of the solution (band at about 360 nm) increased with heating time. After 45 min of heating at 8 A. Suárez-Cruz, I. Molina-Pinilla, K. Hakkou et al. Polymer Degradation and Stability 193 (2021) 109726 Fig. 7. UV-Visible spectra illustrating the photoisomerization of PAAT1 (A), PAAT2 (B), PAAT3 (C) and PAAT4 (D). All spectra were registered in DMSO. Fig. 8. Reversibility of photoisomerization of PAAT1 solution for the trans -tocis and cis -totrans processes. The sample was alternately irradiated with ultraviolet light for 300 s and visible light for 30 s. 60 °C the absorbance reached the initial value. If this same experiment is carried out at room temperature, it would take four days to reach a value close to the initial one. 3.6. Degradation of polymers The sensitivity of these polymers to hydrolysis has been studied under different conditions. As expected, the amide functions present in the structure of these polymers can hydrolyze under relatively drastic conditions, being stable in a physiological environment. The hydrolysis of PAATn polymers has been studied at pH 2.0, 7.4, and 10, and at different tem peratures, 37 and 70 °C, for several months. Likewise, taking advantage of the fact that, in addition to the amide functions, there are also azo functions, a degradation experiment that implied the breaking of this bond and therefore the breaking of the polymer chain was carried out. All these degradation experiments have been studied by following the variation of the molar masses of the samples subjected to degradation by means of size exclusion chromatography. 3.6.1. Buffered degradation The results of the hydrolytic degradation studies are shown in Figs. 9 and 10 , where the decrease in molar mass of the degraded samples is shown against the degradation time. The hydrolytic degradations of polymers PAAT1 and PAAT4 have been studied at 37 °C and in solutions buffered at pH 7.4 and pH 10 for 10 months. It was observed that under these conditions both the numberand mass-average molar masses at the end of study was practically the same as the initial one for PAAT4 ( Fig. 10 ), or it had slightly decreased at the beginning of the degradation to remain practically constant until the end of the study, as in the case of PAAT1 ( Fig. 9 ). However, it was found that if instead of carrying out the hydrolytic degradation under physiological conditions, pH 7.4 and temperature 37 °C, it was done at a higher temperature, 70 °C, the degradation of the polymer occurred, as expected, faster. Under these conditions, the PAAT4 polymer, that did not show degradation under physiological conditions, now did show a continuous decrease in its numberand mass-average molar masses values ( Fig. 10 ). 9