Selenol-Based Nucleophilic Reaction for the Preparation of Reactive Oxygen Species-Responsive Amphiphilic Diblock Copolymers

polymers

Article Selenol-Based Nucleophilic Reaction for the Preparation of Reactive Oxygen Species-Responsive Amphiphilic Diblock Copolymers

Xiaowei An, Weihong Lu, Jian Zhu * , Xiangqiang Pan * and Xiulin Zhu

Jiangsu Key Laboratory of Advanced Functional Polymer Design and Application, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, China; [email protected] (X.A.); [email protected] (W.L.); [email protected] (X.Z.) * Correspondence: [email protected] (J.Z.); [email protected] (X.P.); Tel.: +86-512-6588-0726 (J.Z.); +86-512-6588-3343 (X.P.) Received: 15 February 2019; Accepted: 5 May 2019; Published: 8 May 2019

Abstract: Selenide-containing amphiphilic copolymers have shown signiﬁcant potential for application in drug release systems. Herein, we present a methodology for the design of a reactive oxygen species-responsive amphiphilic diblock selenide-labeled copolymer. This copolymer with controlled molecular weight and narrow molecular weight distribution was prepared by sequential organoselenium-mediated reversible addition fragmentation chain transfer (Se-RAFT) polymerization and selenol-based nucleophilic reaction. Nuclear magnetic resonance (NMR) and matrix-assisted laser desorption/ionization time-to-ﬂight (MALDI-TOF) techniques were used to characterize its structure. Its corresponding nanomicelles successfully formed through self-assembly from the copolymer itself. Such nanomicelles could rapidly disassemble under oxidative conditions due to the fragmentation of the Se–C bond. Therefore, this type of nanomicelle based on selenide-labeled amphiphilic copolymers potentially provides a new platform for drug delivery.

Keywords: RAFT; selenol; amphiphilic polymer; drug delivery

1. Introduction Compared with sulfur, selenium shows versatile properties owing to its larger atomic radius and relatively lower electronegativity [1]. Selenium-containing polymers have attracted a great deal of attention in recent years and have been widely used as photoelectric materials, adaptive materials, and biomedical materials [2,3]. Selenophene polymers have been considered to be effective photoelectric materials which may be widely used in solar cells, molecular switches, thin film transistors, etc. [4–12]. Diselenide-containing adaptive materials were successfully incorporated in the fabrication process under very mild conditions to achieve self-healing properties [13–16]. Selenium-containing polymers show versatile responsive behaviors to multiple stimuli, such as oxidation, reduction, and irradiation [17–23], which makes them potentially useful bio-building blocks. Traditional methods for preparing selenium-containing polymers used step growth polymerization [24–28], radical polymerization [29–34], and ring-opening polymerization [35–37], and our group successfully developed organoselenium-mediated controlled radical polymerization (Se-RAFT) to prepare selenium-containing polymers with both controlled molecular weight and narrow molecular weight distribution [38]. Subsequently, the application of selenol-based nucleophilic substitution and Se-Michael addition reactions for polymer chain end modification was presented [39], so that many functional groups could be introduced to the selenium-containing polymers. Herein, we reported a new application of selenol-based nucleophilic reaction for the design of a reactive oxygen species-responsive

Polymers 2019, 11, 827; doi:10.3390/polym11050827 www.mdpi.com/journal/polymers Polymers 2019, 11, 827 2 of 12 Polymers 2018, 10, x FOR PEER REVIEW 2 of 13 diselenocarbonate-endamphiphilic diblock copolymer. capped polystyrenes Firstly, the diselenocarbonate-end with different molecular capped weights polystyrenes (MWs) withwere di preparedﬀerent throughmolecular the weightsorganoselenium-mediated (MWs) were prepared radical through polyme the organoselenium-mediatedrization of styrene (St). radical After polymerization aminolysis of diselenocarbonateof styrene (St). After with aminolysis hexylamine, of diselenocarbonatenucleophilic attack with of hexylamine,the exposed nucleophilic selenol to attackpoly(ethylene of the exposed selenol to poly(ethylene glycol) methyl ether methacrylate (PEGMA) gave a selenide-labeled glycol) methyl ether methacrylate (PEGMA) gave a selenide-labeled diblock copolymer (denoted as diblock copolymer (denoted as PS-Se-b-PEGMA; see Scheme1). PS-Se-b-PEGMA is able to form PS-Se-b-PEGMA; see Scheme 1). PS-Se-b-PEGMA is able to form micellar aggregates in water which micellar aggregates in water which are disassembled in oxidation conditions. are disassembled in oxidation conditions.

Scheme 1. Selenol-based nucleophilic reaction as a novel reaction forming amphiphilic diblock copolymers. Scheme 1. Selenol-based nucleophilic reaction as a novel reaction forming amphiphilic diblock 2.copolymers. Experimental Section

2. Experimental2.1. Materials Section Styrene was purchased from Shanghai Chemical Reagents Co. Ltd., Shanghai, China, and 1 2.1.purified Materials before use. PEGMA (Mn = 300, 500, and 1000 g mol− ; Aldrich) was passed through anStyrene alumina was column purchased to remove from the Shanghai inhibitor. Chemical After that, Reagents it was dried Co. withLtd., calcium Shanghai, hydride, China, then and distilled under reduced pressure and kept in a refrigerator below 0 ◦C. 2,2’-Azoisobutyronitrile purified before use. PEGMA (Mn = 300, 500, and 1000 g mol−1; Aldrich) was passed through an (AIBN, 98%) was recrystallized from ethanol and then stored in a refrigerator at 4 C. Se-benzyl alumina column to remove the inhibitor. After that, it was dried with calcium hydride,◦ then distilled O-(4-methoxyphenyl) carbonodiselenoate (Se-CTA) was synthesized according to a previously reported under reduced pressure and kept in a refrigerator below 0 °C. 2,2’-Azoisobutyronitrile (AIBN, 98%) method [33]. Tributylphosphane (Bu P, 98%) was purchased from Adamas Reagent Ltd, Shanghai, was recrystallized from ethanol and3 then stored in a refrigerator at 4 °C. Se-benzyl O-(4- China. Dialysis bags (molecular weight cutoff: 1000 Da) were purchased from Sinopharm Chemical methoxyphenyl) carbonodiselenoate (Se-CTA) was synthesized according to a previously reported Reagent Co. Ltd., Shanghai, China. Tetrahydrofuran (THF), N,N-dimethylformamide (DMF), methanol method(MeOH), [33]. and Tributylphosphane other chemicals were (Bu purchased3P, 98%) was from purchased Shanghai Chemicalfrom Adamas Reagents Reagent Co. Ltd. Ltd, Shanghai, Shanghai, China.China Dialysis and used bags without (molecular further treatment.weight cutoff: Doxorubicin 1000 Da) hydrochloride were purchased (DOX fromHCl, Sinopharm 99%) was purchased Chemical · Reagentfrom Sigma, Co. Ltd., Shanghai, Shanghai, China. China. Tetrahydrofuran (THF), N,N-dimethylformamide (DMF), methanol (MeOH), and other chemicals were purchased from Shanghai Chemical Reagents Co. Ltd. Shanghai,2.2. Characterization China and used without further treatment. Doxorubicin hydrochloride (DOX·HCl, 99%) was purchased from Sigma, Shanghai, China. The number-average molecular weight (Mn) and molecular weight distribution (Ð) of the resulting polymers were determined by a TOSOH HLC-8320 size exclusion chromatograph (SEC) equipped 2.2. Characterization with a TSKgel SuperMultiporeHZ-N column (3) (4.6 150 mm) at 40 ◦C. Tetrahydrofuran served as 1 × theThe eluent number-average with a flow rate molecular of 0.35 mL weight min− . SEC(Mn) samples and molecular were injected weight using distribution a TOSOH HLC-8320 (Ð) of the 1 resultingSEC plus polymers autosampler. were Thedetermined molecular by weights a TOSO wereH HLC-8320 calibrated withsize exclusion polystyrene chromatograph (PS) standards. (SEC)H equipped(300 MHz) with NMR a TSKgel spectra SuperMulti were recordedporeHZ-N on a Bruker column Avance (3) (4.6 300 × 150 spectrometer. mm) at 40 Chemical°C. Tetrahydrofuran shifts are 1 servedpresented as the in eluent parts perwith million a flow ( δrate) relative of 0.35 to mL CHCl min3 (7.26-1. SEC ppm samples in H NMR).were injected Transmission using electrona TOSOH microscopy (TEM) was performed with a HITACHI HT7700 microscope operating at a 120-kV HLC-8320 SEC plus autosampler. The molecular weights were calibrated with polystyrene (PS) accelerating voltage. The fluorescence emission spectra (FL) were obtained on a HITACHI F-4600 standards. 1H (300 MHz) NMR spectra were recorded on a Bruker Avance 300 spectrometer. fluorescence spectrophotometer at room temperature. Hydrodynamic diameter (Dh) was determined Chemical shifts are presented in parts per million (δ) relative to CHCl3 (7.26 ppm in 1H NMR). by dynamic light scattering (DLS) using a Brookhaven NanoBrook 90Plus PALS instrument at 25 C Transmission electron microscopy (TEM) was performed with a HITACHI HT7700 microscope◦ operating at a 120-kV accelerating voltage. The fluorescence emission spectra (FL) were obtained on a HITACHI F-4600 fluorescence spectrophotometer at room temperature. Hydrodynamic diameter (Dh) was determined by dynamic light scattering (DLS) using a Brookhaven NanoBrook 90Plus PALS instrument at 25 °C with a scattering angle of 90°. Fourier transform infrared spectroscopy (FT-IR)

Polymers 2019, 11, 827 3 of 12

with a scattering angle of 90◦. Fourier transform infrared spectroscopy (FT-IR) was recorded with the Bruker TENSOR 27 FT-IR instrument using the conventional KBr pellet method. The elemental composition of the surfaces was measured with X-ray photoelectron spectroscopy (XPS) (Thermo Fisher Scientiﬁc ESCALAB 250 XI, Al KR source).

2.3. Typical Procedure of Organoselenium-Mediated Controlled Radical Polymerization A dry 10-mL ampule was filled with styrene (8.0 mL, 80 mmol), Se-benzyl O-(4-methoxyphenyl) carbonodiselenoate (Se-CTA) (0.6147 g, 1.6 mmol), and AIBN (0.0788 g, 0.48 mmol). The solution was degassed using the standard freeze–pump–thaw method (at least 3 cycles). The ampule was flame-sealed and placed into an oil bath, which was thermoset at the desired temperature. At timed intervals, the ampule was immersed into iced water and then opened. The contents were dissolved in 3 mL of tetrahydrofuran (THF) and precipitated into 400 mL of methanol. Carbonodiselenoate-labeled polystyrene (PS-Se) was obtained by filtration and then dried to constant weight at room temperature under vacuum. The conversion of styrene was gravimetrically determined.

2.4. Typical Procedure of Selenol-Based Nucleophilic Addition of PS-Se to PEGMA Without additional notes, a typical procedure for optimization of reaction time is given below as 1 an example. A dry 5-mL ampule was filled with PS-Se-1 (Mn,SEC = 1900 g mol− , 95 mg, 0.059 mmol), 1 PEGMA950 (Mn = 950 g mol− , 0.59 g, 0.59 mmol), and DMF (2.0 mL) with a stir bar. After being thoroughly bubbled with argon for 15 min to eliminate the dissolved oxygen, n-hexylamine (30 µL) was added. Then, the ampule was flame-sealed immediately. After stirring for 1 d at 60 ◦C, the solution was precipitated into 100 mL of methanol. The polymer was obtained by filtration and then dried to constant weight at room temperature under vacuum.

2.5. Fabrication of PS-Se-b-PEGMA Micelles PS-Se-b-PEGMA (2 mg) was dissolved in DMF (2 mL), and then deionized water (0.3 mL) was 1 added to the solution using a syringe pump at the rate of 0.2 mL h− at room temperature. After addition of the water, the suspension was stocked for 1 day to stabilize the aggregates. Then, the suspension was dialyzed in a dialysis bag (molecular weight cutoﬀ: 1000 Da) against deionized water for at least 24 h to remove DMF. After dialysis, the suspension was added to deionized water until the 1 volume increased to 2 mL with a concentration of 1 mg mL− for further tests.

2.6. Chemical Oxidation of PS-Se-b-PEGMA Micelles by H2O2 In brief, 0.5 mL of PS-Se-b-PEGMA micelle suspension was kept in a 1-mL ampule and then placed into H2O2 solution (33 mM). After stirring for 5 h, the micelle suspension was freeze-dried for TEM studies.

2.7. In Vitro Cytotoxicity Study of the Micelles The samples were disinfected under ultraviolet light, and then ﬁve groups of extracts (2, 4, 6, 8, 1 and 10 mg mL− ) were prepared. The blank group (culture medium) and the control group (culture medium and cells) were set up to compare with the experimental group. NIH-3T3 cells were inoculated on 96-well plates at a density of 8 104 mL 1 (8 103 well 1). Cells were cultured in incubators (at × − × − 37 ◦C and 5% carbon dioxide) to become adhered to the 96-well plates. After 24 h, the medium was removed and the extract was added to culture. Then, 24 h later, the extract was removed and 10 µL CCK-8 solution and 90 µL culture medium were added to each pore. Then, cells were incubated at 37 ◦C for 1 h. A microplate reader was used to measure the absorbance of the sample at 450 nm. Polymers 2019, 11, 827 4 of 12

2.8. Drug Loading The following process was carried out in the dark. A mixture of 5.0 mg DOX HCl and 3.6 µL of · triethylamine (TEA) in 1.0 mL of dimethyl sulfoxide (DMSO) was added to a 2-mL ampule. After stirring overnight, excess TEA was removed by rotary evaporation to give the hydrophobic DOX solution. Then, 4 mL of PBS solution (50 mM, pH 7.4) was added dropwise to the mixture of copolymer 1 1 in DMF (1.0 mL, 1 mg mL− ) and DOX base solution (50 µL, 5.0 mg mL− ) with stirring at room temperature. Afterwards, in order to remove both unencapsulated DOX and the organic solvent, the mixture was dialyzed against PBS solution (50 mM, pH 7.4) for 24 h. The amount of DOX was determined by fluorescence (FL4600) measurement (excitation at 480 nm). First, a calibration curve was obtained by measuring the fluorescence intensity of different concentration DOX/DMSO solutions. Second, the fluorescence intensity of DOX-loaded micelles dissolved in DMSO was analyzed. The amount of DOX loaded in the micelles could be determined using the calibration curve. The drug loading content (DLC) and drug loading efficiency (DLE) were calculated using the following formulas: DLC (wt.%) = (weight of loaded drug / weight of (polymer + loaded drug)) 100% × DLE (wt.%) = (weight of loaded drug / weight of drug in feed) 100% × 2.9. Oxidation-Responsive Drug Release

In brief, 33 mM H2O2 was added into freshly prepared self-assembled solution (1.0 mL) of PS-Se-1-b-PEGMA950 in PBS. The reaction mixture was stirred at 25 ◦C for 3 h. Fluorescence spectrophotometry was then used to monitor the change in ﬂuorescence intensity of the micelle solution.

3. Results and Discussion

3.1. Organoselenium-Mediated Controlled Radical Polymerization The diselenocarbonate-end capped polymers were prepared through the organoselenium-mediated polymerization of styrene (St) according to our previous reports [33]. Two polymers, PS-Se-1 and PS-Se-2, with diﬀerent molecular weights and narrow molecular weight distribution (<1.20) were prepared, as detailed in Table1. The structure of PS-Se-1 was characterized by 1H NMR. As shown in Figure1, the signals at δ 3.78 ppm (3H, I3.78 = 3.00) were ascribed to the protons of the methoxy group (–OMe), and the signals around δ 4.50 ppm (1H, I4.43-4.68 = 1.06) were ascribed to the protons of CH-Se. The signals at around δ 7.00 ppm (5H, I6.60-7.11 = 92.1) were ascribed to the protons of the phenyl group. 1 Thus, the molecular weight (Mn, NMR) can be calculated to be 2100 g mol− by the equation Mn, NMR = 104 [(92.1 9) / 5] + 91 + 295. The polymers were also measured by using SEC with coupled × − refractive index (RI) and UV detectors (Figure2). The two curves almost coincide, and the molecular 1 1 weight (Mn,GPC = 1900 g mol− ) was close to the value obtained by H NMR. All evidence proved the high chain end functionality of the diselenocarbonate-ended polystyrene (PS), which ensured further chain end modiﬁcation. Polymers 2018, 10, x FOR PEER REVIEW 5 of 13

Polymers 2019, 11, 827 5 of 12 Polymers 2018, 10, x FOR PEER REVIEW 5 of 13

Figure 1. 1H NMR (in CDCl3) spectra of PS-Se-1.

1 FigureFigure 1. 1.H 1H NMR NMR (in(in CDCl3)3 )spectra spectra of PS-Se-1. of PS-Se-1.

1 Figure 2. SEC curves of PS-Se-1 (Mn, SEC = 1900 g mol− , Ð = 1.20) measured by RI and UV detection at theFigure wavelength 2. SEC curves of 340 of PS-Se-1 nm. (Mn, SEC = 1900 g mol−1, Ɖ = 1.20) measured by RI and UV detection at the wavelength of 340 nm.

Table 1. Polymerization of St with the molar ratio [St]0:[Se-CTA]0:[AIBN]0 = 50:1:0.3 in bulk at 70 ◦C. Figure 2. SEC curves of PS-Se-1 (Mn, SEC = 1900 g mol−1, Ɖ = 1.20) measured by RI and UV detection at a b c the wavelength of 340 nm. Mn,SEC Mn,th Mn,NMR a Entry Time (h) Conv. (%) 1 1 1 Ð (g mol− ) (g mol− ) (g mol− ) 1 (PS-Se-1) 2 27.6 1900 1800 2100 1.20 2 (PS-Se-2) 4 37.5 4200 4200 4400 1.17 a 1 Mn,SEC and Ð were determined by size exclusion chromatography (SEC; using THF as eluent, 1 mL min− , 40 ◦C) using polystyrene calibration. b Theoretical molecular weight. c Molecular weight determined by 1H NMR.

3.2. Reaction Condition Optimization of Selenol-Based Nucleophilic Reaction

As in our previous reports, diselenocarbonate was expected to be converted to selenol by amine 1 compounds [39]. In the present work, after the rapid aminolysis of PS-Se-1 (Mn, SEC = 1900 g mol− ) 1 by n-hexyl amine, the corresponding selenol reacted with PEGMA950 (Mn = 950 g mol− ) to make the block copolymers (Scheme2). We initiated our studies by examining the e ﬀect of time on selenol-based nucleophilic reaction. The results are presented in Table2. Screening experiments indicated that moderate conversion of PS-Se-1 could be obtained after four days (entries 1, 2, 3, and 4). An increase in temperature from 25 ◦C to 60 ◦C resulted in the increase of the conversion of PS-Se-1 from 22.4% to 50% (Table2, entry 5). It was already found that Bu 3P could act as a reducing agent to prevent Polymers 2018, 10, x FOR PEER REVIEW 6 of 13

Table 1. Polymerization of St with the molar ratio [St]0:[Se-CTA]0:[AIBN]0 = 50:1:0.3 in bulk at 70 °C.

Mn,SECa Mn,thb Mn,NMRc Entry Time (h) Conv. (%) Ɖa (g mol-1) (g mol-1) (g mol-1) 1 (PS-Se-1) 2 27.6 1900 1800 2100 1.20 2 (PS-Se-2) 4 37.5 4200 4200 4400 1.17

a Mn,SEC and Ð were determined by size exclusion chromatography (SEC; using THF as eluent, 1 mL min−1, 40 °C) using polystyrene calibration. b Theoretical molecular weight. c Molecular weight determined by 1H NMR.

3.2. Reaction Condition Optimization of Selenol-Based Nucleophilic Reaction As in our previous reports, diselenocarbonate was expected to be converted to selenol by amine compounds [39]. In the present work, after the rapid aminolysis of PS-Se-1 (Mn, SEC = 1900 g mol−1) by n-hexyl amine, the corresponding selenol reacted with PEGMA950 (Mn = 950 g mol−1) to make the block copolymers (Scheme 2). We initiated our studies by examining the effect of time on selenol-based Polymers 2019, 11, 827 6 of 12 nucleophilic reaction. The results are presented in Table 2. Screening experiments indicated that moderate conversion of PS-Se-1 could be obtained after four days (entries 1, 2, 3, and 4). An increase in temperature from 25 °C to 60 °C resulted in the increase of the conversion of PS-Se-1 from 22.4% oxidative coupling of selenol. Without Bu P, the conversion of PS-Se-1 dropped to 14.6% (Table2, entry to 50% (Table 2, entry 5). It was already3 found that Bu3P could act as a reducing agent to prevent 6). Bu3Poxidative also acts coupling as a catalyst of selenol. for Without the subsequent Bu3P, the conversion selenol-Michael of PS-Se-1 addition dropped reaction.to 14.6% (Table Consequently, 2, no moreentry catalyst, 6). Bu such3P also as DBUacts as and a Etcatalyst3N, was for neededthe subseq to beuent added selenol-Michael to this system addition (entries reaction. 7 and 8) [1]. Lastly, theConsequently, eﬀect of molarno more ratio cata onlyst, selenol-based such as DBU and nucleophilic Et3N, was needed reaction to wasbe added examined. to this system The reaction became(entries more smooth7 and 8) as [1]. the Lastly, amount the effect of PEGMA of molar (entries ratio on 9 andselenol-based 10) increased. nucleophilic When reaction the molar was ratio of examined. The reaction became more smooth as the amount of PEGMA (entries 9 and 10) increased. PS-Se-1:PEGMA = 1:10, the reaction reached the highest conversion rate of about 85% after separation. When the molar ratio of PS-Se-1:PEGMA = 1:10, the reaction reached the highest conversion rate of In contrast,about the 85% higher after separation. molar ratio In contrast, of PS-Se-1:PEGMA the higher molar (1:50) ratio resulted of PS-Se-1:PEGMA in an extremely (1:50) resulted viscose in solution, which mayan extremely prevent viscose further solution, reaction. which Moreover, may prevent puriﬁcation further reaction. loss also Moreover, decreases purification the product loss also yield. decreases the product yield.

SchemeScheme 2. Reaction 2. Reaction route route of aminolysis of aminolysis of of diselenocarbonate diselenocarbonate organic organic compound compound with hexylamine with hexylamine and and then selenol-based nucleophilic reaction with PEGMA950. then selenol-based nucleophilic reaction with PEGMA950.

Table 2. Optimization studies for selenol-based nucleophilic reaction of PS-Se-1 with PEGMA950 a. a Table 2. Optimization studies for selenol-based nucleophilic reaction of PS-Se-1 with PEGMA950 .

Time Conv. c Mn,SEC d Entry TimeEntry (d) CatalystCatalystR b R bConv. c (%) M d (g molÐ d1 ) Ð d (d) (%) (gn,SEC mol-1) − 1 1 1 - - 1:1 1:1 24.824.8 3100 31001.14 1.14 2 2 - 1:1 23.0 3400 1.11 2 2 - 1:1 23.0 3400 1.11 3 4 - 1:1 50.0 3500 1.12 4 63 4 - - 1:1 1:1 31.750.0 3500 34001.12 1.11 5 e 4 6 - - 1:1 1:1 22.431.7 3400 37001.11 1.10 6 f 54 e 4 - - 1:1 1:1 14.622.4 3700 37001.10 1.11 7 4 10% DBU 1:1 25.9 3500 1.10 8 4 10% Et3N 1:1 22.8 3400 1.11 9 4 - 1:2 64.0 3500 1.11 10 4 - 1:10 85.0 3700 1.11 11 4 - 1:50 46.0 3500 1.12 a b c The reaction was carried out at 60 ◦C with Bu3P. The ratio of PS-Se-1 with PEGMA950. Conversion of PS-Se-1 determined after separation by NMR analysis with tetramethylsilane (TMS) as the internal standard for chemical d 1 e shifts. Ð was determined by SEC (THF as eluent, 1 mL min− , 40 ◦C) using polystyrene calibration. The reaction f was carried out at 25 ◦C with Bu3P. The reaction was carried out at 60 ◦C without Bu3P.

3.3. Selenol-Based Nucleophilic Reaction of PS-Se-1 and PEGMAs with Different Molecular Weights After studying reaction condition optimization for selenol-based nucleophilic reaction of PS-Se-1 with PEGMA950, PEGMAs with different molecular weights were examined. The results are listed in Table3. PS-Se-1 reacted e fficiently with PEGMA and the conversion rate of PS-Se-1 with PEGMA320 peaked at 95.4%, and the conversion rate of PS-Se-1 decreased with the increase of molecular weight of PEGMA. The Mn of copolymers was determined by SEC. As shown in Figure3, the curves shifted significantly after the nucleophilic reaction, which evidenced the successful modification of PEGMA at the end of PS-Se-1. In the 1H NMR spectra, the proton signals of the corresponding vinyl shifted from 6.10 and 5.54 ppm to 2.57 and 2.49 ppm, and the proton signals of CH groups close to Se shifted from 4.70 and 4.47 ppm to 2.84 ppm, indicating a complete conversion of PEGMA (Figure4). Also, MALDI-TOF mass spectrometry was used to further characterize the structure of the copolymer. As shown in Figure5, the main population at the isotropic peak at 2340.428 m /z matched the theoretical calculation well (2340.152 m/z). Furthermore, two main sequence peaks are very close to the styrene 1 1 (104.15 g mol− ) and CH2CH2O (44.03 g mol− ) units. All the evidence indicated the efficiency of this reaction. Polymers 2018, 10, x FOR PEER REVIEW 7 of 13

6 f 4 - 1:1 14.6 3700 1.11 7 4 10% DBU 1:1 25.9 3500 1.10 8 4 10% Et3N 1:1 22.8 3400 1.11 9 4 - 1:2 64.0 3500 1.11 10 4 - 1:10 85.0 3700 1.11 11 4 - 1:50 46.0 3500 1.12

a The reaction was carried out at 60 °C with Bu3P. b The ratio of PS-Se-1 with PEGMA950. c Conversion of PS-Se-1 determined after separation by NMR analysis with tetramethylsilane (TMS) as the internal standard for chemical shifts. d Ð was determined by SEC (THF as eluent, 1 mL min−1, 40 °C) using

polystyrene calibration. e The reaction was carried out at 25 °C with Bu3P. f The reaction was carried out at 60 °C without Bu3P.

3.3. Selenol-Based Nucleophilic Reaction of PS-Se-1 and PEGMAs with Different Molecular Weights After studying reaction condition optimization for selenol-based nucleophilic reaction of PS-Se- 1 with PEGMA950, PEGMAs with different molecular weights were examined. The results are listed in Table 3. PS-Se-1 reacted efficiently with PEGMA and the conversion rate of PS-Se-1 with PEGMA320 peaked at 95.4%, and the conversion rate of PS-Se-1 decreased with the increase of molecular weight of PEGMA. The Mn of copolymers was determined by SEC. As shown in Figure 3, the curves shifted significantly after the nucleophilic reaction, which evidenced the successful modification of PEGMA at the end of PS-Se-1. In the 1H NMR spectra, the proton signals of the corresponding vinyl shifted from 6.10 and 5.54 ppm to 2.57 and 2.49 ppm, and the proton signals of CH groups close to Se shifted from 4.70 and 4.47 ppm to 2.84 ppm, indicating a complete conversion of PEGMA (Figure 4). Also, MALDI-TOF mass spectrometry was used to further characterize the structure of the copolymer. As shown in Figure 5, the main population at the isotropic peak at 2340.428 m/z matched the theoretical calculation well (2340.152 m/z). Furthermore, two main sequence peaks are very close to the styrene (104.15 g mol−1) and CH2CH2O (44.03 g mol−1) units. All the evidence indicated the efficiency of this reaction. Polymers 2019, 11, 827 7 of 12

Polymers 2018, 10, x FOR PEER REVIEW 8 of 13

1 Figure 3. SEC curves of PS-Se-1 (Mn,SEC = 1900 g mol− , Ð = 1.14), PS-Se-1-b-PEGMA320 (Mn,SEC = 2700 1 −1 1 Figureg mol 3. −SEC, Ð curves= 1.09), of PS-Se-1- PS-Se-1b -PEGMA(Mn,SEC = 5001900(M gn,SEC mol =, Ɖ3200 = 1.14), g mol PS-Se-1-− , Ð =b1.09),-PEGMA and320 PS-Se-1- (Mn,SECb =-PEGMA 2700 g 950 −1 Polymers 2018, 10, x FOR PEER1 REVIEW −1 8 of 13 mol(M, Ɖn,SEC = 1.09),= 3700 PS-Se-1- g molb−-PEGMA, Ð = 1.11).500 (Mn,SEC = 3200 g mol , Ɖ = 1.09), and PS-Se-1-b-PEGMA950 (Mn,SEC = 3700 g mol−1, Ɖ = 1.11).

Figure 4. 1H NMR (in CDCl3) spectrum of PS-Se-1-b-PEGMA320 (Mn,SEC = 2700 g mol−1, Ɖ = 1.09).

Figure1 4. 1H NMR (in CDCl3) spectrum of PS-Se-1-b-PEGMA320 (Mn,SEC = 2700 g mol−1, Ɖ = 1.09).1 Figure 4. H NMR (in CDCl3) spectrum of PS-Se-1-b-PEGMA320 (Mn,SEC = 2700 g mol− , Ð = 1.09).

1 Figure 5. Mass spectra of PS-Se-1-b-PEGMA320 (Mn,SEC = 2700 g mol− , Ð = 1.09).

Figure 5. Mass spectra of PS-Se-1-b-PEGMA320 (Mn,SEC = 2700 g mol−1, Ɖ = 1.09).

Table 3. Selenol-based nucleophilic reaction of PS-Se with different molecular weight PEGMAs.

−1 EntryFigure 5. Mass spectra PEGMA of PS-Se-1- (g molb-PEGMA-1) Conv.320 (M n,SEC(%) =a 2700 M g n,SECmol (g, Ɖmol = 1.09).-1) b Ð b 1 (PS-Se-1-b-PEGMA320) 320 95.4 2700 1.09 Table 3. Selenol-based nucleophilic reaction of PS-Se with different molecular weight PEGMAs. 2 (PS-Se-1-b-PEGMA500) 500 90.2 3200 1.09

3 (PS-Se-1-b-PEGMAEntry 950) PEGMA 950 (g mol-1) Conv. 85.0 (%) a Mn,SEC 3700 (g mol-1) b 1.11 Ð b 1 (PS-Se-1-b-PEGMA320) 320 95.4 2700 1.09 2 (PS-Se-1-b-PEGMA500) 500 90.2 3200 1.09 3 (PS-Se-1-b-PEGMA950) 950 85.0 3700 1.11

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Table 3. Selenol-based nucleophilic reaction of PS-Se with diﬀerent molecular weight PEGMAs.

1 a 1 b b Entry PEGMA (g mol− ) Conv. (%) Mn,SEC (g mol− ) Ð

1 (PS-Se-1-b-PEGMA320) 320 95.4 2700 1.09 2 (PS-Se-1-b-PEGMA500) 500 90.2 3200 1.09 Polymers3 (PS-Se-1- 2018, b10-PEGMA, x FOR PEER950) REVIEW 950 85.0 3700 1.119 of 13 a Conversion of PS-Se-1 determined after separation by NMR analysis with reference to TMS as an internal standard. b 1 Ð wasa Conversion determined of by PS-Se-1 SEC (THF determined as eluent, after 1 mL separation min− , 40 by◦C) NMR using analysis polystyrene with calibration. reference to TMS as an internal standard. b Ð was determined by SEC (THF as eluent, 1 mL min-1, 40 °C) using polystyrene 3.4. Self-Assemblycalibration. of PS-Se-1-b-PEGMA950 Before and After Oxidation

Selenium-containing3.4. Self-Assembly of PS-Se-1-b-PEGMA copolymers have950 Before shown and After redox Oxidation responsiveness in many systems. Some selenide-containing aggregates can respond rapidly to external redox stimuli and subsequently release Selenium-containing copolymers have shown redox responsiveness in many systems. Some the incorporatedselenide-containing species aggregates under mild can conditionsrespond rapidly [26,28 to]. external Here, self-assemblyredox stimuli and behavior subsequently of the three copolymersrelease werethe incorporated investigated. species It was under found mild that conditio PS-Se-1-nsb -PEGMA[26,28]. Here,950 could self-assembly self-assemble behavior spontaneously of the in aqueousthree copolymers solution through were investigated. hydrophobic It was/hydrophilic found that interaction. PS-Se-1-b-PEGMA As shown950 could in Figure self-assemble6, the TEM measurementspontaneously of PS-Se-1- in aqueousb-PEGMA solution950 micelles through showedhydrophobic/hydrophilic spherical aggregates interaction. with an As average shown diameter in of 30Figure nm. The 6, the DLS TEM results measurement are in accordance of PS-Se-1- withb-PEGMA the TEM950 micelles results showed with an spherical average aggregates diameter with of 56 nm (Figurean 7averagea). It is noteworthydiameter of 30 that nm. the The micelles DLS results were quiteare in stableaccordance in an with ambient the TEM environment results with and an could average diameter of 56 nm (Figure 7a). It is noteworthy that the micelles were quite stable in an maintain their structures for at least one month. Then, H2O2 solution was used as the oxidant to study the oxidationambient environment responsiveness and ofcould the maintain selenide their block structures copolymer for at aggregates. least one month. From Then, the TEM H2O images2 solution shown was used as the oxidant to study the oxidation responsiveness of the selenide block copolymer in Figure6c,d, the micellar structure was converted to irregular aggregates after two hours of oxidation aggregates. From the TEM images shown in Figure 6c,d, the micellar structure was converted to process,irregular and theseaggregates irregular after two aggregates hours of oxidation were further process, decomposed and these irregular for another aggregates three were hours. further The DLS resultsdecomposed also proved for that another the morphologythree hours. The change DLS result of thes also aggregates proved that occurred the morphology after adding change H2O of2 solution.the Furthermore,aggregates XPS occurred was alsoafter usedadding to H analyze2O2 solution. these Furthermore, micelles before XPS andwas afteralso used oxidation to analyze treatment. these As shownmicelles in Figure before7b, and the after binding oxidation energy treatment. of Se 3d As5 shifts shown from in Figure 56.22 7b, eV the to 60.81 binding eV, energy suggesting of Se a3d higher5 valencyshifts of from selenium 56.22 eV which to 60.81 is close eV, suggesting to the seleninic a higher acid valency group of [selenium21]. All thewhich results is close proved to the thatseleninic oxidative cleavageacid ofgroup the selenide[21]. All the linkage results leads proved to the that morphology oxidative cleavage change of of the the selenide micelle. linkage leads to the morphology change of the micelle. a) b)

c) d)

FigureFigure 6. TEM 6. TEM images images of self-assembledof self-assembled nanoparticles nanoparticles formed by by PS-Se-1- PS-Se-1-b-PEGMAb-PEGMA (M (n,SECMn,SEC = 3700= g3700 g mol mol1, Ð−1=, Ɖ1.11, = 1.11, 1 1 10× 104−4M) M) atat ((aa)) × 10.010.0 k k and and (b (b) )× 70.070.0 k magnification. k magniﬁcation. TEM TEM images images of nanoparticles of nanoparticles − × − × × 5 h after5 h after adding adding H OH2Osolution2 solution at at ( c(c)) × 10.0 k k and and (d (d) )× 25.025.0 k magnification. k magniﬁcation. 2 2 × ×

PolymersPolymers 20192018,, 1110,, 827x FOR PEER REVIEW 109 ofof 1213

Polymers 2018, 10, x FOR PEER REVIEW 10 of 13

1 4 Figure 7. (a) DLS results of PS-Se-1-b-PEGMA (Mn,SEC = 3700 g mol− , Ð = 1.11, 1 10− M) assemblies Figure 7. (a) DLS results of PS-Se-1-b-PEGMA (Mn,SEC = 3700 g mol−1, Ɖ = 1.11, 1 × 10−4 M) assemblies n,SEC −1 Ɖ ×−4 before andFigure after 7. oxidation (a) DLS results treatment. of PS-Se-1- (b)b XPS-PEGMA analysis (M of = the3700 selenide g mol , polymer = 1.11, 1 × aggregates 10 M) assemblies before and before andbefore after and oxidation after oxidation treatment. treatment. (b) XPS(b) XPS analysis analysis of of the the selenideselenide polymer polymer aggregates aggregates before before and and after oxidation treatment. after oxidationafter oxidation treatment. treatment. 3.5. Cytotoxicity Test 3.5. Cytotoxicity3.5. Cytotoxicity Test Test The nanomicellesThe nanomicelles based based on selenide-labeled on selenide-labeled amphiphilic amphiphilic copolymers potentially potentially provide provide a new a new The nanomicelles based on selenide-labeled amphiphilic copolymers potentially provide a new platformplatform for targeted for targeted drug drug delivery. delivery. We We examined examined the cytotoxicity cytotoxicity of PS-Se-1- of PS-Se-1-b-PEGMAb-PEGMA (Mn,SEC = (3700Mn,SEC = platform for 1targeted−1 drug delivery.−4 4 We examined the cytotoxicity of PS-Se-1-b-PEGMA (Mn,SEC = 3700 3700 g molg mol− , Ð, Ɖ= =1.11, 1.11, 1 × 1010−M).M). As shown As shown in Figure in Figure8, it can8 ,be it seen can that be seenthe PS-Se- thatb the-PEGMA PS-Se- showedb-PEGMA −1 −4 × showedg mol , lowƉlow = 1.11,cytotoxicitycytotoxicity 1 × 10 when M). As compared compared shown inwith with Figure other other 8,selenide-containing it selenide-containing can be seen that polymers the polymers PS-Se- [40].b -PEGMA [The40]. obvious The obviousshowed inhibitory effect on the NIH-3T3 cells was shown when the concentration of the micelles was high, at inhibitorylow cytotoxicity eﬀect on when the NIH-3T3 compared cells with was shownother whenselenide-containing the concentration polymers of the micelles [40]. The was obvious high, at up to 1.6 mg mL−1. inhibitory effect on1 the NIH-3T3 cells was shown when the concentration of the micelles was high, at up to 1.6 mg mL− . up to 1.6 mg mL−1.

1 Figure 8. Cytotoxicity of PS-Se-b-PEGMA (Mn,SEC = 3700 g mol− , Ð = 1.11) at diﬀerent concentrations. n,SEC −1 Ɖ The data areFigure expressed 8. Cytotoxicity as mean of PS-Se-SD,b-PEGMA where n(M= 3. = 3700 g mol , = 1.11) at different concentrations. The data are expressed as mean± ± SD, where n = 3. 3.6. Drug Loading and Oxidation-Responsive Drug Release 3.6. Drug Loading and Oxidation-Responsive Drug Release

The anticancerThe anticancer drug drug doxorubicin doxorubicin (DOX) (DOX) waswas chosen chosen as asa model a model molecule molecule for encapsulation. for encapsulation. The Figure 8. Cytotoxicity of PS-Se-b-PEGMA (Mn,SEC = 37001 g mol−1, Ɖ = 1.11) at different concentrations. The self-assemblyself-assembly of PS-Se-1- of PS-Se-1-b-PEGMAb-PEGMA950950 (1 mg mg mL mL−1)− was) was conducted conducted in DMF in DMFsolution solution in the presence in the presence of 1 of DOXThe (0.1dataDOX mg are (0.1 mLexpressed mg− mL), which−1 ),as which mean has has± a SD, characteristic a characteristicwhere n = 3.maximum maximum emission emission at at590 590 nm. nm. The The DOX-loading DOX-loading micelles weremicelles purified were purified with dialysiswith dialysis membrane, membrane, and and the the DOXDOX concentration was was calculated calculated by the by the fluorescence3.6. Drug fluorescenceLoading emission and emission Oxidation-Responsive spectra. spectra. The drugThe drug loading Drug loading Release content content (DLC) (DLC) was was evaluated evaluated to be to about be about 1.5%, 1.5%, and and drug loading efficiency (DLE) was evaluated to be about 13.3%. Oxidation-triggered drug release drug loading efficiency (DLE) was evaluated to be about 13.3%. Oxidation-triggered drug release The studiesanticancer in vitro drug were doxorubicin investigated (DOX)at pH 7.4 was and chosen 25 °C by as using a model H2O 2molecule. After oxidation for encapsulation. for a specific The studiesself-assemblyintime, vitro ofthewere PS-Se-1- fluorescence investigatedb-PEGMA emission at950 pH of(1 DOXmg 7.4 mLandwas− 1 25)monito was◦C conducted byred, using as shown H in2O inDMF2 .Figure After solution 9a. oxidation The in fluorescence the for presence a specific of time,DOX the(0.1 fluorescencemg mL−1), which emission has ofa characteristic DOX was monitored, maximum as emission shown in at Figure 590 nm.9a. The fluorescenceDOX-loading intensitymicelles were of DOX purified at 590 with nm increased dialysis membrane, gradually, owingand the to DOX the release concentration of hydrophobic was calculated DOX from by thethe drugfluorescence carriers. emission The percentage spectra. The release drug was loading calculated content based (DLC) on was the fluorescenceevaluated to changesbe about at1.5%, 590 and nm. Thedrug percentage loading efficiency release profile (DLE) was was plotted evaluated as a functionto be about of time, 13.3%. as shownOxidation-triggered in Figure9b. drug release studies in vitro were investigated at pH 7.4 and 25 °C by using H2O2. After oxidation for a specific time, the fluorescence emission of DOX was monitored, as shown in Figure 9a. The fluorescence

Polymers 2018, 10, x FOR PEER REVIEW 11 of 13

intensity of DOX at 590 nm increased gradually, owing to the release of hydrophobic DOX from the drug carriers. The percentage release was calculated based on the fluorescence changes at 590 nm. Polymers 2019, 11, 827 10 of 12 The percentage release profile was plotted as a function of time, as shown in Figure 9b.

Figure 9. (a) Fluorescence (FL) spectra of the micelles in PBS solution upon treatment with H2O2 (33 Figure 9. (a) Fluorescence (FL) spectra of the micelles in PBS solution upon treatment with H2O2 (33 mM). (b) Cumulative doxorubicin (DOX) release from DOX-loaded PS-Se-1-b-PEGMA950 micelles. mM). (b) Cumulative doxorubicin (DOX) release from DOX-loaded PS-Se-1-b-PEGMA950 micelles. 4. Conclusions 4. Conclusions In conclusion, a straightforward protocol for the synthesis of an oxidation-sensitive selenide-containing block copolymerIn conclusion, has beena straightforward developed on protocol the basis for of Se-RAFT.the synthesis The diselenocarbonateof an oxidation-sensitive termini selenide- of the polymerscontaining were block readily copolymer converted has to been the selenide-containingdeveloped on the basis block of copolymerSe-RAFT. The via aminolysisdiselenocarbonate and selenol-basedtermini of the nucleophilic polymers reaction.were readily The converted present controlled to the selenide-containing radical polymerization block (CRP)-basedcopolymer via protocolaminolysis offers and a facile selenol-based and straightforward nucleophilic fabrication reaction. ofThe selenide-containing present controlled block radical copolymers polymerization that feature(CRP)-based many monomer protocol accessibilities, offers a facile predictable and straightforward MWs, and programmablefabrication of polymericselenide-containing architectures. block Thesecopolymers copolymers that formed feature micelle-like many monomer nanoparticles accessibilities, of ~56 nm predictable in diameter MWs, that showand insignificantprogrammable cytotoxicitypolymeric of architectures. the micelles at These high concentrationcopolymers formed and could micelle-like be disrupted nanoparticles by H2O2. Theof ~56 biocompatibility nm in diameter andthat targeted show insignificant cytotoxicity cytotoxicity results suggest of the that micelles these oxidation-sensitiveat high concentration selenide-containing and could be disrupted block by copolymersH2O2. The could biocompatibility be developed and for targeted controllable cytotoxicity drug release. results suggest that these oxidation-sensitive selenide-containing block copolymers could be developed for controllable drug release. Author Contributions: X.A. and W.L. performed the experiments; J.Z. analyzed the data and revised the manuscript;Author Contributions: X.P. conceived X.A. and and designed W.L. performed the experiments; the experiments; X.Z. provided J.Z. analyzed suggestion the data for theand designing revised the of experiments.manuscript; X.P. conceived and designed the experiments; X.Z. provided suggestion for the designing of Funding:experiments.This research was funded by the National Natural Science Foundation of China (nos. 21074082, 21302132, and 21874200), the Natural Science Foundation of Jiangsu Province (BK20130296), the Natural Science FoundationFunding: ofThis the research Jiangsu Higherwas funded Education by the Institutions National Natural of China Science (no. 12KJB150021), Foundation of the China Priority (nos. Academic 21074082, Program21302132, Development and 21874200), of Jiangsu the Natural Higher Science Education Foundation Institutions, of Jiangsu and Province the Suzhou (BK20130296), Key Lab of Macromolecularthe Natural Science DesignFoundation and Precision of the Synthesis.Jiangsu Higher Education Institutions of China (no. 12KJB150021), the Priority Academic ConflictsProgram of Development Interest: The authorsof Jiangsu declare Higher no conflictEducation of interest.Institutions, and the Suzhou Key Lab of Macromolecular Design and Precision Synthesis.

ReferencesConflicts of Interest: The authors declare no conflict of interest.

1. Wirth, T. Organoselenium Chemistry: Modern Developments in ORGANIC Synthesis; Springer: Berlin, Germany, References 2000; Volume 208. 2. 1. Xia,Wirth, J.H.; Li,T. Organoselenium T.Y.; Lu, C.J.; Xu, Chemistry: H.P. Selenium-Containing Modern developments Polymers: in ORGANIC Perspectives Synthesis toward; Springer: Diverse Berlin, Applications Germany, in Both2000; AdaptiveVolume 208. and Biomedical Materials. Macromolecules 2018, 51, 7435–7455. [CrossRef] 3. 2. Xu,Xia, H.P.; J.H.; Cao, Li, W.; Zhang,T.Y.; Lu, X. Selenium-ContainingC.J.; Xu, H.P. Selenium-Containing Polymers: Promising Polymers: Biomaterials Perspectives for Controlled toward Release Diverse andApplications Enzyme Mimics. in BothAcc. Adaptive Chem. Res.and 2013Biomedical, 46, 1647–1658. Materials. [CrossRef Macromolecules] 2018, 51, 7435–7455. 4. 3. Nketia-Yawson,Xu, H.P.; Cao, B.;W.; Jung,Zhang, A.R.; X. Sele Nguyen,nium-Containing H.D.; Lee, K.K.; Polymers: Kim, B.;Promising Noh, Y.Y. Biomaterials Difluorobenzothiadiazole for Controlled Release and Selenophene-Basedand Enzyme Mimics. Conjugated Acc. Chem. Polymer Res. 2013 Demonstrating, 46, 1647–1658. an Effective Hole Mobility Exceeding 5 cm(2) V-1 4. s(-1)Nketia-Yawson, with Solid-State B.; Electrolyte Jung, A.R.; Dielectric. Nguyen, H.D.;ACS Appl. Lee, K.K. Mater.; Kim, Interfaces B.; Noh,2018 Y.Y., 10 ,Difluorobenzothiadiazole 32492–32500. [CrossRef] and 5. Liang,Selenophene-Based Z.Q.; Li, M.M.; Conjugated Zhang, X.M.; Polymer Wang, Demonstrating Q.; Jiang, Y.; Tian, an Effective H.K.; Geng, Hole Y.H. Mobility Near-infrared Exceeding absorbing 5 cm(2) V-1 non-fullerenes(-1) with Solid-State acceptors Electrolyte with selenophene Dielectric. as piACS bridges Appl. forMater. effi cientInterfaces organic 2018 solar, 10, 32492–32500. cells. J. Mater. Chem. A 5. 2018Liang,, 6, 8059–8067. Z.Q.; Li, M.M.; [CrossRef Zhang,] X.M.; Wang, Q.; Jiang, Y.; Tian, H.K.; Geng, Y.H. Near-infrared absorbing 6. Kim,non-fullerene H.S.; Song, acceptors E.; Lee, S.B.;with Kang,selenophene I.N.; Cho, as pi K.; bridges Hwang, for D.H.efficient Eff ectorganic of methyl solar cells. substitution J. Mater. on Chem. the A diketopyrrolopyrrole-based2018, 6, 8059–8067. semiconducting polymers for organic thin film transistors. Org. Electron. 2018, 6. 56,Kim, 129–138. H.S.; [ CrossRefSong, E.;] Lee, S.B.; Kang, I.N.; Cho, K.; Hwang, D.H. Effect of methyl substitution on the

Polymers 2019, 11, 827 11 of 12

7. Xu, Z.J.; Shi, G.Z.; Yuan, J.Y.; Glenn, E.; Wang, H.Q.; Ma, W.L. New Semiconducting Polymer Based on

Benzo[1,2-b:4,5-b0]diselenophene Donor and Diketopyrrolopyrrole/Isoindigo Acceptor Unit: Synthesis, Characterization and Photovoltaics. Chin. J. Chem. 2015, 33, 909–916. [CrossRef] 8. Ashraf, R.S.; Meager, I.; Nikolka, M.; Kirkus, M.; Planells, M.; Schroeder, B.C.; Holliday, S.; Hurhangee, M.; Nielsen, C.B.; Sirringhaus, H.; et al. Chalcogenophene Comonomer Comparison in Small Band Gap Diketopyrrolopyrrole-Based Conjugated Polymers for High-Performing Field-Effect Transistors and Organic Solar Cells. J. Am. Chem. Soc. 2015, 137, 1314–1321. [CrossRef] 9. Kang, I.; An, T.K.; Hong, J.A.; Yun, H.J.; Kim, R.; Chung, D.S.; Park, C.E.; Kim, Y.H.; Kwon, S.K. Effect of Selenophene in a DPP Copolymer Incorporating a Vinyl Group for High-Performance Organic Field-Effect Transistors. Adv. Mater. 2013, 25, 524–528. [CrossRef] 10. Chang, H.H.; Tsai, C.E.; Lai, Y.Y.; Liang, W.W.; Hsu, S.L.; Hsu, C.S.; Cheng, Y.J. A New Pentacyclic Indacenodiselenophene Arene and Its Donor-Acceptor Copolymers for Solution-Processable Polymer Solar Cells and Transistors: Synthesis, Characterization, and Investigation of Alkyl/Alkoxy Side-Chain Effect. Macromolecules 2013, 46, 7715–7726. [CrossRef] 11. Lee, W.H.; Son, S.K.; Kim, K.; Lee, S.K.; Shin, W.S.; Moon, S.J.; Kang, I.N. Synthesis and Characterization of New Selenophene-Based Donor-Acceptor Low-Bandgap Polymers for Organic Photovoltaic Cells. Macromolecules 2012, 45, 1303–1312. [CrossRef] 12. Khim, D.; Lee, W.H.; Baeg, K.J.; Kim, D.Y.; Kang, I.N.; Noh, Y.Y. Highly stable printed polymer field-effect transistors and inverters via polyselenophene conjugated polymers. J. Mater. Chem. 2012, 22, 12774–12783. [CrossRef] 13. Ji, S.B.; Cao, W.; Yu, Y.; Xu, H.P. Visible-Light-Induced Self-Healing Diselenide-Containing Polyurethane Elastomer. Adv. Mater. 2015, 27, 7740–7745. [CrossRef] 14. Ji, S.B.; Fan, F.Q.; Sun, C.X.; Yu, Y.; Xu, H.P. Visible Light-Induced Plasticity of Shape Memory Polymers. ACS Appl. Mater. Interfaces 2017, 9, 33169–33175. [CrossRef] 15. Du, W.N.; Jin, Y.; Lai, S.Q.; Shi, L.J.; Fan, W.H.; Pan, J.Z. Near-infrared light triggered shape memory and self-healable polyurethane/ functionalized graphene oxide composites containing diselenide bonds. Polymer 2018, 158, 120–129. [CrossRef] 16. An, X.W.; Aguirresarobe, R.H.; Irusta, L.; Ruiperez, F.; Matxain, J.M.; Pan, X.Q.; Aramburu, N.; Mecerreyes, D.; Sardon, H.; Zhu, J. Aromatic diselenide crosslinkers to enhance the reprocessability and self-healing of polyurethane thermosets. Polym. Chem. 2017, 8, 3641–3646. [CrossRef] 17. Hailemeskel, B.Z.; Addisu, K.D.; Prasannan, A.; Mekuria, S.L.; Kao, C.Y.; Tsai, H.C. Synthesis and characterization of diselenide linked poly(ethylene glycol) nanogel as multi-responsive drug carrier. Appl. Surf. Sci. 2018, 449, 15–22. [CrossRef] 18. Han, P.; Ma, N.; Ren, H.F.; Xu, H.P.; Li, Z.B.; Wang, Z.Q.; Zhang, X. Oxidation-Responsive Micelles Based on a Selenium-Containing Polymeric Superamphiphile. Langmuir 2010, 26, 14414–14418. [CrossRef] 19. Huang, Y.K.; Chen, Q.X.; Lu, H.G.; An, J.X.; Zhu, H.J.; Yan, X.J.; Li, W.; Gao, H. Near-infrared AIEgen-functionalized and diselenide-linked oligo-ethylenimine with self-sufficing ROS to exert spatiotemporal responsibility for promoted gene delivery. J. Mater. Chem. B 2018, 6, 6660–6666. [CrossRef] 20. Salma, S.A.; Patil, M.P.; Kim, D.W.; Le, C.M.Q.; Ahn, B.H.; Kim, G.D.; Lim, K.T. Near-infrared light-responsive, diselenide containing core-cross-linked micelles prepared by the Diels-Alder click reaction for photocontrollable drug release application. Polym. Chem. 2018, 9, 4813–4823. [CrossRef] 21. Sun, T.B.; Zhu, C.Z.; Xu, J. Multiple stimuli-responsive selenium-functionalized biodegradable starch-based hydrogels. Soft Matter 2018, 14, 921–926. [CrossRef] 22. Xia, J.H.; Ji, S.B.; Xu, H.P. Diselenide covalent chemistry at the interface: Stabilizing an asymmetric diselenide-containing polymer via micelle formation. Polym. Chem. 2016, 7, 6708–6713. [CrossRef] 23. Salma, S.A.; Le, C.M.Q.; Kim, D.W.; Cao, X.T.; Jeong, Y.T.; Lim, K.T. Synthesis and characterization of diselenide crosslinked polymeric micelles via Diels-Alder click reaction. Mol. Cryst. Liq. Cryst. 2018, 662, 188–196. [CrossRef] 24. Kroll, H.; Bolton, E.F. Synthesis of Selenium Condensation Polymers Using Bis(2-Hydroxyethyl) Selenide and Bis(2-Aminoethyl) Selenide. J. Appl. Polym. Sci. 1970, 14, 2319–2325. [CrossRef] 25. Tuten, B.T.; Bloesser, F.R.; Marshall, D.L.; Michalek, L.; Schmitt, C.W.; Blanksby, S.J.; Barner-Kowollik, C. Polyselenoureas via Multicomponent Polymerizations Using Elemental Selenium as Monomer. ACS Macro Lett. 2018, 7, 898–903. [CrossRef] Polymers 2019, 11, 827 12 of 12

26. Ma, N.; Li, Y.; Ren, H.F.; Xu, H.P.; Li, Z.B.; Zhang, X. Selenium-containing block copolymers and their oxidation-responsive aggregates. Polym. Chem. 2010, 1, 1609–1614. [CrossRef] 27. Gunther, W.H.H.; Salzman, M.N. Methods in Selenium Chemistry 4. Synthetic Approaches to Polydiselenides. Ann. NY Acad. Sci. 1972, 192, 25–43. [CrossRef][PubMed] 28. Ma, N.; Li, Y.; Xu, H.P.; Wang, Z.Q.; Zhang, X. Dual Redox Responsive Assemblies Formed from Diselenide Block Copolymers. J. Am. Chem. Soc. 2010, 132, 442–443. [CrossRef] 29. Chiefari, J.; Chong, Y.K.; Ercole, F.; Krstina, J.; Jeffery, J.; Le, T.P.T.; Mayadunne, R.T.A.; Meijs, G.F.; Moad, C.L.; Moad, G.; et al. Living free-radical polymerization by reversible addition-fragmentation chain transfer: The RAFT process. Macromolecules 1998, 31, 5559–5562. [CrossRef] 30. Kwon, T.S.; Takagi, K.; Kunisada, H.; Yuki, Y. Synthesis of star polystyrene by radical polymerization with 1,2,4,5-tetrakis(p-tert-butylphenylselenomethyl)benzene as a novel photoiniferter. Eur. Polym. J. 2003, 39, 1437–1441. [CrossRef] 31. Zeng, J.D.; Zhu, J.; Zhang, Z.B.; Pan, X.Q.; Zhang, W.; Cheng, Z.P.; Zhu, X.L. New Selenium-Based Iniferter Agent for Living Free Radical Polymerization of Styrene Under UV Irradiation. J. Polym. Sci. Part A Polym. Chem. 2012, 50, 2211–2218. [CrossRef] 32. Keddie, D.J. A guide to the synthesis of block copolymers using reversible-addition fragmentation chain transfer (RAFT) polymerization. Chem. Soc. Rev. 2014, 43, 496–505. [CrossRef][PubMed] 33. Gao, F.; Pan, X.Q.; Zhu, J.; Zhang, Z.B.; Zhang, W.; Zhu, X.L. Facile synthesis of well-defined redox responsive diselenide-labeled polymers via organoselenium-mediated CRP and aminolysis. Polym. Chem. 2015, 6, 1367–1372. [CrossRef] 34. Cai, Z.X.; Lu, W.H.; Gao, F.; Pan, X.Q.; Zhu, J.; Zhang, Z.B.; Zhu, X.L. Diselenide-Labeled Cyclic Polystyrene with Multiple Responses: Facile Synthesis, Tunable Size, and Topology. Macromol. Rapid Commun. 2016, 37, 865–871. [CrossRef][PubMed] 35. Wei, C.; Zhang, Y.; Yan, B.K.; Du, Z.Z.; Lang, M.D. A Versatile Strategy to Main Chain Sulfur/Selenium-Functionalized Polycarbonates by Macro-Ring Closure of Diols and Subsequent Ring-Opening Polymerization. Chem. Eur. J. 2018, 24, 789–792. [CrossRef][PubMed] 36. Pan, X.Q.; Driessen, F.; Zhu, X.L.; Du Prez, F.E. Selenolactone as a Building Block toward Dynamic Diselenide-Containing Polymer Architectures with Controllable Topology. ACS Macro Lett. 2017, 6, 89–92. [CrossRef] 37. Wei, C.; Xu, Y.; Yan, B.K.; Hou, J.Q.; Du, Z.Z.; Lang, M.D. Well-Defined Selenium-Containing Aliphatic Polycarbonates via Lipase-Catalyzed Ring-Opening Polymerization of Selenic Macrocyclic Carbonate Monomer. ACS Macro Lett. 2018, 7, 336–340. [CrossRef] 38. Zeng, J.D.; Zhu, J.; Pan, X.Q.; Zhang, Z.B.; Zhou, N.C.; Cheng, Z.P.; Zhang, W.; Zhu, X.L. Organoselenium compounds: Development of a universal "living" free radical polymerization mediator. Polym. Chem. 2013, 4, 3453–3457. [CrossRef] 39. Lu, W.H.; An, X.W.; Gao, F.; Zhu, J.; Zhou, N.C.; Zhang, Z.B.; Pan, X.Q.; Zhu, X.L. Highly Efficient Chain End Derivatization of Selenol-Ended Polystyrenes by Nucleophilic Substitution Reactions. Macromol. Chem. Phys. 2017, 218, 1600485. [CrossRef] 40. Zhou, W.; Wang, L.; Li, F.; Zhang, W.; Huang, W.; Huo, F.; Xu, H.P. Selenium-Containing Polymer@Metal-Organic Frameworks Nanocomposites as an Efficient Multiresponsive Drug Delivery System. Adv. Funct. Mater. 2017, 27, 1605465. [CrossRef]

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