ROMP synthesis of benzaldehyde-containing ...

Reactive and Functional Polymers 128 (2018) 1–15

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ROMP synthesis of benzaldehyde-containing amphiphilic block polynorbornenes used to conjugate drugs for pH-responsive release

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Guirong Qiua, Li Zhaoa, Xiong Liua,b, Qiuxia Zhaoa,b, Fangfei Liua,b, Yue Liua,b, Yewu Liua, ⁎ Haibin Gua,b, a b

Key Laboratory of Leather Chemistry and Engineering of Ministry of Education, Sichuan University, Chengdu 610065, China National Engineering Laboratory for Clean Technology of Leather Manufacture, Sichuan University, Chengdu 610065, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Amphiphilic block copolymer Ring-opening metathesis polymerization Polymer-drug conjugates Schiff-base pH-responsive

The development of novel polymer carriers, which have well-defined structures and desired conjugative abiltiy to therapeutic agents, is still an urgent requirement in the field of polymer–drug conjugates (PDCs) used for stimuli-responsive drug delivery systems. Herein, we present the controlled synthesis of a novel amphiphilic polynorbornene-based block copolymer, side-chain containing hydrophobic functionalizable benzaldehyde groups and hydrophilic dentritic triethylene glycol (TEG) moieties, by the ring-opening metathesis polymerization (ROMP) using the 3rd generation Grubbs catalyst 1 as the initiator. The obtained copolymer can selfassemble into spherical micelles with the average size of 84 nm in water, and its remarkable ability as the polymer carrier to fabricate PDCs was fully confirmed by the efficient covalently Schiff-base linking of its sidechain benzaldehyde groups to amino groups of model drugs including O-benzylhydroxylamine (BHA), 1-hexadecanamine, tryptophan and benzocaine. Notably, torispherical micelles with the average size of 90 nm were also obtained by the self-assembly of the formed polymer-BHA conjugate, and the pH-triggered release of BHA molecules from the micelles was observed at acidic environments (for example, pH 4.0 and 2.5). Therefore, the present polynorbornene block copolymer is expected to find potential applications in the stimuli-responsive drug delivery systems as a promising polymer carrier to form PDCs via acid-responsive Schiff-base linkage with amino-containing drugs such as doxorubicin, daunorubicin, epirubicin and pirarubicin.

1. Introduction Since Jatzkewitz's pioneering work in 1955 [1] and Ringsdorf's clear concept in 1975 [2], polymer-drug conjugates (PDCs), in which therapeutic agents are not physically encapsulated but covalently linked to a polymeric macromolecular carrier, have attracted considerable attention as a shining platform for drug delivery in the academic and commercial communities [3–7]. The major advantage of PDCs involve high drug loading [8], increased aqueous solubility of hydrophobic drugs [9], prolonged plasma half-life [10], improved drug bioavailability [11], enhanced metabolic stability [12], modified pharmacokinetic properties and biodistribution [13], pH-responsive or enzymecontrolled drug release at the active site [14], specific accumulation in organs, tissues or cells by targeting agents [15], enhanced permeability and retention (EPR) effect [16], the prevention of the blood brain barrier penetration [12], and so on. The structure of PDCs is normally composed of a water-soluble polymer backbone, hydrophobic drug(s) usually bound to the polymer

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via a stimuli-responsive linker [17], and targeting moiety (not always necessary) [18,19]. The performance of PDCs is thus dramatically influenced by multiple structural factors including the architecture of polymeric macromolecular carriers, linker chemistry, desired target (intracellular, lymphatic system, etc.), and the molecular weight (MW) [20–22]. Especially, a wide range of synthetic and natural water-soluble polymers have been evaluated for the development of PDCs, for example, poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA) [23,24], poly(ethylene glycol) (PEG) [25,26], poly(amidoamine) (PAMAM) [27,28], polyglycerol (PG) [29], poly(L-lysine) (PLL) [30] and polysaccharides [31]. Recently, polynorbornene-based polymeric carriers [32–37] are attracting intensively research interests owing to their well-defined structure and subsequently monodisperse, precise polymeric prodrugs resulted from the living and controlled ringopening metathesis polymerization (ROMP) technique [38,39]. The ROMP method can provide well-defined materials due to the exceptional functional group tolerance of the Grubbs' catalyst employed in this process [40–42]. And because of the distinguished attribute, ROMP

Corresponding author at: Key Laboratory of Leather Chemistry and Engineering of Ministry of Education, Sichuan University, Chengdu 610065, China. E-mail address: [email protected] (H. Gu).

https://doi.org/10.1016/j.reactfunctpolym.2018.03.010 Received 1 February 2018; Received in revised form 21 March 2018; Accepted 25 March 2018 Available online 25 April 2018 1381-5148/ © 2018 Elsevier B.V. All rights reserved.


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2.2. Instruments

has been utilized for the preparation of efficient drug delivery systems including the recently reported PDCs [32–37]. Based on ROMP, for example, Johnson et al. [43–45] constructed polynorbornene-based brush polymer–drug conjugates (BPDCs) with each backbone repeat unit simultaneously carrying a PEG chain and a photocleavable drug (doxorubicin or camptothecin) moiety, and further reported a convergent synthetic platform for single-nanoparticle combination cancer therapy which exhibits ratiometric loading and controlled release of cisplatin, doxorubicin, and camptothecin. Cheng et al. [46] also synthesized well-defined diblock BPDCs for sustained delivery of paclitaxel by using the sequential ROMP method with the 3rd generation Grubbs' catalyst as the initiator. Baumgartner and Cheng et al. [47] reported the synthesis of controlled, high-molecular weight poly(L-glutamic acid) and polynorbornene-based brush polymers and their conjugates with camptothecin. The brush polymers themselves are non-toxic to cells, while the obtained PDCs show cytotoxicity, demonstrating the potential of these carriers for applications in drug delivery [47]. Along this line, polynorbornenes have the great potential to serve as polymeric carriers of PDCs, and it is then of interest to design novel water-soluble polynorbornenes and examine their conjugation capacity with drug molecules. Thus, in this work, we present a novel amphiphilic block copolymer containing polynorbornene backbone and reactive benzaldehyde moiety in the side chain. The benzaldehyde part is designed to conjugate to amino-containing drug molecules by the formation of acid-sensitive revisable Schiff-base linker [48–53], and the release of the bound drug molecules is then expected to be triggered in specific acidic sites such as extracellular matrices of tumor tissues, endosomes and lysosomes within tumor cells [54–57]. The tetraethylene glycol and triethylene glycol (TEG) structures are used to construct the hydrophilic block of the copolymer owing to their excellent water-solubility [58–61], and are expected to have the same advantages (nontoxicity, nonimmunogenicity, stealth effect, etc.) of PEG analogues [25,26]. The TEG block is prepared by using the “graft through” strategy [44,62], namely, a macromonomer containing tetraethylene glycol and triethylene glycol (TEG) moieties is synthesized and then polymerized in a controlled manner by using the powerful ROMP technique. The macromonomer route is attractive owing to the obtained well-defined hydrophilic structure, and there is no possible incomplete grafting problem that is often observed in the normal “graftto” and “graft-from” routes [40,63–65] used to introduce macromolecular PEG units. By using a one-pot two-step sequential ROMP route, the benzaldehyde and TEG-containing amphiphilic block copolymer is synthesized, and its ability as the polymer carrier was well examined to fabricate pH-responsive PDCs by the formation of Schiffbase linker between side-chain benzaldehyde group of the copolymer and amino group of representative model drugs including O-benzylhydroxylamine (BHA), 1-hexadecanamine, tryptophan and benzocaine. The self-assembly behaviors of the amphiphilic block copolymer and its PDCs are detailedly studied, and the pH-triggered release of drugs from the formed micelles is also observed at acidic environments.

1

H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded at 25 °C using a Bruker AC (400 MHz) spectrometer. All the chemical shifts are reported in parts per million (δ, ppm) with reference to tetramethylsilane (TMS). Mass spectra were recorded using an Applied Biosystems Voyager-DE STR-MALDI-TOF spectrometer. The infrared spectra were recorded on an ATI Mattson Genesis series FT-IR spectrophotometer the range 400–4000 cm−1. UV–visible absorption spectra were measured with a Perkin-Elmer Lambda 19 UV–visible spectrometer. Size exclusion chromatography (SEC) measurements were conducted in N, N-dimethyl formamide (DMF) using Shimadzu high performance liquid chromatography (HPLC) system equipped with PLgel 5 μm MIXED-D columns, refractometric and UV detectors, column oven and integrated degasser. Polymer molecular weights were calculated based on the multiangle light scattering data using the Wyatt Astra software, with dn/dc values of the polymers determined from the RI detector using Astra. Column calibration was performed using polystyrene (PS) standards from Polymer Laboratories. Dynamic light scattering (DLS) measurements were made using Malvern Zetasizer Nano-ZS series equipment (Malvern Instruments, UK) at 25 °C at an angle of 90°. Atomic force microscopy (AFM) measurements were conducted using tapping mode operation with a SPM-9600 AFM (SHIMADZU, Japan). Scanning electron microscopy (SEM) observations were conducted with a SM-7500F field emission SEM instrument (JEOL). Samples for SEM were prepared by casting a drop of the solution on a silicon grid, followed by drying at room temperature (r.t., 25 °C). 2.3. Synthesis of 5 3 (1.14 g, 5.94 mmol, 1.2 equiv) and 4 (1.0 g, 4.97 mmol, 1 equiv) and were dissolved dry tetrahydrofuran (THF, 50 ml). CuSO4·5H2O (1.49 g, 5.94 mmol, 1.2 equiv) in water (25 ml) was then added into the solution at r.t. under N2 atmosphere, followed by the dropwise addition of sodium ascorbate (NaAsc, 2.37 g, 11.88 mmol, 2.4 equiv) in water (25 ml). The obtained mixture was stirred for 72 h at r.t. under N2 atmosphere, and vacuumed to remove the THF solvent. The residue was dissolved in the mixture of CH2Cl2 (100 ml) and ammonia water (50 ml), and stirred at r.t. for 30 min. The organic layer was collected, washed with brine, dried over anhydrous Na2SO4 and concentrated to give the crude that was then purified by column chromatography with CH2Cl2/methanol (0% → 10%) as eluent to give 5 as white solid. Yield: 1.7 g, 87%. 1H NMR (400 MHz, (CD3)2SO, 25 °C, TMS), δppm: 9.86 (s, 1H, CHO), 8.07 (s, 1H, C]CH of triazole), 7.86–7.83 (d, J = 7.9 Hz, 2H, ph), 7.11–7.07 (d, J = 8.7 Hz, 2H, ph), 6.29 (d, J = 1.8 Hz, 2H, CH]CH), 4.78 (m, 2H, NCH2), 4.60 (t, J = 6.7 Hz, 2H, CONCH2), 4.49 (t, J = 3.8 Hz, 2H, CH2O-ph), 3.07 (t, J = 4.4 Hz, 2H, ]CHeCH), 2.71 (d, J = 3.1 Hz, 2H, COeCH), 1.23 (m, 2H, CH2-bridge). 13C NMR (100 MHz, CDCl3, 25 °C, TMS), δppm: 190.7 (CHO), 177.3 (CON), 162.5 (O-ph), 142.2 (C]C of triazole), 137.9 (CH]CH of norbornene), 132.1, 130.1, 114.7 (ph), 124.1 (C]C of triazole), 66.5 (OCH2), 49.5 (N] NeNeCH2), 47.9 (CHeCON), 45.4 (]CeCH), 42.8 (CH2-bridge), 33.6 (CONCH2). MS (ESI, m/z), calcd. for C21H20N4O4: 392.42; found: 393.15 (M + H+), 415.13 (M + Na+). IR (KBr, cm−1): 1771 cm−1 (υC]O), 1700 cm−1 (υC]O), 1600 cm−1 (υC]C), 1578, 1508, 1427 cm−1 (ph), 1331 cm−1 (υCeN).

2. Experimental 2.1. Materials Cis-5-norbornene-exo-2, 3-dicarboxylic anhydride (95%), tetraethylene glycol, triethylene glycol monomethyl ether, ethyl vinyl ether (99%, EVE), propargylamine, p-hydroxybenzaldehyde, methyl gallate, BHA, 1-hexadecanamine, tryptophan, benzocaine, and the second generation Grubbs metathesis catalyst were purchased from Energy Chemical, and used directly. Grubbs' 3rd generation catalyst (1) was synthesized following a procedure reported in the literature [42]. All the other chemicals were from commercial sources and used as received. All the solvents used were dried and freshly distilled.

2.4. ROMP synthesis of homopolymer 6 The desired amount of Grubbs' 3rd catalyst 1 was dissolved by a minimum amount of dry CH2Cl2 in a small Schlenk flask under N2 atmosphere. A known amount of monomer 5 in dry CH2Cl2 (1 ml per 100 mg of 5) was added to the catalyst solution under N2 atmosphere with vigorous stirring. The obtained reaction mixture was stirred vigorously until the signal of olefinic protons of monomer 5 at 6.21 ppm 2


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disappeared by checking in situ1H NMR of reaction mixture in CDCl3, which indicates the monomer conversion reached 100%. Then, 0.3 ml of EVE was added to quench the catalyst. All the samples were collected, purified by precipitating from CH2Cl2 with diethyl ether (Et2O) three times, and dried in vacuo until constant weight to get the polymer 6 as white powder. Yield: 95% (6a), 94% (6b), 96% (6c). 1H NMR (400 MHz, (CD3)2SO, 25 °C, TMS) δppm: 9.81 (s, 1H, CHO), 8.02 (s, 1H, C]CH of triazole), 7.79 (s, 2H, ph), 7.37–7.21 (m, end-ph), 7.05 (s, 2H, ph), 5.58 and 5.43 (double broad, 2H, CH]CH), 4.71–4.46 (m, 6H, CONCH2 and NCH2CH2O), 3.07 (broad, 3H, ]CeCH and NCOCH), 2.60 (braod, 1H, NCOCH), 1.91 (broad, 1H, ]CeCHCH2), 1.41 (broad, 1H, ]CeCHCH2). 13C NMR (100 MHz, (CD3)2SO, 25 °C, TMS), δppm: 191.7 (CHO), 178.0 (CON), 163.1 (ph), 142.5 (C]CH of trizaloe), 133.5 (CH]CH), 132.2 (CH]CH and ph), 130.4, 115.4 (ph), 124.1 (C]CH of trizaloe), 65.4 (CH2O), 55.4 (N]N-N-CH2), 51.0 (CHC]O), 49.3 (]CeCHeCH2), 45.2 (]CeCH), 33.7 (CONCH2). IR (KBr, cm−1): 2952 cm−1 (υCH2), 1771 cm−1 (υC]O), 1700 cm−1 (υC]O), 1600 cm−1 (υC]C), 1578, 1508, 1427 cm−1 (ph), 1331 cm−1 (υCeN).

trizaole), 7.63 (broad, 1H, NHCO), 7.23 (s, 2H, ph), 5.68 and 5.46 (double broad, 2H, CH]CH), 5.18 and 5.13 (ds, 6H, 3 × ph-OCH2), 4.51 and 4.45 (ds, 6H, 3 × triazolyl NeCH2), 3.84–3.80 (br, 6H, 3 × triazolyl NeCH2CH2), 3.53–3.46 (m, 40H, NCH2(CH2OCH2)3CH2N and 3 × OCH2CH2OCH2CH2O), 3.29 (s, 9H, 3 × OCH3), 3.01 and 2.64 (br, 4H, ]CHeCH and CHeCO), 2.03 and 1.54 (br, 2H, CH]CHCHCH2). 13C NMR (100 MHz, CDCl3, 25 °C, TMS), δppm: 177.4 (CON), 165.7 (NHCO), 151.0 (ph), 143.1 (triazole), 142.3 (ph), 133.4 and 132.1 (CH]CH), 129.2 (ph), 123.9, 123.7 (triazole), 106.2 (ph), 70.8, 69.4, 69.2, 68.8, 68.4, 68.3, 66.0, 65.2 (OCH2), 61.9 (triazolylNCH2), 58.0 (OCH3), 49.2, 49.0 (ph-OCH2), 42.9 (CHCON), 39.0 (]CHCH), 36.5 (CH2-bridge). Selected IR (KBr, cm−1): 3448 cm−1 (υNH), 2922 cm−1 (υCH2), 1770 cm−1 (υC]O), 1698 cm−1 (υNC]O), 1649 cm−1 (υc]c) 1105 cm−1 (υceoec). 2.7. ROMP synthesis of block copolymer 18 Monomers 5 (13.65 mg, 0.035 mmol, 10 equiv) and 16 (203.85 mg, 0.174 mmol, 50 equiv) were added separately into two small glass tubes, and dissolved in 0.4 ml and 2.0 ml of dry CH2Cl2, respectively. Catalyst 1 (3.08 mg, 0.00348 mmol, 1 equiv) was added into a small Schlenk flask, flushed with N2, and dissolved in 0.03 ml of dry CH2Cl2. First, monomer 5 was transferred to the flask containing the catalyst via a small syringe. The reaction mixture was stirred vigorously for 10 min at r.t. under N2 atmosphere, after which monomer 16 was added into the flask using a small syringe. The obtained reaction mixture was stirred vigorously until the signal of olefinic protons of monomer 16 at 6.22 ppm disappeared by checking in situ1H NMR of reaction mixture, which indicates the monomer conversion reached 100%. Then, 0.5 ml of EVE was added to quench the catalyst. All the samples were collected, purified by precipitating from CH2Cl2 with Et2O three times, and dried in vacuo until constant weight to get the block copolymer 18 as purple-red viscose oily liquid. Yield: 95%. 1H NMR (400 MHz, CDCl3, 25 °C, TMS) δppm: 9.82 (s, 1H, eCHO), 7.95 (broad, 10H, 2 × C]CH of trizaole), 7.85 (s, 5H, C]CH of trizaole), 7.77–7.73, (broad, 3H, C]CH of trizaole and 2H, benzene), 7.40 (s, 5H, NHCO), 7.25 (m, 10H, benzene), 6.93 (m, 2H, benzene), 5.74 and 5.48 (broad, 12H, CH]CH), 5.19–5.16 (m, 30H, pheOeCH2), 4.74 (m, 2H, N]NeNeCH2), 4.66 (m, 2H, N]NeNeCH2CH2), 4.54–4.41 (m, 30H, 3 × N]NeNeCH2), 4.41 (m, 2H, CONeCH2-triazloe), 3.88–3.81 (m, 30H, 3 × N] NeNeCH2CH2), 3.63–3.44 (m, 200H, NCH2(CH2OCH2)3CH2N and 3 × OCH2CH2OCH2CH2O), 3.31 (s, 45H, 3 × OCH3), 3.03 (broad, 12H, ]CHeCH), 2.65 (broad, 12H, CHCON), 1.57 (broad, 12H, CH2 of cyclopentane). 13C NMR (100 MHz, CDCl3, 25 °C, TMS) δppm: 190.8 (CHO), 178.3 (NC]O), 166.7 (NHC]O), 162.8, 132.0, 130.2, 114.9 (ph of aldehyde block), 152.0, 143.3, 130.2, 107.3 (ph of TEG block), 144.1, 143.3, 124.6 (triazole of TEG block), 140.3, 126.2 (triazole of aldehyde block), 132.0 and 130.2 (C]C of norbornene), 71.8, 70.4,70.2, 69.9, 69.4, 69.3, 67.0, 66.3 (OCH2), 65.0 (triazole-CH2CH2 of aldehyde block), 63.0 (triazole-CH2 of TEG block), 58.9 (eOCH3), 52.4 (triazole-CH2 of aldehyde block), 50.2 (pheOeCH2), 46.0 (CHCON), 40.0 (]CHCHCH2), 37.7 (]CHeCH), 32.4 (CH2 of cyclopentane). IR (KBr, cm−1): 3443 cm−1 (υNH), 2922 cm−1 (υCH2), 1768 cm−1 (υC]O), 1697 cm−1 (υNC]O), 1646 cm−1 (υC]C), 1108 cm−1(υCeOeC).

2.5. Synthesis of 16 14 (0.236 g, 0.39 mmol, 1 equiv) and 15 (0.243 g, 1.29 mmol, 3.3 equiv) were dissolved dry THF (20 ml). CuSO4·5H2O (0.321 g, 1.29 mmol, 3.3 equiv) in water (10 ml) was then added into the solution at r.t. under N2 atmosphere, followed by the dropwise addition of NaAsc (0.51 g, 2.58 mmol, 6.6 equiv) in water (10 ml). The obtained mixture was stirred for 72 h at r.t. under N2 atmosphere, and vacuumed to remove the THF solvent. The residue was dissolved in the mixture of CH2Cl2 (50 ml) and ammonia water (10 ml), and stirred at r.t. for 30 min. The organic layer was collected, washed with brine, dried over anhydrous Na2SO4 and concentrated to give the crude that was then purified by column chromatography with CH2Cl2/methanol (0% → 10%) as eluent to give 16 as colorless oil. 1H NMR (400 MHz, CDCl3, 25 °C, TMS), δppm: 7.92 (s, 2H, 2 × C]CH of trizaole), 7.83 (s, 1H, C]CH of trizaole), 7.24 (s, 2H, ph), 7.21 (s, 1H, NHCO), 6.22 (t, J = 3.6 Hz, CH]CH), 5.20 (s, 4H, 2 × pheOCH2), 5.16 (s, 2H, phOCH2), 4.54–4.45 (m, 6H, 3 × N]NeNeCH2), 3.88–3.80 (m, 6H, 3 × N]NeNeCH2CH2), 3.62–3.44 (m, 40H, NCH2(CH2OCH2)3CH2N and 3 × OCH2CH2OCH2CH2O), 1), 3.19 (t, J = 3.4 Hz, 2H, ]CHeCH), 2.62 (d, J = 1.3 Hz, 2H, CHCON), 1.42 (d, J = 9.8 Hz, 1H, CH2-bridge), 1.29 (d, J = 9.8 Hz, 1H, CH2-bridge). 13C NMR (100 MHz, CDCl3, 25 °C, TMS), δppm: 178.1 (CON), 166.8 (NHCO), 152.1 (ph), 144.1, 143.4 (triazole), 140.4 (ph), 137.8 (CH]CH), 130.3 (ph), 124.8, 124.6 (triazole), 107.4 (ph), 71.9, 70.5, 70.3, 70.0, 69.9, 69.4, 69.4, 67.0, 66.4 (OCH2), 63.1 (triazolyl-NCH2), 59.0 (OCH3), 50.3, 50.1 (phOCH2), 47.8 (CHCON), 45.3 (]CHCH), 42.7 (CH2-bridge), 40.1 (CH2NHCO), 37.8 (CONCH2). MS (ESI m/z), calcd. For C54H81N11O18: 1171.58; found: 1194.57 [M + Na]+. IR (KBr, cm−1): 3446 cm−1 (υNH), 2874 cm−1 (υCH2), 1770 cm−1 (υC]O), 1699 cm−1 (υNC]O), 1107 cm−1 (υceoec). 2.6. ROMP synthesis of homopolymer 17 The desired amount of Grubbs' 3rd 1 was dissolved by a minimum amount of dry CH2Cl2 in a small Schlenk flask under N2 atmosphere. A known amount of monomer 16 in dry CH2Cl2 (1 ml per 100 mg of 16) was added to the catalyst solution under N2 atmosphere with vigorous stirring. The obtained reaction mixture was stirred vigorously until the signal of olefinic protons of monomer 17 at 6.22 ppm disappeared by checking in situ1H NMR of reaction mixture, which indicates the monomer conversion reached 100%. Then, 0.3 ml of EVE was added to quench the catalyst. All the samples were collected, purified by precipitating from CH2Cl2 with Et2O three times, and dried in vacuo until constant weight to get the polymer 17 as colorless sticky oil. Yield: 97% (17a), 95% (17b), 97% (17c). 1H NMR (400 MHz, CDCl3, 25 °C, TMS), δppm: 7.95 (s, 2H, 2 × C]CH of trizaole), 7.84 (s, 1H, C]CH of

2.8. General procedure for the synthesis of conjugates of 18 with model drugs Block copolymer 18 (100 mg, 0.016 mmol aldehyde group) and BHA (9.852 mg, 0.08 mmol) were dissolved in 2 ml of dry CH2Cl2. The obtained reaction mixture was stirred at 25 °C for 24 h under N2 atmosphere. The resulting polymer-BHA conjugate 19 was purified by reprecipitation in Et2O for three times and dried to constant weight under vacuum. The grafting ratio was calculated by comparing the intensity of 1 H NMR signal of the Schiff's base CH]N proton at 8.10 ppm with that 3


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N-propargyl-cis-5-norbornene-exo-2, 3-dicarboximide 4. The azide 3 was yielded from the starting p-hydroxybenzaldehyde that firstly reacted with 1, 2-dichloroethane to get 4-(2′-chloroethyoxyl) benzaldehyde, followed the azide reaction with sodium azide, while the norbornene alkynyl derivative 4 was prepared from cis-5-norbornene-exo2, 3-dicarboxylic anhydride in the presence of propargylamine. Fig. 1A shows the 1H NMR spectrum of monomer 5 in (CD3)2SO. The appearance of the signal at 8.07 ppm for the triazolyl proton confirms the success of the “click” reaction between 3 and 4 yielding 5. The peak at 9.86 ppm corresponds to the aldehyde proton, and the two double peaks at 7.78 and 7.73 ppm are assigned to the four phenyl protons. These data demonstrate the integrity of benzaldehyde structure. The peak at 6.21 ppm corresponds to the olefinic protons, while the peak at 1.22 ppm originates from the two characteristic bridgemethylene protons of the cis-norbornene structure. In the 13C NMR spectrum of 5 (Fig. S5), the two peaks at 142.2 and 124.1 ppm are assigned to the two carbon atoms of the triazolyl unit. The peak at 190.7 ppm corresponds to the carbon of the aldehyde group, while that located at 177.4 ppm originates from the carbonyl carbon of the cisnorbornene backbone, and that located at 137.9 ppm its olefinic carbon atoms. All the other peaks of the 1H and 13C NMR spectra are clearly assigned and match well with the structure of 5. The high-resolution mass spectrum of 5 (Fig. S6) provides the molecular peak at 393.16 Da, in good agreement with the theoretical value of 392.42 Da. The UV–vis spectrum of 5 (Fig. S7) shows a maximum absorption (λmax) at 271 nm assigned to the π → π* anti bond orbital transition of phenyl group. The IR spectrum (Fig. S8) also gives complementary information for the structure of 5. As shown in Scheme 1, the polymerization of 5 was then conducted in dry CH2Cl2 at r.t. through the ROMP technique using Grubbs' thirdgeneration catalyst 1 possessing dramatic tolerance toward various functional groups of substrates [40–42]. The adopted feed molar ratios are 10:1, 25:1, and 50:1, respectively, yielding the corresponding aldehyde-containing polymers 6 with various polymerization degrees. A kinetic study was carried out to monitor the polymerization of 5 at the biggest feed mole ratio of 50:1. At different intervals, a small sample of the reaction mixture was taken out, quenched with EVE, dried under reduced pressure, and checked the in situ1H NMR spectrum in CDCl3. The monomer conversion was estimated by comparing the intensities of the 1H NMR signals of the olefinic protons between monomer 5 (δ = 6.21 ppm) and polymers 6 (δ = 5.57 ppm and 5.43 ppm, Fig. 1B). Actually, the ROMP of 6c is complete with nearly 100% monomer conversion after only 10 min of stirring (Fig. S11). The fast polymerization rate indicates that the presence of aldehyde group has no negative effect on the activity of catalyst 1. In the 1H NMR spectrum of polymer 6a in (CD3)2SO (Fig. 1B), the disappearance of the peak at 6.21 ppm corresponding to the olefinic protons of monomer 5 (Fig. 1A) and the appearance of two new broad peaks at 5.57 ppm and 5.43 ppm assigned to the olefinic protons of polymer 6 demonstrate the successful ROMP of the monomer 5. Moreover, after polymerization, the other peaks of the cis-norbornene backbone, which are sharp signals in monomer 5, change to broad signals. The peak at 9.08 ppm corresponds to the proton of the aldehyde group, while the peak at 8.02 ppm originates from the triazolyl proton. The two broad peaks at 7.79 and 7.05 ppm arise from the phenyl protons in the benzaldehyde structure, while the signals for the end-phenyl protons are concentrated in 7.39–7.21 ppm. These data clearly demonstrate the integrity of the triazole and benzaldehyde moieties. In the 13C NMR spectrum of 6a (Fig. 2A), the two carbons of the triazolyl unit is observed at 142.47 and 124.10 ppm, respectively, while the peak at 191.74 ppm originates from the carbon atom of the aldehyde group. The peak at 178.0 ppm corresponds to the carbonyl carbons of polynorbornene, and the two peaks at 133.5 and 132.2 ppm are assigned to olefinic carbons. All the other peaks of the 1H and 13C NMR spectra are clearly assigned and match well with the structure of 6. The UV–vis spectrum of 6 (Fig. 3a) also exhibits an absorption band with λmax at

of the characteristic proton of aldehyde group at 9.80 ppm. Similar procedures were adopted for the preparation of conjugates 20–22 with 1-hexadecanamine, tryptophan and benzocaine, respectively. 2.9. Determination of critical micelle concentration (CMC) The pyrene fluorescence probe technique [66] was adopted to determine the CMCs of 18 and its conjugate 19 with BHA. Concretely, a known amount of pyrene in acetone was added to a battery of 10 ml volumetric flask to give a pyrene concentration of 2.0 × 10−6 M, followed the volatilization of acetone in vacuum condition. A measured amount of the micelle aggregate solutions with concentration ranging from 2.0 to 2.5 × 10−4 mg ml−1 was then added to the above each flask, and stirred at r.t. overnight to equilibrate the micelle aggregates and pyrene. The fluorescence spectra were detected by using a fluorescence spectrophotometer (RF-5310PC, SHIMADZU, Japan). The excitation wavelength was 339 nm. The plot of I3/I1 intensity ratios versus concentration of aggregate solutions was drawn to estimate the CMC by the tangent method [66]. 2.10. Preparation of micelles of 18 and PDC 19 in water The dialysis method has been adopted to fabricate the copolymer micelle aggregates. Concretely, 2.5 mg of copolymer 18 was absolutely dissolved in 0.5 ml DMF, followed by the addition of the deionized water 4.5 ml with vigorous stirring for 2 h. The obtained solution was then carefully injected into a dialysis bag with molecular weight cutoff (MWCO) of 35,000. The micellar solution was obtained after completely remove of DMF solvent through immersing the bag into 1000 ml of deionized water for 48 h, accompanying with the change of dialysate every 4 h. And similar procedure was used for the fabrication of micelles of the PDC 19 containing BHA. 2.11. The BHA release from micelles of PDC 19 For release experiment, 50 ml of micelles solution of 19 (2 mg ml−1) was injected into a dialysis bag (MWCO, 35000) and dialyzed against buffered solution of pH 7.4, 6.0, 4.0 and 2.5 (300 ml), respectively, with continuous stirring at r.t. for 48 h. At a predetermined interval, all dialysate were taken out from the Bunsen beaker for UV–visible analysis and replaced by fresh buffered solution with the same volume to keep the system volume constant. According to the standard curve of BHA and the corresponding formulation y = 2.7637× + 0.0075 (x is the concentration of BHA, and y is the absorption) in Fig. S58A, the BHA amount released (M) was quantitative calculated by using the release medium outside the dialysis bag through detecting the ultraviolet absorption (Abs) at 261 nm, and the calculation equation is as following:

M (mg ) =

Abs − 0.0075 × 300 2.7637

The value of 300 is the volume (ml) of dialysate used. The accumulative BHA release was then calculated by follows:

Cumulative BHA release /% =

Mt × 100% M0

Mt represents the released amount of BHA at time t, M0 indicate the amount of BHA loaded at the copolymer. 3. Results and discussion 3.1. Synthesis and ROMP of monomer 5 A novel norbornene monomer 5 (Scheme 1) containing the benzaldehyde group was synthesized by the CuAAC “click” reaction between 4-(2′-azidoethyoxyl) benzaldehyde 3 and the key intermediates 4


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Scheme 1. Synthesis route and ROMP of monomer 5 containing benzaldehyde group.

Fig. 1. 1H NMR spectra of monomer 5 (A) and polymer 6a (B) in (CD3)2SO.

271 nm corresponding to the π → π* anti bond orbital transition of phenyl and triazolyl groups. IR spectrum (Fig. S9) also provides the structure of 6. The MW distribution of the final polymers 6 was characterized by end-group analysis method and SEC technique. End-group analysis by 1 H NMR of the polymers 6a and 6b in (CD3)2SO (Tables S2 and S3) were conducted to estimate their polymerization degrees by comparing the signal integration of the five end-phenyl protons (7.21–7.39 ppm) with that of aldehyde proton (9.81 ppm), triazolyl proton (8.02 ppm), phenyl protons (7.79 and 7.05 ppm), olefinic protons (5.57 and 5.43 ppm), and methylene protons (4.48 and 4.72 ppm), respectively. The obtained values are 10 ± 0.5 for 6a and 25 ± 2 for 6b (Table 1). These results are in good agreement with the theoretical polymerization degrees calculated according to the molar feed ratios and the corresponding monomer conversions from 1H NMR, and indicate the controlled characteristic of ROMP of 5. However, end-group analysis is infeasible for 6c due to the weak signal of end-phenyl protons in the 1H NMR spectrum. Furthermore, SEC provides Mn value of 18,400 Da with 1.11 of polydispersity index (PDI) for 6aversus PS standard in DMF (Table 1). The determined value is larger than the corresponding theoretical value owing to the large structural difference between the PS standards and the benzaldehyde-containing polymer 6. Even so, the

small PDI value, reflected by unimodal and symmetric SEC trace (Fig. 4a); strongly demonstrate the controlled ROMP of the benzaldehyde-containing monomer 5. 3.2. Synthesis and ROMP of monomer 16 Scheme 2 shows the synthesis route of the dendronized macromonomer 16 that is designed to contain a long hydrophilic tetraethylene glycol unit linking the polymerizable cis-norbornene backbone and the dendron terminated by three water-soluble triethylene glycol (TEG) moieties. The tetraethylene glycol and TEG structures are expected to give 16 excellent hydrophilic properties, and the former as a long linker also has the ability to relieve the shell effect occurring during the polymerization process of the bulky dendronized macromonomers [40]. The intermediate 10 containing the long linker with amino end is prepared by the reaction of cis-5-norbornene-exo-2,3-dicarboxylic anhydride with 1,11-diamine-3,6,9-trioxaundecane 9 whose synthesis is detailedly described in the Supporting Information. The key norbornene derivative 14 containing three terminal alkynes is then synthesized by the amidation reaction between 10 and 3,4,5-tripropargyloxybenzoyl chloride 13 [40]. The targeted monomer 16 is finally successfully prepared by the “click” CuAAC reaction between 14 and 25


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Fig. 2.

13

C NMR spectra of homopolymer 6a (A, in (CD3)2SO) and 17 (B, in CDCl3), and block copolymer 18 (C, in CDCl3). Table 1 MW data for polymers 6, 17b and 18. Polymer

6a

6b

17b

18

[M]:[C]a Convb(%) np1c np2d M1e M2f PDIg

10:1 > 99 10 10 ± 0.5 4028 18,414 1.11

25:1 > 99 25 25 ± 2 – – –

50:1 > 99 50 – 58,683 76,494 1.07

10:50:1 > 99 10 10 ± 0.5 62,607 89,838 1.14

> 99 50 50 ± 3

a

[M]:[C]: feed molar ratio of monomer to catalyst 1. Monomer conversion determined by 1H NMR spectroscopy. c Polymreization degree obtained from 1H NMR data using the monomer conversion. d Polymreization degree determined by end-group analysis using 1H NMR spectroscopy. e Theoretical MW calculated by the conversion of monomer. f MW determined by SEC using polystyrene as the standard. g PDI calculated from the SEC data. b

Fig. 3. UV–vis curves of polymers 6 (a), 17 (b) and 18 (c).

(2-(2-methoxyethoxy)ethoxy)ethyl azide 15. In the 1H NMR spectrum of 16 in CDCl3 (Fig. 5A), the three triazolyl protons are observed at 7.92 and 7.83 ppm, proving the success of the “click” reaction between 14 and 15. The characteristic peak corresponding to three terminal methoxy groups is located at 3.30 ppm, while the methylene protons in the TEG termini and tetraethylene glycol linker are concentrated in 4.54–4.45, 3.88–3.80 and 3.62–3.44 ppm, respectively. The peak at 7.24 ppm is attributed to the two phenyl protons, and the peak at 7.21 ppm is assigned to the characteristic acylamino proton. The triple peak at 6.21 ppm corresponds to the two characteristic olefinic protons of cis-norbornene backbone,

while the two peaks at 3.19 and 2.62 ppm arise from its CeH protons, and its characteristic bridge-methylene protons are located at 1.42 and 1.29 pm, respectively. These peaks confirm the integrity of the cisnorbornene unit. In the 13C NMR spectra of 16 (Fig. S33), the four peaks at 144.1, 143.9, 124.8 and 124.6 ppm corresponds to the carbons of three triazolyl units. The carbon of acylamino group is located at 166.78 ppm, while the olefinic and carbonyl carbons of cis-norbornene 6


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theoretical value of 1195.3 Da. The UV–vis spectrum of 16 (Fig. S35) shows two peaks at 228 and 258 nm corresponding to the π → π* anti bond orbital transition of phenyl and triazolyl groups. The IR spectrum (Fig. S36) also provided complementary information of functional groups for the structure of 16. ROMP of the macromonomer 16 was also conducted in dry CH2Cl2 at r.t. using catalyst 1, and three molar feed ratios were adopted to produce the dendronized polymers 17 with various polymerization degrees. The polymerization reaction was monitored by in situ1H NMR spectroscopy, and the kinetic study results are shown in Fig. 6. After these polymerizations are activated, the olefinic protons of 16 shift from 6.26 to 5.73 and 5.49 ppm (Fig. 5B) and significantly broaden, and the monomer conversions are determined by comparing the integral area of the corresponding peaks. It took 45 min to accomplish the ROMP of 16 with nearly 100% conversion when the molar feed ratio was 10:1 (Fig. 6a), while for the increased feed ratios of 50:1 and 100:1, it was necessary to prolong the reaction time to 24 and 100 h (Fig. 6b and d), respectively, to achieve the quantitative conversions. This slow rate is mainly taken into account by the possibly reversible coordination of triazole units to the ruthenium (Ru) atom of the catalyst 1 [40,67], leading to its partial inhibition. Furthermore, with the increase of molar feed ratio, the prolonged reaction rates are attributed to the decreased initial concentrations of catalyst 1 [61,68]. In the 1H NMR spectrum of the dendronized polymer 17 (Fig. 5B), the disappearance of the characteristic peak at 6.26 ppm arising from the olefinic protons of monomer 16 and the appearance of the double peaks at 5.73 and 5.49 ppm corresponding to the olefinic protons of polynorbornene demonstrated the successful formation of polymer 17. The two peaks at 7.95 and 7.84 ppm are assigned to the three triazolyl protons in the dendron structure, while the peak at 3.29 ppm

Fig. 4. SEC curves of polymers 6a (a), 17b (b) and 18 (c).

are observed at 137.8 and 178.1 ppm, respectively, and the peaks at 152.1, 140.4, 130.3 and 107.4 ppm are assigned to the phenyl carbons. The peak at 59.0 ppm is attributed to the carbons of characteristic OCH3 group. All the other peaks of the 1H and 13C NMR spectra are clearly assigned and match well with the structure 16. The ESI mass spectrum of 16 (Fig. S34) provides the ion peak at 1194.56 Da ([M + Na]+), the molecular weight of 16 plus sodium ion, in good agreement with the

Scheme 2. Synthese route of TEG-contaning monomer 16 and polymer 17. 7


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Fig. 5. 1H NMR spectra of monomer 16 (A) and polymer 17 (B) in CDCl3.

also in agreement with the formation and structure of polymer 17. MALDI-TOF mass spectroscopy and SEC technique were adopted to characterize the MW distribution of polymer 17. Becuase the 1H signals of end-phenyl protons are mixed with that of phenly protons in the dendron structure, it is infeasible to estimate the polymerization degree of 17 by using the end-group analysis method. The MALDI-TOF mass spectrum of polymer 17a shows well-defined individual peaks for polymer fragments that are separated by 1172 ± 1 Da, which exactly corresponds to the mass of a unit of monomer 16 (Fig. 7). SEC data for the resulting polymer 17b provides a Mn value of 76,500 Da with PDI = 1.07 versus PS standards (Table S4). The result is larger than the theoretical value calculated by using the molar feed ratio and the corresponding monomer conversion, owing to the large structural difference between the PS standard and the dendronized polymer. However, the small PDI of obtained from the SEC trace (Fig. 4b), which are < 1.10, further demonstrated the controlled polymerization of 16.

Fig. 6. Kinetic curves for the synthesis of polymers 17a (a), 17b (b), 18 (c) and 17c (d).

corresponds to the characteristic terminal methoxy groups. The broad peak at 7.63 ppm originates from the acylamino proton, and the peak at 7.23 ppm is attributed to the phenyl protons, mixed with the signals for the end-phenyl and CDCl3 solvent protons. The two broad peaks at 5.18 and 5.13 correspond to methylene protons between triazolyl and phenyl rings, while the peaks at 4.51 and 4.45, 3.84 and 3.80, and 3.53–3.46 ppm are assigned to the methylene protons in the linker and TEG-containing dendron. In the 13C NMR spectrum of 17 (Fig. 2B), the four peaks at 143.1, 142.3, 123.7 and 123.9 ppm are assigned to the carbons of the trizaolyl rings, while the peak at 58.0 ppm is attributed to the carbons of OCH3 group. The peak at 177.4 ppm originates from the carbonyl carbons of polynorbornene, while the two peaks at 133.5 and 132.1 ppm correspond to its characteristic olefinic carbons. The carbon of the acylamino group is observed at 165.7 ppm, and the phenyl carbons are located at 151.0, 142.3, 129.2 and 106.2 ppm. All the other peaks of the 1H and 13C NMR spectra are clearly assigned and match well with the structure 17. The UV–vis spectrum of polymer 17 (Fig. 3b) shows an absorption band with λmax at 229 and 258 nm, respectively, similar to that of monomer 16. The IR spectrum (Fig. S37) is

3.3. ROMP synthesis of diblock copolymer 18 The amphiphilic diblock copolymer 18 containing the pendant aldehyde group and dendronized TEG units was synthesized by chain extension of the aldehyde-containing homopolymer 6 with Ru-end to the second dendronized macromonomer 16via one-pot two-step sequential ROMP using the catalyst 1 at r.t. in dry CH2Cl2. The order of monomer introduction in this ROMP synthesis of 18 was guided by the much faster reaction rate observed during the polymerization of monomer 5 than that of 16, and the satisfactory polymerization of 16 using the Ru-ended homopolymer 6 as the first block. The feed molar ratio of [monomer 5]: [monomer 16]: [catalyst 1] is 10:50:1. As shown in Scheme 3, the ROMP of monomer 5 was first conducted in dry CH2Cl2, and the obtained aldehyde-containing homopolymer 6 with the Ru-end was then used as a macromolecular initiator to initiate the ROMP of monomer 16 in dry CH2Cl2. It took 10 min to complete the ROMP of 5 with 100% conversion, and 16 in dry CH2Cl2 was then added, and a kinetic study was conducted to monitor the polymerization of 16 in CH2Cl2 through the in 8


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Fig. 7. MALDI-TOF MS spectrum of polymer 17a.

situ1H NMR analysis method. At different intervals, a little of the reaction mixture was taken out, quenched by EVE and dried, and its 1H NMR spectrum was recorded in CDCl3. When the peak at 6.26 ppm corresponding to the olefin protons of monomer 16 disappeared, the conversion rate was believed to be 100%. As expected, unlike the case of homo-polymerization of 16 at the feed molar ratio of 50:1, for which 24 h is needed to achieve the quantitative conversion, it took a prolonged reaction time, namely, 42 h to complete the ROMP of the second monomer 16 with 100% conversion (Fig. 6c). The decreased reaction

O

1 N

N N

CH 2 Cl 2 , r. t. 10 min

Ru

O

O

N

O

CH 2 Cl 2 , r. t. 42 h

N N

n

Ph

16

O

N

m

O

N

N N

N

R NH 2

O

O

N

o

CH 2 Cl 2 , 25 C

O

m

O

N

O 3

O

O

CHO

CHO

CHO

C

5

6

HN O

O

N

N N

N

N

N N

O

N

19

R NH 2 =

21

R NH 2 =

H C N H

NH 2

COOH

O

O

20

R NH 2 = CH 3 (CH 2 ) 15

22

R NH 2 = CH 3 CH 2 OOC

O O

19-22 NH 2

NH 2

Scheme 3. Synthesis route of diblock copolymer 18 and PDCs 19–22 with various amino-containing model drugs. 9

O O

18 NH 2

N O

O

O

O

CH 2 O

N N O

O

m = 50 O

N

O

O

O

N

N N

O

n = 10

O O

N

R

N

O

O

HC

O

N

C

O

O N

O

N

HN O

N

N

O N

n

Ph

3

N

O

n

Ph

rate is primarily attributed to the increased feed molar ratio of monomers and catalyst (60:1). Fig. 8 shows the 1H NMR spectrum of 18 in CDCl3, and its comparison with those of the two homopolymers of 6 (Fig. 1B) and 17 (Fig. 5B) leads to the structure of 18. The successful introduction of the second TEG-containing block is well confirmed by the appearance of several characteristic signals. For example, the two peaks at 7.95 and 7.85 ppm correspond to the triazolyl protons in the TEG block, and its phenyl protons are observed at 7.25 ppm. The broad peak at 7.40 ppm


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Fig. 8. 1H NMR spectrum of block copolymer 18 in CDCl3.

polymerization degree of the second block by comparing the signal intensity of aldehyde proton in the first block with that of characteristic protons in the second TEG block using the 1H NMR spectrum of 18 in CDCl3 (Fig. 8). Namely, the integration of aldehyde proton at 9.82 ppm was compared with those of the triazolyl protons at 7.96 and 7.85 ppm, acylamino proton at 7.39 ppm, methylene protons of OCH2 group at 5.19–5.16 and 4.54–4.41 ppm, and the methoxy protons at 3.31 ppm, respectively. The calculated polymerization degree is 50 ± 3, exhibiting an excellent agreement with the theoretical value of 50 for the second TEG block from the 1H NMR conversion. SEC was also adopted to determine the MWs of the diblock polymers 18. It indicates a Mn value of 89,800 Da with PDI = 1.14 versus PS standards (Table S5). The obtained MW is larger than the theoretical value calculated by using the molar feed ratios and the corresponding monomer conversions, attributed to the large structural difference between the PS standard and the diblock polymer. Howerver, an obvious forward shift of retention time (Fig. 4c) is observed in comparison to the SEC curves of the corresponding homopolymers 6a and 17b with molar feed ratios of 10:1 and 50:1, respectively, which convincingly confirms the increased MW of the prepared diblock copolymer 18. More importantly, the small PDI of obtained from the SEC trace (Table S5), which are < 1.15, further demonstrated the controlled polymerization of the two blocks in 18. Furthermore, the random copolymer 24 was also prepared, for the comparison in the following applications, by adding the two monomers at the same time into the CH2Cl2 solution of catalyst 1 at the feed molar ratio of [monomer 5]: [monomer 16]: [catalyst 1] = 25:50:1, and its synthesis, kinetic study, structural and MW characterization were detailed in SI. Notably, the final polymer 24 exhibits excellent water-solubility in spite of the increased proportion of hydrophobic aldehydecontaining monomer 5.

is assigned to the proton of acylamino group, while the two peaks at 5.19 and 5.16 ppm originate from the protons of OCH2 between benzene and triazole rings. The characteristic protons of terminal OCH3 groups are observed at 3.31 ppm, and the OCH2 protons in the long linker and TEG units are located at 4.54–4.41, 3.88–3.81 and 3.63–3.44 ppm. For the first aldehyde-containing block, the peak at 9.82 ppm arises from the CHO proton, while the broad peak at 7.77 ppm corresponds to the triazolyl and phenyl protons, and the broad peak 6.93 ppm is assigned the other phenyl protons. The two broad peaks at 4.74 and 4.41 ppm are attributed to methyl protons in the side chain of the first block. Thus, all the above results full demonstrate the integrity of the aldehyde-containing block. In the 13C NMR spectrum of 18 in CDCl3 (Fig. 2C), the carbon of acylamino group is located at 166.8 ppm, and the triazolyl carbons in the second block are observed at 144.1, 143.4, 124.8 and 124.6 ppm, respectively. The two peaks at 50.3 and 50.1 ppm are assigned to the carbons of OCH2 group between benzene and triazole rings, while the peak at 58.9 ppm is attributed to the carbons of OCH3 group. For the first block, its aldehyde carbon is found at 190.8 ppm, and the peaks at 140.3 and 126.3 ppm originate from the carbons of its triazole ring. These data also provides strongly proofs for the formation of the second TEG block, and the integrity of the first aldehyde block. Furthermore, the two peaks at 132.0 and 130.2 ppm correspond to the characteristic olefinic carbons of polynorbornene backbone; mixing with the signals from phenyl carbons, while the peaks at 178.3 and 178.2 carbonyl carbons of polynorbornene. All the other peaks of the 1H and 13C NMR spectra are clearly assigned and match well with the structure of 18. The UV − vis spectrum in CH2Cl2 of the diblock copolymer 18 (Fig. 3c) shows also two λmax, as expected, at 228 and 263 nm corresponding to the π → π* anti bond orbital transition of phenyl and triazolyl groups. The location of the second peak is between 271 nm for the first aldehyde block and 258 nm for the second TEG block, providing also supplementary information for the formation of the block copolymer 18. The IR spectrum (Fig. S43) further gives helpful information about the functional groups in the first and second block of 18. The MW distribution of the diblock copolymer 18 was also determined by using the end-group analysis and SEC methods, respectively. The polymerization degree of the first block, namely homopolymer 6a, were first calculated by end-group analysis (vide supra) using the 1H NMR spectra of polymers 6a in (CD3)2SO (Fig. 1A), and the obtained value of 10 ± 0.5 was then adopted to estimate the

3.4. Preparation of PDCs 19–22 The hydrophobic block of the designed diblock copolymer 18 contains the reactive aldehyde functional group that is well known for its reaction with amine compounds to form reversible Schiff base covalent bond (CH]N) [48,69–71]. Thus, the present copolymer is expected to be especially useful for the covalent attachment of biologically relevant amino-containing molecules and drugs for controlled release [72–75]. To confirm the conjugation ability of 18, several commercial available amine molecules as shown in Scheme 3 were chosen as model drugs to 10


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Fig. 9. The partial 1H NMR in CDCl3 of block copolymer 18 (A) and its PDCs 19–22 with BHA (B), 1-hexadecanamine (C), tryptophan (D) and benzocaine (E), respectively.

completely disappear, and the grafting ratio is only 26.3% (0.695 mg of benzocaine per 100 mg of 18) estimated according to the integral ratio of the new Schiff base proton at 8.33 ppm and the aldehyde proton. The incomplete conjugation is attributed to the inert reactivity of benzocaine as an aromatic amine to benzaldehyde group in 18 [48].

form PDCs. The selected model drugs contain BHA, 1-hexadecanamine, tryptophan and benzocaine. The conjugation reaction (Scheme 3) was trippingly conducted by dissolving the block copolymer 18 in CH2Cl2, followed by the addition of superfluous model drugs and string at 25 °C for 24–72 h. The grafting ratio is determined by comparing the signal intensity of the Schiff base CH]N proton with that of the characteristic aldehyde (CHO) proton in the 1H NMR spectra of resulting PDCs 19–22 (Figs. 9 and S53–S56), and then the corresponding drug-loading content is calculated by using the grafting ratio. For example, in the 1H NMR spectrum of the conjugate 19 containing BHA in CDCl3 (Figs. 9B and S53), the peak at 9.82 ppm corresponding the proton of aldehyde group thoroughly disappears, while a new signal peak assigned to the Schiff base CH]N proton appears at 8.10 ppm. The methylene protons of BHA are located at 5.15 ppm, and its phenyl protons were observed at 7.34 ppm. The other peaks are well assigned and matched well with the structure of 19, indicating the structural integrity of the two blocks. Thus, these results indisputably confirm the successful conjugation between 18 and BHA, and the grafting ratio is 100%. The drug-loading content for BHA is 1.97 mg per 100 mg of 18. As shown in Fig. 9C and D, 1-hexadecanamine and tryptophan can also be successfully conjugated to 18 with the grafting ratio of 100%, indicating the applicability and generality of this method for aliphatic amine molecules. The drug-loading contents are 3.86 mg of 1-hexadecanamine per 100 mg of 18, and 3.26 mg of tryptophan per 100 mg of 18, respectively. However, for benzocaine (Fig. 9E), even after 72 h of stirring at 25 °C, the peak at 9.82 ppm for aldehyde proton had no

3.5. Micelles of diblock copolymer 18 in water The block copolymer 18 was obtained as purple red viscous oil liquid after precipitation from CH2Cl2 with Et2O. It is soluble in common organic solvents including CH2Cl2, chloroform, THF and methanol, and strong polar solvents such as DMF and dimethylsulfoxide (DMSO). More importantly, it shows good solubility in water because of the presence of hydrophilic TEG block. Thus, owing to this amphiphilic characteristic, the diblock copolymer 18 is expected to self-assemble into micelles in aqueous solution by virtue of their hydrophilic/hydrophobic balance. The pyrene fluorescence probe technique [66] was adopted to determine the CMC of 18 and its self-assembly behavior. With the increase of concentrations, block copolymer 18 self-assembles into micelles, and the hydrophobic pyrene can be encapsulated into the hydrophobic microdomains of 18, leading to the significant change of its fluorescence excitation spectra and the increase of intensity ratios (I3/I1) of pyrene at peaks of 383 and 372 nm, respectively. The plot of I3/I1 intensity ratios versus logarithmic concentrations of 18 was drawn, and the CMC is estimated following the known procedure [66] by using this

Fig. 10. Plots of the I3/I1 ratios versus logarithmic concentrations of block copolymer 18 (A) and PDC 19 (B) and the corresponding calculated CMCs. 11


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Fig. 11. Micellar morphology and size of 18 in water (0.5 mg ml−1) (A) SEM image of the micelles; (B) DLS curve of the micelles; (C) size distribution (number %) of the micelles by SEM; (D) amplitude trace of the micelles by AFM; (E) height profile of the micelles by AFM; (F) statistical evaluation of the diameters by AFM.

curve. Fig. S51a shows the fluorescence emission spectra of block copolymer 18 in water with pyrene, and Fig. 10A is the corresponding plot of the I3/I1 ratios versus logarithmic concentrations of 18. The determined CMC is 0.355 mg ml−1 for 18, while 0.437 mg ml−1 for the random copolymer 24 (Fig. S51d). The higher CMC value of 24 is due to its better water-solubility than the block copolymer 18. The random distribution of hydrophilic and hydrophobic components in random copolymers is advantageous for its dissolution in water, but unfavourable for the self-assembly and subsequent drug-loading, thus no further investigation was conducted for the random copolymer 24. The dialysis method was used to prepare micellar aggregates of block copolymer 18. The deionized water was added into the DMF solution of 18 with vigorous stirring for 2 h, and the Tyndall effect was clearly observed as shown in Fig. S52, indicating the formation of aggregates through of the self-assembly of 18. Using the dialysis bag with MWCO of 35,000 g mol−1, the dialysis treatment against deionized water was then carried out for 48 h to remove DMF completely, and the obtained samples were investigated by SEM, AFM and DLS (Fig. 11). Nearly spherical micelles was observed (Fig. 11A), as expected, with the size of 84 ± 10 nm by SEM (Fig. 11C). In a typical micelle, the darker color is observed in the core constructed by the hydrophobic benzaldehyde-containing block, while the periphery shows the brighter color attributing to the self-assembly of hydrophilic dendronized TEG tails. Also, AFM analysis was conducted on air-dried samples. The test sample was prepared by the dialyzed solution of 18 drop-casted onto clean mica substrate and left to air-dry for 24 h before tapping mode AFM analysis. Fig. 11D–F show the typical AFM images and statistical diameter analysis. The AFM images of amplitude profiles in different areas indicate that all the aggregates exhibit obvious particles with spherical morphologies, and the statistical analysis [76] of these AFM image was conducted to yield the average particle diameter (DavAFM) of 51 ± 5 nm, close to the size by SEM. Moreover, DLS (Fig. 11B)

provides a hydrodynamic diameter of 143 nm with a PDI of 0.222 for these micelles. The size obtained by DLS are typically larger than those determined using SEM and AFM, attributed to their different determination conditions. The size distribution by DLS reflects the dimensions of both the swollen core and the stretched TEG shell, while the sizes checked by SEM and AFM provide only the conformation in the dry state [77–79]. Whatever, all these results demonstrate the satisfactory self-assembly property of 18 into micelles on the nanoscale. 3.6. Micelles of PDC 19 in water To confirm the self-assembly ability of the present PDCs, 19 conjugating BHA was taken as an example to be investigated. The PDC 19 is expected to spontaneously self-assemble into a micellar-like structure in water with the BHA-containing block as a hydrophobic inner core and TEG block as a hydrophilic outer shell. To test this hypothesis, the CMC of 19 was first studied by using the pyrene fluorescence probe method, and Fig. 10B provides the value of 0.134 mg ml−1, which is lower than that for the original block copolymer 18, probably owing to the higher hydrophobicity of the conjugated BHA part and the resulting increased size of hydrophobic block in 19. Even so, the yielded PDC 19 still has good water-solubility, and shows the similar amphiphilic characteristic as the original block copolymer 18. The fabrication of micelles from the PDC 19 was also conducted in water by using the dialysis method, and the adopted procedure is the same as that for 18, and the obtained micelles were also characterized by SEM, AFM and DLS (Fig. 12). Like the case for 18, the SEM image (Fig. 12A) also provides the torispherical micelles of 19 with the size of 90 ± 10 nm (Fig. 12C), slightly larger than that of 18. In a typical micelle, the darker color is observed in the core constructed by the hydrophobic block containing BHA, while the periphery shows the brighter color attributing to the 12


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Fig. 12. Micellar morphology and size of 19 in water (0.5 mg ml−1) (A) SEM image of the micelles; (B) DLS curve of the micelles; (C) size distribution (number %) of the micelles by SEM; (D) amplitude trace of the micelles by AFM; (E) height profile of the micelles by AFM; (F) statistical evaluation of the diameters by AFM.

can be decomposed at the acidic conditions, but remain stable in alkaline solution [80]. For example, the imine (CH]N) bond formed between a primary amine and an aldehyde group can be completely broken in an aqueous solution with a pH value below 6.5 [81]. Following this line, the present micelles are expected to show pH-sensitivity, and the drugs encapsulated could be released at acidic environment. To verify this point, in vitro drug release experiments were carried out with the micelles self-assembled by PDCs 19 and 21. Figs. S58 and 59 shows the UV–visible absorption spectra of dialysate of 19 and 21 at different interval and pH conditions. The release amount of BHA was calculated by equations in 2.11 according to the standard curve of BHA drawn at its λmax of 261 nm (Fig. S58A). As expected, the cumulative release of BHA was completely suppressed under the physiological condition (pH 7.4), and even under acidic condition of pH 6.0, no BHA was detected in the dialysate after three days of dialysis. However, under more acidic conditions (pH 4.0 and 2.5), the BHA was released much faster than at subacid and physiological pH environments. For example, about 75% of BHA was released from the micelles at pH 4.0

self-assembly of hydrophilic TEG tails. Furthermore, DLS was carried out to provide indirect evidence for the micellar structure formation of 19. As the result shown in Fig. 12B, it could be concluded that the PDC 19 self-assembles into particles with the hydrodynamic diameter of 488 nm (PDI = 0.239). The obtained size is dramatically larger than that determined by SEM, which is attributed to the slight aggregation in the micellar system. This point is also demonstrated by the result of AFM analysis. In the Fig. 12D–F, the AFM image shows obvious particles with spherical morphologies. The average particle diameter DavAFM is 414 ± 80 nm, in agreement with the average diameter detected by DLS, and the large error is resulted from the mild agglomeration of micelles. Thus, all the above results indicate the satisfactory self-assembly property of the polymer-BHA conjugate 19 into micelles on the nanoscale.

3.7. In vitro pH-responsive drug release Schiff base bond is well known as a pH-responsive linker [48–53]. It

Fig. 13. UV–visible spectra of dialysates of the micelles 19 and 21 at different intervals of in vitro release experiments (A) 19 at pH 4.0; (B) 19 at pH 2.5; (C) 21 at pH 2.5. 13


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within 24 h, and after 2 days, all the conjugated BHA was released (Fig. 13A). Moreover, the BHA release was remarkably accelerated by decreasing the pH of release media to 2.5. After only 30 min of dialysis, the release ratio of BHA reaches 74%, and all of the loaded BHA was released from the micelles in 120 min (Fig. 13B). This pH-responsive release behavior may be attributed to the acid-labile benzoic-imine linkage between the model drug BHA and block copolymer 18, which is stable at subacid and physiological pH, and labile at highly acidic pH condition. Furthermore, the release behavior of tryptophan in the micelles of PDC 21 was also investigated by in the release media of pH 2.5. As shown in Fig. 13C, about 53% of the loaded tryptophan was released after immersing at pH 2.5 for 4 h and the cumulative release achieved 95% after 24 h, and the 2 days of immersion led to the release of all the encapsulated tryptophan. These results indicate the significantly slower release rate of tryptophan compared with BHA at the same pH condition.

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4. Conclusions In summary, we present a new amphiphilic block copolymer containing polynorbornene backbone with side-chain hydrophobic benzaldehyde groups and hydrophilic dendronized TEG branches by the controlled polymerization method of ROMP with the aid of Grubbs' very efficient third generation ruthenium catalyst. Nearly spherical micelles with the size of 84 ± 10 nm were formed as a new potential drug delivery system by the self-assembly of the bock copolymer in water. More importantly, amino-containing model drugs including BHA, 1-hexadecanamine, tryptophan and benzocaine were covalently linked to the benzaldehyde groups of the copolymers through pH-sensitive Schiff-base bonds to form well-defined polymer-drug conjugates. Especially, the BHA-loaded copolymer formed torispherical micelles with the size of 90 ± 10 nm in aqueous media, and the in vitro experiments confirm the pH-responsive release behavior. Thus, the nice conjugation ability of the benzaldehyde-containing copolymer in this work is expected to be extended to the other amino-containing drugs including doxorubicin, daunorubicin, epirubicin, pirarubicin, and so on. Future work will focus on the evaluation of hemocompatibility and cytotoxicity the polymeric carrier, too. Consequently, the exploited polynorbornene copolymer is a promising polymer carrier to form polymer-drug conjugates via acid-responsive Schiff-base linkage for the application of controlled drug delivery [82–84]. Conflict of interest The authors declare no conflict of interest. Acknowledgment Financial support from Sichuan University (C2018101859) is gratefully acknowledged. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.reactfunctpolym.2018.03.010. References [1] H. Jatzkewitz, Peptamin (glycyl-L-leucyl-mescaline) bound to blood plasma expander (polyvinylpyrrolidone) as a new depot form of a biologically active primary amine (mescaline), Z. Naturforsch. 10b (1955) 27–31. [2] H. Ringsdorf, Structure and properties of pharmacologically active polymers, J. Polym. Sci. Polym. Symp. 51 (1975) 135–153. [3] J. Khandare, T. Minko, Polymer–drug conjugates: progress in polymeric prodrugs, Prog. Polym. Sci. 31 (2006) 359–397. [4] C. Li, S. Wallace, Polymer-drug conjugates: recent development in clinical

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