Polymer Functional Nanodiamonds by Light ... - ACS Publications

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Polymer Functional Nanodiamonds by Light-Induced Ligation Kilian N. R. Wuest,†,‡,∥ Vanessa Trouillet,⊥ Anja S. Goldmann,†,‡ Martina H. Stenzel,*,∥ and Christopher Barner-Kowollik*,†,‡ †

Preparative Macromolecular Chemistry, Institut für Technische Chemie und Polymerchemie (ITCP), Karlsruhe Institute of Technology (KIT), Engesserstr. 18, 76131 Karlsruhe, Germany ‡ Institut für Biologische Grenzflächen (IBG) and ⊥Institute for Applied Materials (IAM-ESS) and Karlsruhe Nano Micro Facility (KNMF), Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany ∥ Centre for Advanced Macromolecular Design (CAMD), The University of New South Wales, Sydney, NSW 2052, Australia S Supporting Information *

ABSTRACT: A light-triggered strategy to functionalize nanodiamonds (NDs) with well-defined functional polymers is presented. The employed grafting approach is based on o-methylbenzaldehydes, which upon UV irradiation form oquinodimethanes that undergo Diels−Alder reactions with dienophiles. A series of well-defined maleimide end-group functional polymers, i.e., poly(styrene) (Mn = 5800 g mol−1; Đ = 1.2), poly(N-isopropylacrylamide) (Mn = 5800 g mol−1, Đ = 1.2), and poly(2-(2′,3′,4′,6′-tetra-O-acetyl-α-D-mannosyloxy)ethyl methacrylate) (Mn = 24 300, 39 000, and 58 800 g mol−1, Đ ≤ 1.3), were prepared via reversible addition−fragmentation chain transfer (RAFT) polymerization of protected maleimide functional RAFT agents. After deprotection of the furan-protected maleimide end groups, the polymers were photografted to omethylbenzaldehyde functional NDs and characterized in detail via infrared (IR) spectroscopy, X-ray photoelectron spectroscopy (XPS), and thermogravimetric analysis (TGA). The grafting density decreases with increasing polymer chain length (6.9−3.8 μmol g−1). Moreover, the binding of the glycopolymer functional NDs to the lectin Concanavalin A was demonstrated with a turbidity assay.

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molecules.8 Nevertheless, a more stable covalent surface coating is often desired to avoid premature detachment in biological media. Synthetic polymer shells on nanoparticles can significantly influence their properties, such as solubility, stability, and biological activity. Generally, two synthetic approaches have been applied to covalently graft polymers onto the surface of NDs, referred to as the “grafting-from” and “grafting-to” approach. In a grafting-from approach, NDs were for instance decorated with chain transfer agents, bromine groups, or carboxyl groups for subsequent reversible addition− fragmentation chain transfer (RAFT) polymerization,7,9 atom transfer radical polymerization (ATRP),10 and ring-opening polymerization11 from the surface, respectively. A high grafting

INTRODUCTION The development of novel nanodiamond (ND) systems for biological applications has developed into an active field of research during the past years.1−3 The unique properties of NDs, such as nontoxicity,4 possibility to introduce nonbleaching fluorescence,5 and ease of surface functionalization,6 make them promising candidates for drug delivery and imaging applications. For instance, the decoration of NDs with cisplatin increased the cytotoxicity in ovarian cancer cells, and their cellular uptake could readily be monitored by fluorescence microscopy.7 Moreover, the industrial production of detonation NDs provides ready access to high quantities, meeting an important requirement for the commercialization of new ND materials.3 Different surface functionalization strategies to introduce new properties and (biological) functions to ND systems have been investigated. A simple access route to functionalized NDs is the noncovalent attachment of © XXXX American Chemical Society

Received: December 1, 2015 Revised: February 6, 2016

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DOI: 10.1021/acs.macromol.5b02607 Macromolecules XXXX, XXX, XXX−XXX

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and 2-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-3a,4,7,7a-tetrahydro-1H4,7-epoxyisoindole-1,3(2H)-dione23 were prepared according to literature procedures. Detonation nanodiamonds (pristine NDs, 97% trace metals basis, Sigma-Aldrich), EDC·HCl (99+%, Roth), D(+)-mannose (99+%, Acros), BF3·OEt2 (98+%, Alpha), acetic anhydride (99+%, Acros), 4-cyano-4-((phenylcarbonothioyl)thio)pentanoic acid (>97%, Sigma-Aldrich), BH3·THF (1.0 M in THF, Sigma-Aldrich), H2SO4 (96%, Roth), HNO3 (67%, Roth), HCl (37%, Roth), N,N′-dicyclohexylcarbodiimide (DCC, 99%, Acros), 2,2′-azobis(isobutylonitrile) (AIBN, VWR), (3-aminopropyl)triethoxysilane (APTES, 98%, abcr), 2-hydroxyethyl methacrylate (97%, Sigma-Aldrich), 4-(dimethylamino)pyridine (DMAP, 99%, abcr), 2-thiazoline-2-thiol (98%, Sigma-Aldrich), chloroform-d1 (CDCl3, 99.8%, EURISO-TOP), dimethyl-d6 sulfoxide (DMSO-d6, 99.8%, EURISO-TOP), and 4-(dimethylamino)pyridine (DMAP, 99%, Acros) were used as received. N,N-Dimethylformamide (DMF), tetrahydrofuran (THF), dichloromethane (DCM), diethyl ether, ethyl acetate, and cyclohexane were purchased as analytical grade (Sigma-Aldrich) and used as received. Styrene (Sigma-Aldrich) was passed through a column of basic aluminum oxide (Acros). NIsopropylacrylamide (NIPAM, TCI, 98%) was recrystallized twice from hexane and stored at −20 °C prior to usage. Oxidation of NDs (ND-COOH). According to the literature,24 pristine nanodiamonds (500 mg) were dispersed in 10 mL of H2SO4 (96%)/HNO3 (67%) (9/1, v/v) and stirred at 90 °C for 3 days. The NDs were washed with water and ultrasonicated with a high power sonotrode (Branson Sonifier W450 with microtip) for 1 h. The abovedescribed procedure was repeated once again. Subsequently, the NDs were dispersed in 20 mL of 0.5 M NaOH and stirred at 90 °C for 1 h. After centrifugation, the NDs were dispersed in 20 mL of 0.1 M HCl and stirred at 90 °C for 1 h. The NDs were centrifuged and redispersed in 20 mL of water and sonicated with a high power sonotrode for 2 h. After centrifugation and drying under reduced pressure the oxidized NDs (ND-COOH) were obtained. Reduction of NDs (ND-OH). Oxidized NDs (400 mg) were dispersed in 20 mL of anhydrous THF. Subsequently, 5 mL of 1 M BH3·THF was added, and the reaction mixture was stirred at 70 °C for 24 h. The reaction mixture was quenched with 5 mL of 2 M HCl and washed with acetone/water (1/1, v/v). Silanization of NDs (ND-NH2). The reduced NDs (235 mg) were dispersed in 40 mL of anhydrous THF. APTES (2.10 mL) was added and stirred under nitrogen at ambient temperature for 48 h. The NDs were centrifuged, and the precipitate was washed extensively with acetone and dried under reduced pressure to yield amine functional NDs (ND-NH2). Photoenol Functionalization of NDs (ND-PE). ND-NH2 (150 mg) were dissolved in 50 mL of dry THF and ultrasonicated in an ultrasound bath for 1 h. Methyl 4-((2-formyl-3-methylphenoxy)methyl)benzoic acid (91.2 mg, 0.337 mmol, 1.00 equiv), EDC·HCl (77.6 mg, 0.405 mmol, 1.20 equiv), and DMAP (16.5 mg, 0.135 mg, 0.400 equiv) were added, and the reaction mixture was stirred at ambient temperature for 65 h. After centrifugation, the precipitate was washed twice with water and twice with THF in consecutive washing/ centrifugation cycles to yield photoenol-functional NDs (ND-PE). Monomer Synthesis. Synthesis of 1,2,3,4,6-Penta-o-acetyl-Dmannose. According to a procedure adopted from the literature,25 mannose (5.00 g, 27.8 mmol, 1.00 equiv) was suspended in acetic anhydride (26.2 mL, 28.3 g, 278 mmol, 10.0 equiv) at 0 °C. Two drops of 96% sulfuric acid were added, and the mixture was stirred for 18 h at ambient temperature. Subsequently, 100 mL of water was added, and the resulting mixture was extracted with dichloromethane. The organic phase was washed consecutively with NaHCO3 and water until the aqueous phase was neutral and dried over Na2SO4. The solvent was removed under reduced pressure to yield penta-acetylated mannose (10.44 g, 26.7 mmol, 96%). 1H NMR (400 MHz, chloroform-d) δ/ppm = 6.08 (s, 1 H), 5.49−5.38 (m, 1H), 5.28− 5.24 (m, 1 H), 5.37−5.33 (m, 2 H), 4.33−4.24 (m, 1 H), 4.16−4.01 (m, 2 H), 2.17, 2.16, 2.08, 2.04, 2.00 (5 s, each 3 H, acetyl groups). Synthesis of 2-(2′,3′,4′,6′-Tetra-o-acetyl-β-D-mannosyloxy)ethyl Methacrylate (ManAc). According to a procedure adopted from the

density can be achieved; however, the detailed characterization of the surface-grafted polymer and the distinction between covalently attached and adsorbed polymer are challenging. On the other hand, in the grafting-to approach, premade functional polymers are employed for the direct conjugation to the surface. For instance, esterification on NDs,12 as well as radical trapping reactions,13,14 and dopamine functionalization15 were employed to graft polymers to NDs. An advantage of the grafting-to approach is that a wide range of well-defined and thoroughly characterized polymers can be prepared and attached to the surface in a modular fashion. Moreover, cografting of different polymer chains with known characteristics allows for the preparation of more complex surface structures. Light-induced ligations represent an elegant method for the grafting of (macro)molecules to surfaces. The utilization of light to trigger the grafting step allows mild reaction conditions, i.e., ambient temperature and catalyst-free conditions, suitable for biological applications. A very versatile and efficient conjugation reaction for the covalent conjugation of polymers to surfaces is photoenol chemistry. o-Methylbenzaldehyde or o-methylbenzophenone derivatives form oquinodimethanes (photoenols) upon UV irradiation, which can be trapped from dienophiles in a Diels−Alder reaction.16 In early studies, the light-induced photoenol reaction was employed e.g. for the preparation of polymeric nanoparticles and the functionalization of planar and nonplanar surfaces.17−21 The current contribution highlights the application of photoenol chemistry for the light-induced grafting of a variety of polymers to nanodiamonds (Scheme 1). Photoenol groups Scheme 1. General Schematic Representation of the Grafting of Maleimide End-Group Functional Polymers, i.e., PS, PNIPAM, and Glycopolymer, to o-Methylbenzaldehyde Functional NDs (ND-PE) to Obtain Polymer-Coated NDs (ND@polymer)

were covalently attached via an amidation reaction to silanized, amine functional NDs. Subsequently, maleimide end-group functional poly(styrene) (PS), poly(N-isopropylacrylamide) (PNIPAM), and glycopolymers with lateral mannose units were grafted to the ND surfaces. The binding of the prepared mannose functional NDs to the lectin ConA demonstrates the applicability of the employed grafting-to strategy for the preparation of bioactive NDs.

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EXPERIMENTAL SECTION

Methyl 4-((2-formyl-3-methylphenoxy)methyl)benzoic acid,19 4-(2hydroxyethyl)-10-oxa-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione,22 B


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Macromolecules literature,25 penta-acetylated mannose (3.00 g, 7.69 mmol, 1.00 equiv) and 2-hydroxyethyl methacrylate (1.12 mL, 1.20 g, 9.22 mmol, 1.20 equiv) were dissolved in 30 mL of anhydrous dichloromethane under a nitrogen atmosphere and cooled to −10 °C. BF3·OEt2 (4.74 mL, 5.45 g, 38.4 mmol, 5.00 equiv) was added dropwise. The reaction mixture was stirred at ambient temperature for 20 h. After adding 20 mL of water, the mixture was extracted with dichloromethane, washed with water, NaHCO3, and water again, and dried over Na2SO4. The solvent was removed under reduced pressure. The crude product was purified by column chromatography (cyclohexane/ethyl acetate, 7/3, v/v) yielding a white solid (1.97 g, 4.28 mmol, 56%). 1H NMR (400 MHz, chloroform-d): δ/ppm = 6.17−6.08 (m, 1 H), 5.61−5.58 (m, 1 H), 5.34 (dd, J = 10.0, 3.4 Hz, 1 H), 5.31−5.23 (m, 2 H), 4.87 (d, J = 1.8 Hz, 1 H), 4.38−4.31 (m, 2 H), 4.26 (dd, J = 12.2, 5.4 Hz, 1 H), 4.08 (dd, J = 12.2, 2.4 Hz, 1 H), 4.01 (ddd, J = 9.9, 5.4, 2.4 Hz, 1 H), 3.96− 3.86 (m, 1 H), 3.81−3.71 (m, 1 H), 2.15, 2.09, 2.03, 1.98 (4 s, each 3 H, acetyl groups), 1.95 (dd, J = 1.3 Hz, J = 1.3 Hz, 3 H). 13C NMR (75 MHz, chloroform-d): δ/ppm = 170.75, 170.13, 169.98, 169.82, 167.23, 136.09, 126.18, 97.66, 69.56, 69.08, 68.75, 66.24, 66.04, 63.26, 62.54, 21.00, 20.84, 20.81, 18.41. ESI-MS: [M + Na]+, [C20H28O12Na]+, theoretical: 483.147; experimental: 483.148. Synthesis of 2-(Dodecylthiocarbonothioylthio)-2-methylpropionic Acid 2-(3,5-Dioxo-10-oxa-4-aza-tricyclo[5.2.1.02,6]dec-8en-4-yl)ethyl Ester (CTA1). According to the literature,26 2-(((dodecylthio)carbonothioyl)thio)-2-methylpropanoic acid (DMP) (1.50 g, 4.12 mmol, 1.00 equiv), 4-(2-hydroxyethyl)-10-oxa-4-azatricyclo[5.2.1.02,6]dec-8-ene-3,5-dione (1.29 g, 6.18 mmol, 1.50 equiv), and DMAP (0.101 g, 0.823 mmol, 0.20 equiv) were dissolved at 0 °C in 75 mL of anhydrous THF. Subsequently, EDC·HCl (2.37 g, 12.36 mmol, 3.00 equiv) was added, and the reaction mixture was stirred at ambient temperature for 18 h. The solvent was removed under reduced pressure. The residue was dissolved in dichloromethane, washed with NaHCO3, brine, and water, and dried over Na2SO4. After removal of the solvent under reduced pressure, the residue was purified by column chromatography (cyclohexane/ethyl acetate, 1/1, v/v). A yellow solid was obtained (897 mg, 1.61 mmol, 39%). 1H NMR (400 MHz, chloroform-d): δ/ppm = 6.50 (s, 2H), 5.25 (s, 2 H), 4.24 (t, J = 5.4 Hz, 2 H), 3.77 (t, J = 5.4 Hz, 2 H), 3.24 (t, J = 7.4 Hz, 2 H), 2.86 (s, 2 H), 1.68−1.50 (m, 8 H), 1.38−1.05 (m 18H), 0.88 (t, J = 6.8 Hz, 3 H). 13C NMR (75 MHz, chloroform-d): δ/ppm = 221.80, 175.91, 172.83, 136.67, 80.99, 62.31, 56.08, 47.67, 37.70, 37.17, 32.04, 29.76, 29.68, 29.58, 29.47, 29.23, 29.10, 27.94, 25.26, 22.82, 14.26. ESI-MS: [M + Na]+, [C27H41NO5S3Na]+, theoretical: 578.204; experimental: 578.205; [2M + Na]+, [C54H82N2O10S6Na]+, theoretical: 1133.419; experimental: 1133.422. Synthesis of 2-Cyano-5-((2-(2-(2-(1,3-dioxo-1,3,3a,4,7,7a-hexahydro-2H-4,7-epoxyisoindol-2-yl)ethoxy)ethoxy)ethyl)amino)-5oxopentan-2-yl Benzodithioate (CTA2). 4-Cyano-4-((phenylcarbonothioyl)thio)pentanoic acid (650 mg, 2.33 mmol, 1.00 equiv), mercaptothiazoline (277 mg, 2.33 mmol, 1.00 equiv), DCC (577 mg, 2.80 mmol, 1.20 equiv), and DMAP (28.5 mg, 0.233 mmol, 0.10 equiv) were dissolved in 10 mL of anhydrous dichloromethane and stirred at ambient temperature for 14 h. Subsequently, 2-(2-(2-(2aminoethoxy)ethoxy)ethyl)-3a,4,7,7a-tetrahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione (655 mg, 2.13 mmol, 0.95 equiv) in 4 mL of anhydrous dichloromethane was added dropwise (over 1 h to minimize aminolysis of the RAFT agent) and stirred at ambient temperature for 5 h. The precipitate was filtered off, and the solvent was removed under reduced pressure. The residue was purified by column chromatography (ethyl acetate/cyclohexane, 1/4, v/v gradient to pure ethyl acetate) yielding a red solid (205 mg, 0.368 mmol, 16%). 1 H NMR (400 MHz, chloroform-d): δ/ppm = 7.90 (dd, J = 8.5, 1.3 Hz, 2 H), 7.59−7.53 (m, 1 H), 7.39 (dd, J = 8.4, 7.4 Hz, 2 H), 6.62 (s, 1H), 6.50 (s, 2 H), 5.25 (s, 2 H), 3.86−3.41 (m, 10 H), 2.86 (s, 2 H), 2.78−2.36 (m, 4 H), 1.95 (s, 2 H), 1.25 (s, 3 H). 13C NMR (75 MHz, chloroform-d): δ/ppm = 222.81, 176.53, 170.80, 144.72, 136.70, 133.10, 128.69, 126.81, 118.90, 81.09, 70.49, 70.16, 69.78, 67.53, 47.64, 46.30, 39.54, 38.69, 34.33, 31.73, 29.83, 24.29. ESI-MS: [M + Na]+, [C27H31N3O6S2Na]+, theoretical: 580.155; experimental: 580.156.

Polymerizations. RAFT Polymerization of Styrene. Styrene, CTA1, and AIBN (molar ratio 1000:10:1) were mixed in a Schlenk tube (monomer concentration = 8.74 mol L−1) and degassed via three consecutive freeze−pump−thaw cycles. Subsequently, the polymerization mixture was stirred at 60 °C for 14 h. The polymerization was quenched by cooling with liquid nitrogen and exposing the mixture to oxygen. The polymerization mixture was precipitated twice in methanol. The obtained polymer was dried under reduced pressure to obtain a yellowish solid (Mn = 6200 g mol−1; Đ = 1.2). 1H NMR (300 MHz, chloroform-d): δ/ppm = 7.40−6.28 (m, aromatic H, vinylic H), 5.21 (s, 2 H, HC−O−CH), 3.78−3.21 (m, CH2), 2.78 (s, 2 H, NCOCH), 2.39−1.13 (m, CH3, CH2, CH), 0.89 (t, J = 6.5 Hz, 3 H, CH2CH3). RAFT Polymerization of N-Isopropylacrylamide. 1.00 g of NIPAM, CTA1, and AIBN (molar ratio 1000:10:1) were dissolved in 5 mL of DMF (monomer concentration = 1.77 mol L−1) and degassed via three consecutive freeze−pump−thaw cycles. Subsequently, the polymerization mixture was stirred at 60 °C for 8 h. The polymerization was quenched by cooling with liquid nitrogen and exposing the mixture to oxygen. The polymerization mixture was precipitated twice in diethyl ether. The obtained polymer was dried under reduced pressure to obtain a yellowish solid (Mn = 4700 g mol−1; Đ = 1.2). 1H NMR (400 MHz, chloroform-d): δ/ppm = 7.00− 5.81 (bs, NH), 6.52 (s, 2 H, vinylic H), 5.25 (s, 2 H, HC−O−CH), 3.99 (s, NHCH), 3.76 (s, 2 H, CH2), 3.34 (s, 2 H, CH2), 2.90 (s, 2 H, NCOCH), 2.49 (s, CH polymer backbone), 2.34−0.93 (m, CH2 polymer backbone, CH2 dodecyl moiety), 0.87 (t, J = 6.8 Hz, 3 H, CH2CH3). RAFT Polymerization of ManAc. In a typical procedure, 200 mg of ManAc, CTA2, and AIBN (for PManAc I and II: molar ratio 250:5:1; for PManAc III: molar ratio 500:5:1) were dissolved in 1 mL of DMF (monomer concentration = 0.434 mol L−1). The polymerization mixture was degassed by three consecutive freeze−pump−thaw cycles and polymerized at 60 °C for 14 h (PManAc II: 22 h; PManAc III: 16 h). The crude product was purified by multiple precipitations in cold diethyl ether and dried under reduced pressure to afford a pale red solid (I: Mn = 12 900 g mol−1, Đ = 1.2; II: Mn = 20 700 g mol−1, Đ = 1.3; III: Mn = 42 000 g mol−1; Đ = 1.2). 1H NMR (400 MHz, chloroform-d): δ/ppm = 7.95−7.36 (m, 5 H, aromatic H), 6.54 (s, 2 H, vinylic H), 5.45−5.16 (m, mannose CH), 4.90 (s, anomeric H), 4.37−3.33 (m, O−C2H4−O, mannose CH), 2.89 (s, 2 H, NCOCH), 2.14, 2.10, 2.05, 1.98 (4 s, each 3 H, acetyl groups), 1.43−0.80 (m, CH2 and CH polymer backbone). Retro-Diels−Alder Deprotection of Polymers. The retro-Diels− Alder reaction of PS, PNIPAM, and the glycopolymers (PManAc I− III) was performed in bulk under reduced pressure at 110 °C for 6 h, at 95 °C for 10 h, and at 110 °C for 15 h, respectively. Photoreactions. In a typical procedure, 5 mg of ND-PE was dissolved in 5 mL of THF and ultrasonicated in an ultrasound bath for 1 h. Subsequently, the maleimide end-group functional polymer was added, and the dispersion was purged with nitrogen for 10 min. The reaction mixture was irradiated with an Arimed B6 lamp in a custombuilt photoreactor at a distance of 15 cm (1.57 mW cm−2, for emission spectrum see Figure S13) and at ambient temperature for 6 h to ensure maximal grafting densities. The dispersion was centrifuged and washed via four consecutive ultrasound-assisted redispersion (THF)/ centrifugation cycles and dried under reduced pressure. Kinetic Study. The kinetic study was conducted with MalPNIPAM and Mal-PManAc I using the following general procedure: ND-PE were dispersed in THF (1 g L−1) and ultrasonicated in an ultrasound bath for 1 h. Polymer was added to obtain a 0.210 mmol L−1 solution. The dispersion was degassed by purging with nitrogen for 10 min and divided into several air-sealed vials and irradiated with an Arimed B6 lamp in a custom-built photoreactor at a distance of 15 cm (1.57 mW cm−2, for emission spectrum see Figures S13) at ambient temperature for different time periods (Mal-PNIPAM: 30, 60, 120, 240, and 360 min; Mal-PManAc: 30, 60, 120, and 900 min). Subsequently, the reaction mixtures were purified as described above and analyzed by TGA. C


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Macromolecules

Scheme 2. Schematic Representation of the Synthetic Route from Pristine NDs to Photoenol Functional NDs (ND-PE)

Deacetylation of the Glycopolymer Shell. The glycopolymer grafted NDs were deacetylated directly after the photoreaction by adding 130 μL of 25% sodium methylate solution in methanol. The reaction mixture was stirred at ambient temperature for 2 h and washed with four consecutive ultrasound-assisted redispersion/ centrifugation cycles (2 × water and 2 × THF) and dried under reduced pressure.

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RESULTS AND DISCUSSION Preparation of Photoreactive NDs. The ND surface was modified in four synthetic steps (Scheme 2). Pristine NDs, as received from the supplier, possess a variety of functional groups on their surface, e.g., hydroxyl, carboxylic acid, lactone, ester groups, and sp2 carbon structures.6 In order to obtain a homogeneous surfacein terms of functionalitieson the NDs, a literature known purification procedure was followed.24 The pristine NDs were treated with a mixture of concentrated sulfuric and nitric acid at elevated temperatures to yield oxidized NDs (ND-COOH). In order to break large aggregates, the NDs were ultrasonicated several times with a high-power sonotrode (see Experimental Section for details). Subsequently, ND-COOH were reduced with borane in THF to obtain reduced NDs (ND-OH). The reduced ND surfaces were silanized with APTES to obtain surface-expressed amine groups. In the last step, the carboxylic acid functional photoenol compound (PE) was attached to the ND surface via an EDCmediated amidation reaction. The success of the photoenol surface decoration was verified by UV−vis spectroscopy and XPS measurements. In the UV−vis spectrum of ND-PE the characteristic absorption bands of the photoenol at 1512 and 831 cm−1 are visible (Figure 1). Moreover, the shift of the carbonyl absorption band from 1682 to 1792 cm−1 indicates a structural change, probably due to the formation of the amide bond. The presence of a Si 2p signal at 101.9 eV in the XP spectrum after silanization additionally confirms the success of the functionalization route (Figure 5). Preparation of Polymers. Upon UV irradiation at 320 nm the o-methylbenzaldehydes on ND-PE form o-quinodimethanes, which react with dienophiles in a Diels−Alder reaction. Maleimides, for instance, are highly reactive dienophiles suitable for the photoenol conjugation. Thus, polymers terminated with maleimide end groups were designed. The maleimide functional polymers were prepared

Figure 1. FT-IR spectra of the prepared NDs at different stages of functionalization. The abbreviations are assigned to schematic structures in Scheme 2.

by RAFT polymerization with furan-capped maleimide functional RAFT agents and subsequent retro-Diels−Alder deprotection. The maleimide functionality was incorporated as part of the leaving group of the RAFT agent to avoid hydrolytic cleavage of the maleimide group and to ensure a high end-group fidelity. Three different monomers were polymerized to show the versatility and modularity of the light-induced grafting-to approach (Scheme 3). Initially, styrene and Nisopropylacrylamide were polymerized using a trithiocarbonatebased RAFT agent (CTA1). The retro-Diels−Alder reaction was performed at elevated temperatures (T ≥ 95 °C) under reduced pressure. The molecular weight distributions of the polymers (Figure 2A,B) are narrow (Đ ≤ 1.2), the proton resonances of the RAFT termini groups are still visible in the 1 H NMR spectra (see Supporting Information), and no decoloration of the samples occurred. Thus, the polymers withstood the high-temperature treatment. The SEC data for all prepared polymer samples are summarized in Table 1. Since the expected weight loss during the retro-Diels−Alder reaction is not significant (68 Da) and resonance overlap complicates D


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Scheme 3. Polymers Prepared by RAFT Polymerization with Furan-Protected Maleimide Functional RAFT Agents (AIBN, 60 °C)a

a The maleimide end groups were obtained after a retro-Diels−Alder reaction at elevated temperatures (T ≥ 95 °C). Glycopolymers of three different chain lengths were synthesized (Mal-PManAc I−III; for characterization date see Table 1). The protected glycopolymer was deacetylated after the grafting-to step on the surface of the nanodiamonds.

Figure 2. Size-exclusion chromatograms (A, C measured in THF and B in DMAC at 40 and 50 °C, respectively) of the furan protected polymers (solid line) and deprotected maleimide functional polymers (dashed lines; A: Mal-PS; B: Mal-PNIPAM, C: Mal-PManAc I−III, black, blue, green). The molecular weights and dispersities are collated in Table 1.

the integration, the Mn(NMR) values from the furan-protected maleimide polymers were used for further calculation. The peracetylated mannose methacrylate monomer was synthesized in two synthetic steps according to a modified literature procedure.25 First, mannose was peracetylated using acetic anhydride. Subsequently, a BF3·OEt2 mediated glycosylation reaction with 2-hydroxyethyl methacrylate was conducted. The deacetylation of the acetylated glycopolymers is usually performed with nucleophiles, e.g., sodium methylate. However, the methylate nucleophile is not selective toward

acetate groups and cleaves the methacrylate ester. Yet, after polymerization these ester bonds of the repeating units are stable under deacetylation conditions. Thus, the deacetylation of the mannose units was performed as the last synthetic step, after the grafting to the NDs. The above employed RAFT agent (CTA1) contains an ester linkage between the maleimide group and the RAFT group, which is not stable under nucleophilic deacetylation conditions. Therefore, a novel RAFT agent with a stable amide linkage (CTA2) was designed for the polymerization of the mannose monomer. 4-Cyanopentanoic E


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Macromolecules Table 1. Characterization Data of the Prepared Polymersa polymer

Mn (SEC) (g mol−1)

Đ (SEC)

Mn (NMR) (g mol−1)

DP (NMR)

PS mal-PS PNIPAm mal-PNIPAm PManAc I mal-PManAc I PManAc II mal-PManAc II PManAc III mal-PManAc III

6200 5800 6200 5800 23400 24300 38000 39000 56200 58800

1.2 1.2 1.2 1.2 1.2 1.2 1.3 1.3 1.2 1.2

6200

55

4700

37

12900

27

20700

44

42000

90

for the PNIPAM and the glycopolymer system, respectively (Figure 3). The glycopolymer grafted NDs (ND@PManAc I−

Mn = number-average molecular weight; Đ = dispersity. Mn (NMR) and the degree of polymerization DP (NMR) were determined using the integral of the furan-protected maleimide end group as a reference and by comparing it to the integral of the proton signals of the repeating units (PS: aromatic protons; PNIPAM: tertiary isopropyl proton; PManAc: proton of anomeric center).

a

Figure 3. Kinetic study of PNIPAM and PManAc I system. ND-PE/ polymer mixtures were irradiated with UV light for different time periods (for details see Experimental Section). After several washing cycles, the grafting densities were determined by TGA.

acid dithiobenzoate (CPADP)a well suitable chain transfer agent for methacrylateswas reacted with an amine functional furan-protected maleimide compound. It is crucial to minimize aminolysis side reactions during the CTA synthesis. Benicewicz and colleagues have demonstrated the successful amidation of CPADP on amine functional nanoparticles.27 Inspired from this work, the amine was slowly introduced to a solution of 2mercaptothiazoline activated CPADP. The slow addition ensures a low amine concentration and reduces the possibility of aminolysis reactions. The resulting RAFT agent was then employed to control the polymerization of 2-(2′,3′,4′,6′-tetra-oacetyl-β-D-mannosyloxy)ethyl methacrylate (ManAc). In order to investigate the correlation between the polymer chain length and the grafting density, three glycopolymers with varying molecular weights (Mn = 12 900−42 000 g mol−1, Đ ≤ 1.3) were synthesized (PManAc I−III; for characterization data see Figure 2, Table 1, and Supporting Information). Photografting. The light-induced grafting of the polymers to ND-PE was performed in a custom-built photoreactor with a commercially available UV lamp (λmax = 320 nm, for spectrum see Figure S13). First, the nanodiamonds were dispersed in THF in an ultrasound bath. Subsequently, the specific polymer dissolved in THF was added, and the stirred dispersions were irradiated with UV light (λmax = 320 nm). In order to evidence that the polymer on the nanodiamonds is covalently grafted and not physically adsorbed on the ND surface, a control experiment with the most hydrophobic polymer (Mal-PS) of the prepared polymers was performed. Therefore, a mixture of ND-PE and Mal-PS in THF was prepared and divided into two separate vials. One sample was irradiated at 320 nm while the second sample was stirred in the dark (control sample). Unreacted polymer was removed with four washing/redispersion cycles. Thermogravimetric analysis (TGA) of the control sample and the irradiated sample evidenced that only the irradiated sample contained polymer (see Figure S12). Next, the kinetics of the grafting-to approach were investigated for the Mal-PNIPAM and Mal-PManAc. ND-PE dispersions in THF were irradiated with UV light in the presence of polymer for different time periods. After the photoreactions, the NDs were washed in multiple, consecutive ultrasound assisted redispersion/centrifugation cycles to remove any unreacted polymer and analyzed by TGA. The kinetic study revealed that the amount of grafted polymer reached a plateau after 2 or 5 h

II) were deacetylated directly after the photoreaction by addition of a sodium methylate/methanol solution yielding ND@PMan. Only one acetylated glycopolymer−ND system (ND@PManAc II) was isolated for characterization. Dynamic light scattering (DLS) experiments were performed in water to obtain hydrodynamic diameters of the prepared nanodiamond polymer hybrid particles. Number-average hydrodynamic diameters between 170 and 290 nm (Figure S14) were calculated for the polymer coated nanodiamonds. It should be noted that the particles consist of aggregated NDs with primary sizes smaller than 10 nm. The two glycopolymer coated NDs with shorter polymer chain lengths (ND@PMan I and II) have smaller hydrodynamic volumes than the ND precursor (NDPE), probably because of a better dispersibility of the glycopolymer-coated NDs. All other samples showed an increase of hydrodynamic volume after photografting. Subsequently, the obtained polymer-coated NDs were qualitatively and quantitatively characterized by IR spectroscopy, XPS, and TGA. IR Spectroscopy. FT-IR spectra were recorded for all ND/ polymer hybrid particles and compared to the spectra of NDPE and the corresponding polymers. After the photografting, the characteristic IR absorption bands of ND-PE vanished as depicted in Figure 4. The aromatic C−H vibrations of PS (754 and 695 cm−1) are visible in the IR spectrum of ND@PS. The characteristic PNIPAM bands at 1635, 1540, and 1460 cm−1 in the IR spectrum of ND@PNIPAM can be assigned to the amide I, amide II, and symmetric −C(CH3)2 deformation band, respectively.28 The characterization of the grafting of the glycopolymers to ND-PE is shown with Mal-PManAc II in Figure 4. The IR spectrum of ND@PManAc II shows the characteristic absorption bands of the acetate protecting groups at 1738, 1637, and 1215 cm−1, which disappear after the deacetylation step. XPS. X-ray photoelectron spectroscopy (XPS) is an excellent characterization method to obtain information about the chemical composition of the prepared nanoparticles at their surface. Compared to TGA, XPS does not deliver bulk information because the information depth is only close to 10 nm. The grafting of Mal-PNIPAM to ND-PE leads to an F


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Macromolecules

Figure 4. IR spectra (A−C) and TGA results (D, E) of the prepared samples. Exemplarily, only characterization data from grafting PManAc II are shown (C and F, data from PManAc I and III are in the Supporting Information).

286.5 eV)/C−H, C−C (at 285.0 eV) ratio. The deacetylation of the glycopolymer coating is accompanied by a slight decrease of carbonyl and an increase of hydroxyl groups, which is in agreement with the C 1s signal intensities observed in the XP spectra. TGA. The grafting density of the polymers on the NDs was determined by comparing the TGA determined weight loss

increase in the intensity of the signal N 1s at 399.6 eV attributed to the amide (Figure 5).29 The nitrogen concentration increases significantly from 2.0 to 3.0 at. % and is in the expected range. The grafting of the oxygen-rich glycopolymers to ND-PE introduces oxygen containing groups to the ND material. As expected, the analysis of the deconvoluted C 1s signal intensities indicates an increase of the C−O, C−N (at G


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Macromolecules

Figure 5. Si 2p, N 1s, and C 1s X-ray photoelectron spectra of selected ND samples. The silanization is evidenced by the appearance of the Si 2p signal (left). The success of the grafting of Mal-PNIPAM can be shown with the increase of the intensity of the N 1s signal (middle). The grafting of the glycopolymer to ND-PE and the subsequent deacetylation leads to a stepwise increase of the C−O, C−N/C−H, C−C signal intensity ratio (right). For a better visualization, all spectra are normalized to maximum intensity.

can be explained by a not fully deacetylated polymer. Moreover, the measured grafting densities were used to estimate a polymer chain footprint. The calculated footprints are in the order of magnitude of previously reported footprints where polymers were grafted from NDs (2.2 nm2)7 and to gold nanoparticles (0.71−5.52 nm2).30 Furthermore, it was shown that the grafting density correlates with the polymer chain length (Figure 6); i.e., the

profiles of ND-PE and polymer grafted NDs. The thermogravimetric experiments were conducted in the presence of oxygen, where the NDs decompose at 615 °C. As shown in Figure 4, all polymers have similar degradation profiles with the temperature of maximal degradation at around 350 °C. The grafting density (wt %, mass polymer/mass sample) was calculated from the difference of the weight loss of ND-PE and ND@polymer at the decomposition step of the polymer. In addition, the amount of polymer per sample mass (in mol g−1) was calculated using the number-average molar mass of the grafted polymer determined by 1H NMR spectroscopy (Table 1). The TGA results are summarized in Table 2. Grafting densities between Table 2. Grafting Densities of the Prepared ND@polymer Particles Determined by Thermogravimetric Analysisa grafting density sample

wt %

μmol g−1

av footprint (nm2 chain−1)

ND@PS ND@PNIPAM ND@PMan I ND@PManAc II ND@PMan II ND@PMan III

3.8 5.0 5.5 10.9 7.9 9.3

6.4 11.1 6.9 5.9 6.4 3.8

2.7 1.5 3.8 3.1 4.2 7.9

a

The wt % is the percentage of the polymer mass contributing to the mass of the sample. The wt % was converted into (1) μmol g−1 using the NMR-determined number average molecular weight from Table 1 and (2) a polymer footprint (see Supporting Information for details).

Figure 6. Correlation between grafting densities of glycopolymer functionalized NDs determined by TGA and the degree of polymerization of the grafted polymers (grafting density in wt % (■) and μmol g−1 (▲)).

3.8 and 10.9 wt % were reached. The molar grafting densities are between 3.8 and 11.1 μmol g−1. The TGA of the glycopolymer grafted NDs reveals a grafting density of 10.9 wt % and after deacetylation 7.9 wt %. The weight loss upon deacetylation is due to the loss of the acetate groups. The molar grafting density was calculated based on the assumption that all mannose units are deacetylated. The discrepancy between the molar grafting density of ND@PManAc II and ND@PMan II

wt % of the grafted glycopolymer samples increases with the polymer chain length. Nevertheless, the molar grafting density (in μmol g−1) decreases, which is equivalent to increasing polymer footprints. The steric hindrance of long polymer chains on the ND surface prevent a high molar surface coverage. H


Article

Macromolecules Lectin Binding. Finally, the binding of the glycopolymer coated nanoparticles to Concanavalin A (ConA) was investigated. ConA is a carbohydrate binding protein (lectin), which selectively binds to α-D-mannosyl and α-D-glucosyl groups. The binding of synthetic glycopolymers to cell surface expressed lectins can promote cellular uptake of glycopolymer particles to specific cells and thereby enhance the therapeutic efficacy of drug delivery systems. Herein, ConA serves as a model lectin to evaluate the activity of the prepared glycopolymer particles toward lectins. A qualitative assay to investigate the lectin binding is the turbidity assay. ConA has four binding pockets, and thus the interaction with glycopolymers leads to a cross-linking induced turbidity, which can be measured by UV−vis spectroscopy. For all three samples the turbidity increased upon addition of a ConA/ HEPES buffer solution, which indicates the binding of the glycopolymer functional nanodiamonds with ConA (Figure 7).

mannose functional glycopolymerswere efficiently grafted to photoenol decorated nanodiamonds. The light-induced photoenol grafting was performed with a commercially available UV lamp at ambient temperature and without any catalyst. The successful photografting was confirmed by TGA, IR, and XP spectroscopy. A correlation between the grafting density and the polymer chain length was found: polymer chains with higher molecular weight are grafted with a lower molar grafting density to the ND surfaces due to steric hindrance. Moreover, the binding of the glycopolymer functional nanodiamonds to the lectin ConA was shown with a turbidity assay. The introduced light-triggered modular strategy for the preparation of functional nanodiamonds offers a high potential for the development of novel hybrid nanomaterial for biological applications. Moreover, employing light to modify NDs has the advantage of a high spatial resolution, which is highly interesting for the development of novel ND patterned surfaces and ND-based 3D structures.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.5b02607. Information regarding the employed materials and instrumentation as well as additional characterization data and figures (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail [email protected] (C.B.-K.). *E-mail [email protected] (M.H.S.). Notes

Figure 7. Turbidity assay to investigate the ConA binding behavior of glycopolymer functional NDs (A: ND@PMan I; B: ND@PMan II; C: ND@PMan III). A solution of ConA in HEPES buffer was added to the ND dispersion, mixed, and immediately placed into a UV−vis spectrometer to record the time-dependent absorbance at 420 nm.

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS A.G., M.S., and C.B.-K. acknowledge the German Research Council (DFG) for funding this project. K.W. thanks the Fonds der Chemischen Industrie for a scholarship funding part of his PhD studies. C.B.-K. acknowledges continued support from the Karlsruhe Institute of Technology (KIT) via the Helmholtz BioInterfaces program. Dr. Astrid Hirschbiel (KIT) is thanked for providing the methyl 4-((2-formyl-3-methylphenoxy)methyl)benzoic acid. The authors acknowledge the staff at the NMR and TEM facilities of UNSW, Sydney.

The binding of ND@PMan nanoparticles to ConA is an additional proof for the successful deacetylation of the glycopolymers on the surface of the NDs. The ConA binding to the nanodiamonds with the shortest grafted glycopolymer chains (ND@PMan I, A) leads to the highest increase followed by a linear decrease in absorbance. Because of the short glycopolymer chains on ND@PMan I, the possibility for interparticle cross-linking is higher, probably leading to the precipitation of the NDs and a decrease of absorbance after 4 min. In the systems with longer glycopolymer chains (B, C), no significant sedimentation is observed, probably because the ConA binds primarily with mannose residues from the same nanoparticle and thus aggregation associated with sedimentation is inhibited.

■

ABBREVIATIONS ND, nanodiamond; PE, photoenol; RAFT, reversible addition− fragmentation chain transfer; IR, infrared; XPS, X-ray photoelectron spectroscopy; TGA, thermogravimetric analysis; DLS, dynamic light scattering; HEPES, (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid); ConA, Concanavalin A.

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■

CONCLUSIONS A light-induced method for the covalent grafting of maleimide end-group functional polymers to NDs is introduced. The maleimide end-group functional polymers were readily accessible via RAFT polymerization with furan-protected maleimide functional RAFT agents and subsequent retroDiels−Alder deprotection. To demonstrate the versatility of the strategy, three different polymerspolystyrene, PNIPAM, and

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