Photochemical Design of Stimuli-Responsive Nanoparticles Prepared

Article pubs.acs.org/Macromolecules

Photochemical Design of Stimuli-Responsive Nanoparticles Prepared by Supramolecular Host−Guest Chemistry Astrid F. Hirschbiel,†,‡ Bernhard V. K. J. Schmidt,∥ Peter Krolla-Sidenstein,§ James P. Blinco,⊥ and Christopher Barner-Kowollik*,†,‡,⊥ †

Preparative Macromolecular Chemistry, Institut für Technische Chemie und Polymerchemie (ITCP), Karlsruhe Institute of Technology (KIT), Engesserstr. 18, 76128 Karlsruhe, Germany ‡ Soft Matter Synthesis Laboratory, Institute for Biological Interfaces (IBG), and §Institute of Functional Interfaces (IFG), Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany ∥ Max Planck Institute of Colloids and Interfaces, 14424 Potsdam, Germany ⊥ School of Chemistry, Physics and Mechanical Engineering, Queensland University of Technology (QUT), 2 George St., 4001 Brisbane, Queensland, Australia S Supporting Information *

ABSTRACT: We introduce the design of a thermoresponsive nanoparticle via sacrificial micelle formation based on supramolecular host−guest chemistry. Reversible addition−fragmentation chain transfer (RAFT) polymerization was employed to synthesize well-defined polymer blocks of poly(N,N-dimethylacrylamide) (poly(DMAAm)) (Mn,SEC = 10 700 g mol−1, Đ = 1.3) and poly(N-isopropylacrylamide) (poly(NiPAAm)) (Mn,SEC = 39 700 g mol−1, Đ = 1.2), carrying supramolecular recognition units at the chain termini. Further, 2-methoxy-6methylbenzaldehyde moieties (photoenols, PE) were statistically incorporated into the backbone of the poly(NiPAAm) block as photoactive cross-linking units. Host−guest interactions of adamantane (Ada) (at the terminus of the poly(NiPAAm/PE) chain) and β-cyclodextrin (CD) (attached to the poly(DMAAm chain end) result in a supramolecular diblock copolymer. In aqueous solution, the diblock copolymer undergoes micellization when heated above the lower critical solution temperature (LCST) of the thermoresponsive poly(NiPAAm/PE) chain, forming the core of the micelle. Via the addition of a 4-arm maleimide cross-linker and irradiation with UV light, the micelle is crosslinked in its core via the photoinduced Diels−Alder reaction of maleimide and PE units. The adamantyl−cyclodextrin linkage is subsequently cleaved by the destruction of the β-CD, affording narrowly distributed thermoresponsive nanoparticles with a trigger temperature close to 30 °C. Polymer chain analysis was performed via size exclusion chromatography (SEC), nuclear magnetic resonance (NMR) spectroscopy, and dynamic light scattering (DLS). The size and thermoresponsive behavior of the micelles and nanoparticles were investigated via DLS as well as atomic force microscopy (AFM).

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constitutions, e.g., micellar configurations19,20 or single-chain nanoparticles.21,22 Polymeric micelles are often obtained by the self-assembly of amphiphilic block copolymers.23−25 These micelles can subsequently undergo a cross-linking reaction to result in more robust structures for specialized applications, whereby the linkage can either occur in the shell26−28 or the core.29,30 Common reactions employed for the cross-linking step include copper-catalyzed azide−alkyne cycloadditions (CuAAC),31,30 photoinduced reactions,32−34 or via hydrogen bonding,35 to name a few examples.36,37 In particular, stimuliresponsive nanocapsules and micelles are intensively investigated as their properties, such as size, shape, and their interactions can be changed by an external trigger.38−40 For example, pH,41,42

INTRODUCTION Research into nanoparticles is an area of major importance to several scientific fields. Nanoparticles find broad applicability, ranging from drug delivery systems1−4 to nanoreactors,5,6 molecular imaging agents,7−9 and even microelectronic sensors.10−12 According to Kreuter,13 the first ideas of nanocapsules reach back to Ehrlich in the 1960s, who envisaged the generation of miniaturized delivery systems for medical applications. Further, Speiser and co-workers were the first to fabricate nanoparticles for drug delivery purposes.13,14 Since then, a plethora of studies have been conducted, concerning the development and application of nanosized particles, which continues to the present day. The dimensions of nanoparticles usually range between 10 and 100 nm, and they can be composed of inorganic15,16 or organic materials.17,18 Primarily polymeric nanostructures have gained strong attention because their synthesis is very versatile and can lead to a wide variety of © XXXX American Chemical Society

Received: May 1, 2015 Revised: June 8, 2015

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DOI: 10.1021/acs.macromol.5b00923 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules Scheme 1. Schematic Overview of the Nanoparticle Formation Approach Followed in the Current Study

temperature,29,43,44 voltage,45 or light43,46,47 can act as stimulus to release substances from such nanocontainers. Herein, a novel and effective tool to design thermoresponsive nanoparticles is introduced, critically enhancing the existing avenues for nanoparticle syntheses. Formation of the nanoparticle occurs through a sacrificial micellar scaffold, which is shaped by the self-assembly of a supramolecular diblock copolymer. The diblock copolymer consists of poly(N,Ndimethylacrylamide) (poly(DMAAm)) and poly(N-isopropylacrylamide) (poly(NiPAAm)) with additional photoreactive units incorporated in its side chain. Poly(NiPAAm) has been widely studied as a thermoresponsive material48,49 and is therefore often employed in the formation of stimuli-responsive micelles.50,20,51 It was possible to make use of the unique features of poly(NiPAAm) and to add further functionalities by copolymerizing the acrylamide with a photoreactive moiety, a monomeric derivative of 2-methoxy-6-methylbenzaldehyde (abbreviated as PE for photoenol). Reversible addition− fragmentation chain transfer (RAFT) polymerization, a powerful technique to obtain polymers of defined molecular weight with narrow size distributions (Đ), was employed for the synthesis of poly(DMAAm) and poly(NiPAAm/PE), as it can simultaneously introduce specific groups at a polymer chain end. Thus, poly(DMAAm) was equipped with a β-cyclodextrin (β-CD) and poly(NiPAAm/PE) with an adamantyl function, which results in supramolecular diblock copolymer formation via β-CD host− guest interactions with the adamantyl units. CD-based host− guest interactions are an important tool for noncovalent conjunctions and have been widely employed for the construction of complex macromolecular architectures.52−54 For example, Wintgens et al. utilized the inclusion complex formation of alkyne groups, attached to modified dextrans, with a β-CD polymer to form temporary networks.55 Furthermore, the preparation of well-defined poly(2-methyl-2-oxazoline) and poly(NiPAAm), end-functionalized with adamantane and βCD, respectively, was demonstrated by Voit and co-workers:56 A supramolecular diblock structure was formed and further selfassembled into reversible aggregates by adjusting the temperature. Consequently, CDs are also utilized in micelle formation, since they create dynamic linking points via their high complexation capacity with many hydrophobic molecules.27,57−60 Supramolecular block copolymers have been utilized in a similar way as their covalent analogues, e.g., in

hierarchical self-assembly in the bulk.61 In the present contribution block copolymer self-assembly is exploited for the synthesis of core cross-linked micelles. Importantly and critical to our approach, the supramolecular nature of the block junctions allows for the removal of the sacrificial micellar coronawhich only serves as a scaffold for micellizationfreeing the core nanoparticles. Scheme 1 represents the overall approach for the formation of the thermoresponsive nanoparticles via micellization of the supramolecular diblock copolymer. Micellar assembly occurs after heating the aqueous solution of the diblock copolymer above the lower critical solution temperature (LCST) of the poly(NiPAAm/PE). Subsequently, photoinduced cross-linking of the core via a linking molecule results in a nanoparticle. The nanoparticle is encapsulated in the micelle and finally released by the removal of the β-CD equipped arms, which solely serve as scaffolds. Atomic force microscopy (AFM), dynamic light scattering (DLS), and nuclear magnetic resonance (NMR) studies are employed for the analysis of the micelles and final nanoparticles.

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EXPERIMENTAL SECTION

Materials. Ascorbic acid (99%, Acros), Quantofix 100 peroxide dipsticks (1−100 mg L−1 H2O2, Roth), CuBr (99.9%, Acros), N,N,N′,N′,N″-pentamethyldiethylenetriamine (PMDETA, 99.9%, Merck), 2-(dodecylthiocarbonothioylthio)propionic acid (DoPAT, Orica), 4-(dimethylamino)pyridine (DMAP, 99%, Acros), N,N′dicyclohexylcarbodiimide (DCC, 99%, Acros), HCl (37%, Roth), NaHCO3 (≥99%, Roth), MgSO4 (≥99%, Roth), K2CO3 (VWR), 18crown-6 (99%, Acros), 4-arm PEG-maleimide (2K, Creative PEGWorks), and trifluoroacetic acid (TFA, 99%, ABCR) were used as received. N-Isopropylacrylamide (NiPAAm, 98%, TCI) was recrystallized in hexane prior to use and stored at −19 °C. Azobis(isobutyronitrile) (AIBN, 99%, Fluka) was recrystallized twice in methanol and stored at −19 °C. N,N-Dimethylacrylamide (DMAAm, 99%, TCI) and 4-vinylbenzyl chloride (90%, Sigma) were passed through a column of basic alumina (Acros) and subsequently stored at −19 °C. Milli-Q water was obtained from a purification system by VWR (TKA Micro-Pure; 0.055 μS cm−1), N,N-dimethylformamide extra dry (DMF, 99.8%, Acros), tetrahydrofuran (THF), dichloromethane (DCM), acetone, ethyl acetate (EA), cyclohexane, and 1,4-dioxane were purchased as analytical grade (Aldrich) and used as received. Characterization. Size Exclusion Chromatography (SEC). SEC measurements were performed with N,N-dimethylacetamide (DMAC) as eluent containing 0.03 wt % LiBr on a Polymer Laboratories PL-GPC B


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Macromolecules 50 Plus Integrated System, comprising an autosampler, a PLgel 5 μm bead-size guard column (50 × 7.5 mm) followed by three PLgel 5 μm MixedC columns (300 × 7.5 mm), and a differential refractive index detector at 50 °C with a flow rate of 1.0 mL min−1. The SEC system was calibrated against linear poly(styrene) standards with molecular weights ranging from 160 to 6 × 106 g mol−1. Calculations for the molecular weight of poly(DMAAm) and poly(NiPAAm/PE) were carried out according to a poly(styrene) calibration, i.e., K = 14.1 × 10−5 dL g−1, α = 0.70 (PS).62 The molecular weight dispersity is abbreviated as Đ. Electrospray Ionization−Mass Spectrometry (ESI-MS). Mass spectra were recorded on an LXQ mass spectrometer (ThermoFisher Scientific, San Jose, CA) equipped with an atmospheric pressure ionization source operating in the nebulizer-assisted electrospray mode. The instrument was calibrated in the m/z range 195−1822 using a standard containing caffeine, Met-Arg-Phe-Ala acetate (MRFA), and a mixture of fluorinated phosphazenes (Ultramark 1621) (all from Aldrich). A constant spray voltage of 3.5 kV, a dimensionless sheath gas of 8, and a sweep gas flow rate of 2 were applied. The capillary voltage, the tube lens offset voltage, and the capillary temperature were set to 60 V, 120 V, and 300 °C, respectively. UV−Vis Spectrometry. UV−vis spectra were measured on a Cary 300 Bio UV−vis spectrophotometer (Varian) at either 25 or 10 °C, depending on the sample. Cloud points were measured on the same instrument at 600 nm. The heating rate was set to 0.32 °C min−1 and the concentration at 0.06 mmol L−1. For the determination of the cloud point the point of inflection of the transmittance vs temperature plot was used. Nuclear Magnetic Resonance (NMR) Spectroscopy. NMR measurements for structure confirmation were carried out on a Bruker Ascend 400 spectrometer with 400 MHz for hydrogen nuclei and 100 MHz for carbon nuclei. Samples were dissolved in CDCl3, DMSO-d6, or D2O. The δ-scale was referenced with tetramethylsilane (δ = 0.00) as internal standard. Abbreviations used below in the description of the materials syntheses include singlet (s), broad singlet (bs), doublet (d), triplet (t), quartet (q), broad multiplet (bm), and unresolved multiplet (m). The 2D NOESY (nuclear Overhauser enhancement spectroscopy) spectrum was measured on a 600 MHz Bruker Avance III spectrometer equipped with a 1H, 13C, 15N TCI inversely detecting cryoprobe at 10 °C (283 K). The mixing time was set to 300 μs, and the concentration of the sample was 50 mg mL−1 in D2O. Dynamic Light Scattering (DLS). Samples were prepared by dissolving the samples in Milli-Q water at a constant concentration of 0.06 mmol L−1. The concentration for the micelles and the nanoparticles were at 1 mg mL−1. The solutions were filtered via a 0.2 μm syringe filter to remove dust particles. Before the measurement the solution of the diblock was left for 30 min, so the equilibrium between β-CD and adamantyl units could re-establish. Hydrodynamic diameters where determined with dynamic light scattering (Nicomp 380 DLS spectrometer from Particle Sizing Systems, Santa Barbara, CA; laser diode: 90 mW, 658 nm). Measurements were performed in automatic mode and evaluated with a standard Gaussian and an advanced evaluation method, the latter proceeding via an inverse Laplace algorithm to analyze for multimodal distributions. The values provided in the study are the number-weighted average values as calculated in the NICOMP evaluation. All measurements were determined at an angle of 90° to the incident beam. The associated autocorrelation functions can be found in the Supporting Information. Atomic Force Microscopy (AFM). Sample preparation: Freshly prepared and dried micelles as well as nanoparticles (∼1 mg) were dissolved in Milli-Q water (1 mL). 10 μL of 1:10000 stock solutions (stepwise diluted with Milli-Q water) were pipetted onto freshly cleaved mica disks of 12 mm diameter. The samples were covered and spreaddried at elevated temperature (hot plate being hand warm). Sample measurements: AFM analysis was performed on a NanoScope IIIa controlled MultiMode 2 AFM (Bruker) equipped with a scanner type “E” at a scan size of 1 × 1 μm, at a scan speed of 0.5 Hz, with a resolution of 512 lines and points and a scan angle of 0°. A silicon nitrate cantilever was used with a typical resonance frequency of 75 kHz and force constant of approximately 0.2 N/m (MikroMasch, HQ: NSC18/No

Al). The image data were processed with the NanoScope Analysis 1.40 software (Bruker). Synthesis. Prop-2-yn-1-yl 2-(((ethylthio)carbonothioyl)thio)-2methylpropanoate (1),63 mono-(6-azido-6-desoxy)-β-cyclodextrin (βCD-N3) (4),64 2-hydroxy-6-methylbenzaldehyde,65 and N-((3s,5s,7s)adamantan-1-yl)-6-hydroxyhexanamide (7)66 were synthesized according to literature procedures. Synthesis of Alkyne-Functionalized Poly(DMAAm) (2). Prop-2-yn1-yl 2-(((ethylthio)carbonothioyl)thio)-2-methylpropanoate, 1 (0.21 g, 0.79 mmol, 1.0 equiv), DMAAm (12.00 g, 121.05 mmol, 152.71 equiv), and AIBN (0.012 g, 0.07 mmol, 0.01 equiv) were added into a Schlenk tube, equipped with a stirring bar and dissolved in DMF (36.0 mL). Oxygen was removed from the reaction mixture via three freeze− pump−thaw cycles. Subsequently, the tube was placed in an oil bath at 60 °C, and the reaction was quenched after 2 h by cooling with liquid nitrogen. The mixture was dialyzed against deionized water with a SpectraPor3 membrane (MWCO = 1000 Da) for 3 days at ambient temperature. The water was removed by lyophilization, and the polymer was obtained as a yellow solid (5.40 g, Mn,theo = 15 400 g mol−1, Mn,SEC (DMAC) = 10 100 g mol−1, Đ = 1.1, Mn,NMR = 10 050 g mol−1). Synthesis of Hydroxyl-Terminated, Alkyne-Functionalized Poly(DMAAm) (Alkyne-Poly(DMAAm)-OH) (3). Freshly distilled THF (50 mL), in a 250 mL round-bottom flask, was heated to 60 °C in an oil bath, and AIBN (0.33 g, 1.98 mmol, 20.00 equiv) was added under vigorous stirring. Subsequently, the alkyne-functionalized RAFT polymer poly(DMAAm) (2) (Mn,NMR (DMAC) = 10 050 g mol−1, 1.00 mg, 0.09 mmol, 1.00 equiv) was added to the solution which turned light yellow. The mixture was stirred at 60 °C under ambient oxygen until complete discoloration of the solution, which indicated full conversion. The temperature was reduced to 40 °C, and ascorbic acid (0.11 g, 0.59 mmol, 6.00 equiv) was added to quench the generated peroxides. Peroxide dipsticks (Quantofix Peroxid 100, 1−100 mg L−1 H2O2) were employed to check for the presence of peroxides. After the removal of the peroxides with the ascorbic acid, the solvent was reduced in vacuo, and the residue was dialyzed against deionized water, filtered, and dried. The product was obtained as white powder (920.00 mg, Mn,SEC (DMAC) = 11 700 g mol−1, Đ = 1.1, Mn,NMR = 10 200 g mol−1). Synthesis of β-Cyclodextrin-Functionalized Poly(DMAAm) (Poly(DMAAm)-β-CD) (5). Alkyne-poly(DMAAm)-OH (3) (Mn,NMR = 11 000 g mol−1, 0.60 g, 0.06 mmol, 1.00 equiv), β-CD-N3 (4) (2.07 g, 1.79 mmol, 30.00 equiv), and N,N,N′,N′,N″-pentamethyldiethylenetriamine (PMDETA) (0.20 g, 1.15 mmol, 20.00 equiv) were placed into a Schlenk tube and dissolved in DMF (48 mL). The mixture was degassed by three freeze−pump−thaw cycles, and the CuBr (0.18 g, 1.25 mmol, 20.00 equiv) was added under a stream of argon. The reaction was stirred at room temperature for 2 days and subsequently dialyzed against deionized water for 3 days. After lyophilization, the product was obtained as white solid (0.43 g, Mn,SEC (DMAC) = 10 700 g mol−1, Đ = 1.3, Mn,NMR = 11 000 g mol−1). Synthesis of 6-(((3s,5s,7s)-Adamantan-1-yl)amino)-6-oxohexyl 2(((Dodecylthio)carbonothioyl)thio)propanoate (DoPAT-Ada) (8). DoPAT (1.00 g, 2.85 mmol, 1.00 equiv), N-((3s,5s,7s)-adamantan-1yl)-6-hydroxyhexanamide (7) (0.72 g, 2.85 mmol, 1.00 equiv), and DMAP (0.07 g, 0.06 mmol, 0.02 equiv) were dissolved in 25 mL of dry DCM. The solution was cooled to 0 °C in an ice bath, and DCC (0.88 g, 4.27 mmol, 1.50 equiv), dissolved in 5 mL of dry DMF, was added dropwise to the mixture. After 1 h, the ice bath was removed, and the reaction stirred overnight at ambient temperature. The solvent was removed under reduced pressure, and the residue dissolved in diethyl ether. After filtration, the organic layer was washed with 5% HCl, saturated NaHCO3 solution, and deionized H2O and dried over MgSO4. Subsequently, the solvent was removed on a rotary evaporator, and the crude product was purified via column chromatography (silica gel, cyclohexane/ethyl acetate 5:1, Rf = 0.31) to give a yellow oil, which was stored in the fridge (1.19 g, 70%). 1H NMR (400 MHz, CDCl3): δ = 5.08 (s, 1 H, NH), 4.81 (q, 1 H, HHJ3 = 7.4 Hz, S−CH−CH3−CO), 4.18−4.08 (m, 2 H, CO2−CH2−CH2), 3.35 (dt, 2 H, HHJ3 = 7.3 Hz, CH2−CH2−S), 2.10−2.06 (m, 5 H, CH2−CH2−CO, Ad−H), 1.99 (s, 6 H, Ad−H), 1.73−1.59 (m, 9H, CH2−CH2−S, OC2H2−CH2−CH2− CH2−CH2CO), 1.43−1.35 (m, 2 H, OC2H2−CH2−CH2−CH2− C


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Macromolecules

Scheme 2. Synthetic Strategy for the Formation of the Polymer Building Blocks along with the Diblock Copolymer Formation

CH2CO), 1.26 (s, 18 H, CH3−C9H18−CH2), 0.88 (t, 3 H, HHJ3 = 6.7 Hz, CH3−C9H18) ppm. 13C NMR (400 MHz, CDCl3): δ = 171.87 (OCH− NH), 171.21 (CH3−CH−CO), 65.75 (O−CH2−CH2), 51.86 (NH− CAda), 48.07 (S−CH−CH3), 41.77 (CAda), 37.60 (CAD), 37.30 (CH2− CO−NH), 36.42 (CH2−S3), 31.96 (CH3−CH2−CH2), 29.60 (O− CH2−CH2), 29.50 (CH3−C2H4−CH2), 29.38 (C6H12−C2H4S), 27.94 (CAda), 25.49 (OC2H2−CH2−C3H5ONH), 25.32 (CH2−C2HONH), 22.73 (CH3−CH2), 16.96 (S−CH−CH3), 14.17 (CH3−C9H18) ppm. MS (ESI) m/z calculated for C32H55NO3S3 [M + Na]+: 620.32; found 620.44. Synthesis of 2-Methyl-6-((4-vinylbenzyl)oxy)benzaldehyde (Vinyl Benzyl Photoenol) (9). In a three-necked round-bottom flask, equipped with a reflux condenser, 2-hydroxy-6-methylbenzaldehyde (500.00 mg, 3.67 mmol, 1.00 equiv), K2CO3 (761.29 mg, 5.51 mmol, 1.5 equiv), and 18-crown-6 (14.55 mg, 0.06 mmol, 0.015 equiv) were evacuated with subsequent addition of nitrogen. The reagents were emulsified in dry THF (10 mL) and stirred for 4 h at 80 °C. The slightly yellow reaction mixture turned light green. Subsequently, 4-vinylbenzyl chloride (616.53 mg, 4.04 mmol, 1.10 equiv) was slowly added to the reaction and stirring was continued at 80 °C for another 17 h. The solvent was evaporated under reduced pressure, and the residue was dissolved in DCM, washed with deionized H2O (4 × 100 mL), and dried over MgSO4. After evaporation of the solvent, the crude product was purified via column chromatography (cyclohexane/ethyl acetate, 10:1, Rf = 0.41) to yield a white solid (877.20 mg, 47%). 1H NMR (400 MHz, CHCl3): δ = 10.74 (s, 1 H, CHO), 7.38−7.27 (m, 5 H, CHAr), 6.89 (d, 1 H, HHJ3 = 8.4 Hz, HCAr−CO), 6.83 (d, 1 H, HHJ3 = 7.6 Hz, HCAr−C−CH3), 6.77− 6.69 (m, 1 H, CHCH2), 5.77 (d, 1 H, HHJ3 = 16.9 Hz, CHcisHtrans), 5.28 (d, 1 H, = 11.6 Hz, CHcisHtrans), 5.15 (s, 2 H, Ar−CH−O), 2.59 (s, 3 H, Ar−CH3) ppm. 13C NMR (400 MHz, CHCl3): δ = 192.29 (CHO), 162.30 (CAr−O), 142.16 (CAr−CH3), 137.59 (CAr−CHCH2), 136.32 (CAr−CH2−O), 135.73 (CHCH2), 134.39 (HCAr), 127.52 (HCAr− CAr−CArH), 126.52 (HCAr−CAr−CArH), 124.45 (CAr−CHO), 123.69 (CAr−CAr−CH3), 114.37 (CHCH2), 110.46 (CAr−CAr−O), 70.40 (CAr−CH2−O), 21.53 (CAr−CH3) ppm. Synthesis of Adamantyl-Functionalized Poly(NIPAAm/Photoenol) (Poly(NiPAAm/PE)-Ada) (10). 6-(((3s,5s,7s)-Adamantan-1-yl)amino)6-oxohexyl 2-(((dodecylthio)carbonothioyl)thio)propanoate (8) (20.34 mg, 0.034 mmol, 1.00 equiv), NIPAAm (1.00 g, 8.837 mmol, 259.82 equiv), 2-methyl-6-((4-vinylbenzyl)oxy)benzaldehyde (9)

(66.89 mg, 0.265 mmol, 7.79 equiv), and AIBN (0.55 mg, 0.003 mmol, 0.10 equiv) were added to a Schlenk tube and dissolved in 1,4dioxane (4 mL). Oxygen was removed from the mixture via four freeze− pump−thaw cycles. Subsequently, the tube was placed in an oil bath at 67 °C for 24 h. The mixture was dialyzed against deionized water with a SpectraPor3 membrane (MWCO = 1000 Da) for 3 days at 4 °C. The solvent was removed by lyophilization, and the residue was precipitated in cold diethyl ether from a THF solution. After filtration the polymer was obtained as white powder (633 mg, Mn,theo = 30 000 g mol−1, SEC (DMAC): Mn,SEC = 39 700 g mol−1, Đ = 1.2, Mn,NMR = 24 100 g mol−1, calc. (NMR): 3% photoenol units incorporated). Formation of the Supramolecular Diblock Copolymer (11). Poly(NIPAAm/PE)-Ada (10) (Mn,NMR = 24 100 g mol−1, 100.00 mg, 1.00 equiv) was placed into a vial and dissolved in DMF (10 mL). In a second vial poly(DMAAm)-β-CD (5) (Mn,NMR = 11 000 g mol−1, 65.56 mg, 1.00 equiv) was also dissolved in DMF (5 mL). Subsequently, the solution of the poly(DMAAm)-β-CD (5) was added to the DMF solution of poly(NIPAAm/PE)-Ada (10) and stirred at ambient temperature for 30 min. Afterward, the mixture was dialyzed with a SpectraPor3 membrane (MWCO = 1000 Da) against a gradient of deionized H2O/DMF (70:30, 80:20, 90:10, and finally 100% H2O). The formed complex was analyzed via 2D NOESY NMR in D2O and DLS measurements in Milli-Q water with a concentration of 0.06 mmol L−1 (Dh ≈ 19 nm). Spectra and graphs are shown in the Results and Discussion section. For later stoichiometric calculation, the sum of the molecular weight distributions of poly(NiPAAm/PE)-Ada and poly(DMAAm)-β-CD is employed for the Mn of the diblock copolymer. Formation of the Cross-Linked Micelle (13). The supramolecular diblock copolymer 11 (Mn = 39 900 g mol−1, 10.60 mg, 0.0003 mmol, 0.006 mmol L−1, 1.00 equiv) and 4-arm PEG-maleimide 12 (Mn = 2090 g mol−1, 3.04 mg, 0.0012 mmol, 0.025 mmol L−1, 4.00 equiv) were added into a headspace vial, dissolved in Milli-Q water (4.5 mL), and sealed. The vial was placed in an ice bath, and the mixture was purged with nitrogen for 1 h. Meanwhile, a photoreactor was equipped with five Arimed B6 low pressure mercury lamps (320 nm, 36 W) and turned on for 1 h before adding the sample, so a reactor temperature close to 50 °C was established. Prior to irradiation of the sample, the solution was heated above the LCST of the diblock copolymer, resulting in a slightly turbid emulsion. Subsequently, the vial was positioned in the photoreactor and irradiated at 50 °C for 6 h. The sample was filtered D


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Macromolecules through a 0.45 μm syringe filter and freeze-dried. The cross-linked micelles were analyzed via DLS (Dh ≈ 50 nm) and AFM. The data is discussed in the Results and Discussion section. Release of the Nanoparticle (14). Trifluoroacetic acid (TFA) (0.20 mL) was added to a dispersion of the cross-linked micelle (10) (5.64 mg) at a concentration of 1 mg mL−1 in Milli-Q water (5.64 mL), stirred for 1 h at ambient temperature, and stored in the fridge for 2 days. To remove the cleaved micelle arms, the sample was centrifuged stepwise. Therefore, the solution was distributed into four Eppendorf Safe-Lock Tubes (500 μL) and placed in a preheated centrifuge (40 °C) for 5 min. Subsequently, the samples were centrifuged at 40 °C for 4 min with 3000 rpm. Subsequently, 400 μL of the supernatant fluid was withdrawn from the Safe-Lock Tubes and collected in a vial. Then, another 500 μL of the nanoparticle solution was added to the same tube and centrifuged at 40 °C for 4 min at 5000 rpm. Following the removal of the supernatant fluid, the sample was washed by the addition of 400 μL of Milli-Q water and subsequent centrifugation. The washing was repeated two times. The last centrifugation step was carried out at 40 °C with 13 000 rpm for 4 min. The nanoparticle was analyzed via NMR, DLS (Dh ≈ 35 nm), and AFM; please refer to the Results and Discussion section.

attached to the alkyne-terminated poly(DMAAm) in a CuAAC click reaction employing CuBr and PMDETA to obtain the βCD-polymer 5. Figure 1 depicts the proton NMR spectra of pure

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RESULTS AND DISCUSSION Nanoparticles, which react when exposed to an external stimulus, can be designed for a multitude of applications. In the current contribution a supramolecular diblock copolymer, which is formed via β-CD/adamantyl host−guest interaction, undergoes micellization at elevated temperature in aqueous solution. Subsequently, the micelle is cross-linked in its core, and the βCD functionalized polymer units, forming the arms of the micelle, are cleaved via addition of a strong acid to yield the nanoparticle. Preparation of the Polymer Blocks and Formation of the Supramolecular Diblock. The syntheses of the cyclodextrin-functionalized polymer block, the adamantyl-bearing block, and the supramolecular diblock are described (Scheme 2) along with a detailed characterization. The β-CD-terminated polymer chain was synthesized as previously reported by us.67 Poly(DMAAm) was synthesized in a RAFT polymerization with the chain transfer agent (CTA) 1 and AIBN in DMF at 60 °C, which introduces an alkyne function at the chain end. With the alkyne unit at the end of poly(DMAAm), the polymer can be readily modified via conjugation of the desired functionality to the polymer chain. The polymer was characterized via SEC and NMR (Mn,SEC = 10 100 g mol−1, Đ = 1.1, Mn,NMR = 10 050 g mol−1); refer to the Supporting Information Figures S1and S2. Further calculations for synthetic approaches were based on the NMR derived values of Mn due to the fact that the SEC traces are calibrated relative to PS standards, which reflect relative molecular weight information only. In an intermediate step the trithiocarbonate was cleaved from the polymer, since it was noted that the click CuAAC reaction proceeds in a more effective fashion without the presence of RAFT groups.68 Therefore, poly(DMAAm) 2 was dissolved in THF at elevated temperatures under the addition of AIBN and vigorous stirring in the presence of ambient oxygen. Discoloration of the slightly yellow solution indicated complete removal of the trithiocarbonate. The subsequent addition of ascorbic acid reduced the generated hydroperoxide functional polymer to the hydroxyl-functionalized polymer 3 (Figures S1 and S2). A detailed analysis of the transformation mechanism was described by our team.69 The SEC trace of the hydroxyl-terminated polymer 3 reveals a shoulder in the higher molecular weight region, which is probably due to small amounts of coupling products, resulting from the heating of the alkyne polymer in the presence of AIBN. In a final step, the azide functional β-CD 4 was

Figure 1. Overview of 1H NMR spectra (DMSO-d6, 400 MHz) following the end-group modification of poly(DMAAm): (A) poly(DMAAm)-β-CD 5 with characteristic resonances at 8.05, 5.71, 4.83, and 3.64 ppm; (B) alkyne-terminated hydroxyl functional poly(DMAAm) 3; (C) pure β-CD (the protons assignment applies for all seven glucose units).

β-CD (C), the alkyne-terminated hydroxyl functional polymer 3 (B), and poly(DMAAm)-β-CD 5 (A). The overview illustrates the success of the synthetic sequence by the proton resonances of the triazole click product at 8.05 ppm (a) and the inner and outer protons of the β-CD at 5.71, 4.83, and 3.64 ppm (b, c, d). For the RAFT polymerization of the thermoresponsive, guestbearing polymer chain 10 a novel CTA 8 was synthesized. 2(Dodecylthiocarbonothioylthio)propionic acid (DoPAT) was linked with the adamantane derivative 7 via Steglich esterification. The Supporting Information entails all relevant characterization data as well as a kinetic investigation (Figures S11−S13) of the controlling ability of the CTA 8, confirming the living character of the polymerization. In addition, to introduce a cross-linker into the polymer side chain, a photoactive 2methoxy-6-methylbenzaldehyde (photoenol, PE) monomer was designed by etherification of the 2-hydroxy-6-methylbenzaldehyde precursor with the styrene derivative 4-vinylbenzyl chloride. The product 9 was characterized via proton and carbon NMR spectroscopy (see Figures S9 and S10). The photoactive reactions of photoenol have been thoroughly investigated by our group in a large variety of macromolecular designs, e.g., surface modifications,70 block copolymer linkages,71 and in threedimensional polymer structures created by direct laser writing.72 2-Methoxy-6-methylbenzaldehyde forms a reactive diene upon irradiation with UV light, which can undergo Diels−Alder reactions with active double bonds. Since no further addition of reagents is necessary for the photoreaction and its synthesis is straightforward, the photoenol compound is very attractive for chemical bond formation. Based on a publication by Nichifor and Zhu on the influence of the copolymerization of styrene and Nacrylamides on their thermoresponsive behavior, a styrene derivative was chosen for the synthesis of the photoenol monomer 9.73 Subsequently, the statistical copolymer 10 of NiPAAM and 2-methyl-6-((4-vinylbenzyl)oxy)benzaldehyde (9) (vinyl benzyl photoenol) was synthesized. The freshly E


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Figure 2. Characterization of the thermoresponsive copolymer 10. (A) SEC traces recorded in DMAC at 50 °C (Mn = 39 100 g mol−1, Đ = 1.2). (B) LCST measurement in water (concentration 0.06 mmol L−1). (C) 1H NMR (CDCl3, 400 MHz) of poly(NiPAAm/PE)-Ada (Mn,NMR = 24 100 g mol−1) with 3% PE units incorporated in the side chain.

Figure 3. (A) DLS measurements at 10 °C, showing the number-weighted size distributions of poly(NiPAAm/PE)-Ada 10, poly(DMAAm)-β-CD 5, and the resulting supramolecular diblock 11 in Milli-Q water at 10 °C with a polymer concentration of 0.06 mmol L−1. (B) LCST measurement of the single poly(NiPAAm/PE)-Ada block 10 and after the diblock formation 11 with poly(DMAAm)-β-CD.

produced CTA 8 was employed to introduce the adamantyl guest molecule at the chain end. The ratio of NiPAAm to PE units was calculated from the proton NMR spectrum of 10 (illustrated in Figure 2A) to be 30:1 (NiPAAm/PE), which is equivalent to approximately 200 NiPAAm units and approximately 7 vinyl benzyl photoenol units (3%). For the calculations, the resonance of the aldehyde proton (a) and the methylene protons of the photoenol monomer (h) were evaluated against the single proton of the isopropyl unit of NiPAAm (i). Additionally, the molecular weight was calculated from the NMR spectrum with the aforementioned proton resonance as reference for the integration of the protons signals resulting from the polymer backbone (Mn,NMR = 24 100 g mol−1). Furthermore, the SEC traces of the copolymer were recorded in DMAC at 50 °C. According to the SEC shown in Figure 2A, the polymer has a number-average molecular weight of 39 100 g mol−1 and a Đ of 1.2. Here, as well as for poly(DMAAm)-β-CD, the Mn values based on the NMR measurements are considered for further calculations. The copolymer showed thermoresponsive behavior with an LCST at approximately 20 °C, as depicted in Figure 2A. The LCST of poly(NiPAAm/PE)-Ada was measured via UV−vis

turbidity measurements in Milli-Q water with a polymer concentration of 0.06 mmol L−1, in the temperature range from 5 to 40 °C. Because of the amide functionality of the poly(NiPAAm), the UV−vis traces show hysteresis of the LCST between heating and cooling of the sample. The amide functions lead to additional inter- and intramolecular interactions above the LCST of poly(NiPAAm/PE)-Ada 10, which hinder the rehydration of the polymer and therefore lead to the observed behavior.74 β-CD with its cage-like structure and adamantane are known to form strong inclusion complexes in aqueous solution. For the formation of the diblock copolymer, the two building blocks 5 and 10 were dissolved in DMF and dialyzed against deionized water. In DMF, as a nonselective solvent for poly(NiPAAm/PE)Ada and poly(DMAAm)-β-CD, the guest group features better accessibility for β-CD and via the slow exchange of the solvent the supramolecular complex is formed. Dynamic light scattering (DLS) was performed to analyze the diblock copolymer formation. The graph depicted in Figure 3A shows the number weighted distribution of poly(NiPAAm/PE)-Ada (10, Dh = 9.0 nm), the β-CD functional poly(DMAAm) (5, Dh = 12.5 nm), F


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Macromolecules Scheme 3. Synthetic Strategy for the Design of the Nanoparticles

and the distribution of the resulting diblock (11, Dh = 19 nm). The diblock copolymer 11 shows a clear shift to higher hydrodynamic diameter, thus indicating the block formation (refer to Figures S14−S16 for the corresponding autocorrelation functions). Because of the encapsulation of the hydrophobic adamantyl molecule in β-CD, additional LCST measurements (Figure 3B) indicate a shift to higher temperatures, confirming the DLS measurements.75 The connection of the polymer blocks via β-CD/adamantyl host−guest interactions was analyzed with nuclear Overhauser enhancement spectroscopy (NOESY). NOESY-NMR is an excellent method to investigate supramolecular inclusion complexes by recording the homonuclear correlation of protons in close proximity. Therefore, host−guest interactions are verified by corresponding cross-correlation peaks in the spectrum. Figure S19A depicts the NOESY spectrum of the supramolecular diblock copolymer 11, showing the crosscorrelation peaks of the adamantyl moiety at 1.73, 1.99, and 2.05 ppm with the inner protons of β-CD at 3.64 ppm, thus evidencing the successful formation of the inclusion complex. However, additional cross-correlation peaks between the inner CD protons (3.64 ppm) and the dodecyl protons of the DoPAT molecule at 0.88 and 1.31 ppm are present. The data reveal that not only adamantyl is enclosed in the β-CD but also the long alkyl rest which is attached to the other chain end of poly(NiPAAm/PE)-Ada 10. The inclusion of the C12H25 chain was already observed by Bertrand et al., who employed a similar DoPAT derivative to synthesize an adamantane end-capped poly(acrylic acid) chain to prepare supramolecular comb-shaped polymers.76 Nevertheless, these authors state that the association constant of the alkyl chain with β-CD (Kassoc ≈ 102 M−1) is about

2 orders of magnitude weaker compared to the constant for adamantane with β-CD (Kassoc ≈ 104 M−1), and therefore alkyl complexes exist only to a minor degree.76 Moreover, for our application it is irrelevant from which side of the thermoresponsive polymer chain the β-CD-bearing chain is attached. Therefore, the system was applied in the nanoparticle formation without further optimization. Thermoresponsive Micellization, Photoinduced CrossLinking, and Nanoparticle Design. After the successful formation of the supramolecular diblock copolymer 11, the system was prepared for micellization and simultaneous crosslinking. Thus, polymer 11 and the 4-arm cross-linker 12 were dissolved in Milli-Q water with a polymer concentration of 0.06 mmol L−1, cooled in ice water, and purged with nitrogen for 1 h. Meanwhile, a house-built photoreactor (Figure S21) was equipped with five compact low-pressure mercury lamps emitting at λ = 320 nm (36 W, see Figure S20 for the UV−vis emission spectrum). The reactor was left running for 1 h prior to use with the ventilation turned off, so the reacting chamber heated up to a constant temperature of 50 °C. Subsequently, the sample was warmed above its LCST, placed in the photoreactor, and irradiated. A detailed overview of the micelle cross-linkage and nanoparticle release is illustrated in Scheme 3. During micellization the cross-linker 12 is encapsulated in the micelle structure where it reacts with the photoenol units in the polymer side chain of 10 when irradiated with UV light. It is known that the equilibrium constant of the supramolecular complex is reduced with increasing temperature.77 Schmidt et al. observed the degradation of a supramolecular three-armed star polymer at 70 °C.66 Therefore, the temperature of the photoreactor might G


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Macromolecules weaken the equilibrium constant of the present supramolecular copolymer yet will not lead to its destruction. According to prior experiences in our group, photoreactions with the present system react rather slowly in water, and crosslinked micelles were obtained after an irradiation time of 6 h. The micelles were characterized via AFM and DLS which is discussed in the next section. Subsequently, the β-CD-units were destroyed by adding trifluoroacetic acid (TFA) to the aqueous solution of the cross-linked micelles. The TFA hydrolyses the α-1,4 glucose unit of the β-CD, resulting in a linear seven-membered glucose chain.78 Furthermore, the mixture of nanoparticles and schismatic micelle arms was centrifuged at elevated temperatures to separate the two units. Therefore, the sample and the centrifuge were heated to 40 °C with low rotation, so the thermoresponsive nanoparticles would agglomerate and the nonresponsive poly(DMAAm)-arms stayed in solution. A detailed description of the centrifugation process is given in the Experimental Section. The successful separation of micelle arms and nanoparticles via centrifugation was verified via NMR with the disappearance of the proton signals, originating from poly(DMAAm) between 2.75 and 3.08 ppm, shown in the NMR spectra collection in Figure 4. Acidic cleavage of the arms is a fast

Figure 5. DLS measurements at 10 °C in Milli-Q water, showing number weighted size distributions of the diblock copolymer 11, of the micelles 13 and the nanoparticles 14 at a concentration of 1 mg mL−1.

micelles 13, and the final nanoparticles 14. A strong indication for the formation of the micelles 13 is the shift of the diblock copolymer 10 from 19 to approximately 50 nm after fixation of the micelle via photoinduced cross-linking. With the cleavage of the micelle arms and after purification of the sample, the hydrodynamic diameter decreases to 34 nm. To avoid agglomeration of the nanoparticles, due to their hydrophobic shell, the sample was sonicated for several seconds before the DLS measurement. Nevertheless, after longer measuring times, agglomeration of the particles occurred. In accordance with the NMR spectra in Figure 4, the shift to lower Dh is indicative of the successful degradation of micelles to nanoparticles (refer to Figures S17 and S18 for the corresponding autocorrelation functions). Furthermore, the thermoresponsive behavior of micelles and nanoparticles were investigated via DLS, as illustrated in Figure 6. The change in Dh with changing temperature is observed for the micelles as well as the nanoparticles and refers to the temperature-induced contraction and relaxation of the stimuli-responsive species. Furthermore, AFM was performed to image and characterize the size differences between micelles and nanoparticles. Therefore, diluted solutions of the samples in Milli-Q water (0.1 μg mL−1) were adsorbed on freshly cleaved mica surfaces, dried, and measured with a scan size of 1 × 1 μm. The two- and three-dimensional topographic maps of the micelles (A) and the nanoparticles (B) are depicted in Figure 7 (higher magnification AFM images are shown in Figure S24). Because of the inherent disperse nature of the polymer samples 5 and 10 the AFM images of both samples show dispersity in particle size as well. In addition, particle analysis was performed for each sample employing the NanoScope Analysis tool and is illustrated in the Supporting Information (Figures S22 and S23). Comparing the samples, the particle analysis reveals a decrease in diameter on averagewhen going from micelles to nanoparticles (see table in Figures S23 and S24). The data are in agreement with the decrease of Dh in the DLS experiment and the disappearance of the poly(DMAAm) proton signals in the NMR spectrum, thus also confirming the degradation of the micelles to the nanoparticles. In general, the diameter values obtained from the AFM measurements contain uncertainties due to tipconvolution effects. Furthermore, the particles prepared for the

Figure 4. 1H NMR spectra (CDCl3, 400 MHz): (A) nanoparticle 14 received after centrifugation, not showing poly(DMAAm) proton resonances in the spectrum; (B) isolated substance obtained from the supernatant fluid after centrifugation; (C) cross-linked micelle 13; (D) supramolecular diblock copolymer 11; (E) poly(DMAAm)-β-CD 5; (F) poly(NiPAAm/PE)-Ada 10.

and efficient method to release the nanoparticle from the micellar scaffold. An alternative way to hydrolyze the CD units is via the addition of the enzyme α-amylase, which similar to TFA cleaves the glucose units. Since TFA is a small molecule compared to αamylase, it was more suitable for our applications. Other alternative methods to remove the arms are for example adding either an excess of β-CD or an access of the adamantane guest molecule to the micelle suspension to suppress the diblock formation between the polymer chains and subsequent dialysis against DMF. However, centrifugation was the easiest and fastest way to separate the arms from the nanoparticles, which is validated by our data. Atomic force microscopy (AFM) and DLS were employed to investigate the formation and size of micelles and nanoparticles. The DLS results are depicted in Figure 5 and unambiguously reveal the distribution of the supramolecular block 11, the H


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Figure 6. Temperature sequenced DLS measurements, showing contraction and relaxation of the thermoresponsive species: (A) micelles and (B) nanoparticles.

Figure 7. 2D and 3D AFM topography images of (A) micelles and (B) nanoparticles. The samples were measured on freshly cleaved mica surfaces and cast from a 0.1 μg mL−1 solution in Milli-Q water.

buildings blocks of poly(DMAAm)-β-CD and thermoresponsive poly(NiPAAm/PE)-Ada were prepared via RAFT polymerization and analyzed with 1H NMR and SEC. An adamantyl bearing CTA was synthesized for the copolymerization of NiPAAm as well as a new photoactive monomer. The small molecules were characterized via 1H and 13C NMR plus a polymerization kinetic of the novel CTA was performed. β-CD/ adamantyl host−guest interactions of poly(DMAAm)-β-CD and poly(NiPAAm/PE)-Ada led to the formation of a supramolecular diblock copolymer which self-assembled into a micelle at elevated temperature in aqueous solution. NOESY and DLS were employed to evidence the host−guest interactions and the formation of the diblock copolymer. The micellar structure was

AFM measurements are air-dried on a mica surface, which might result in particle shrinking effects,74 whereas the particles in the DLS measurement are swollen in solution. Thus, the particle size data obtained from DLS and AFM are not directly comparable, and AFM rather serves to visualize the micelle and nanoparticle formation.

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CONCLUSIONS

In summary, we introduce a novel synthetic approach for the formation of thermoresponsive nanoparticles via a sacrificial micellar scaffold, employing supramolecular host−guest chemistry and photoinduced Diels−Alder reactions. The polymer I


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cross-linked in its core while adding a 4-armed maleimide linker molecule and irradiation with UV light. Importantly, the β-CD units were hydrolyzed to cleave of the sacrificial micelle arms and release the nanoparticle. Cross-linked micelles and released nanoparticles were investigated with 1H NMR, DLS, and AFM. It was possible to evidence the successful transformation of the micelle to the nanoparticle as well as its contractions responding to a thermal stimulus. Regarding future applications of the nanoparticles, the adamantyl guest molecules, which remain on the surface of the nanoparticle, play an important role. The guest functionalized nanoparticles can thus be refunctionalized with β-CD bearing moieties, such as varying polymer chains or even proteins, which are in the focus of upcoming studies.

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ASSOCIATED CONTENT

S Supporting Information *

1 H NMR, 13C NMR, and SEC traces of the following compounds: 2, 3, and 5; 1H and 13C NMR spectra of 8 and 9; a kinetic study of polymerizations employing CTA 8; autocorrelation functions of the DLS measurements; the emission spectrum of the Arimed B6 low-pressure fluorescent lamp and an illustration of the photoreactor; a NOESY spectrum of the supramolecular diblock copolymer. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.5b00923.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (C.B.-K.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS C.B.-K. is grateful to the German Research Council (DFG) for cofunding the current project. In addition, C.B.-K. is grateful for continued support for this project from the Karlsruhe Institute of Technology (KIT) in the context of the Helmholtz BIF program and the Ministry of Science and Arts of the State of BadenWürttemberg. C.B.-K. and J.B. thank the German Academic Exchange Service for supporting a visit of A.F.H. to the laboratories of J.B. The authors are additionally grateful to David Schulze-Suenninghausen (KIT) for the measurement of the NOESY-NMR spectrum.

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REFERENCES

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