Versatile Synthesis of Amine-Reactive Microgels by Self-Assembly of

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Versatile Synthesis of Amine-Reactive Microgels by Self-Assembly of Azlactone-Containing Block Copolymers Xu Wang,*,†,‡ Jesse L. Davis,‡ Bethany M. Aden,‡ Bradley S. Lokitz,∥ and S. Michael Kilbey, II*,‡,§ †

National Engineering Research Center for Colloidal Materials, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, P. R. China ‡ Department of Chemistry and §Department of Chemical and Biomolecular Engineering, University of Tennessee, Knoxville, Tennessee 37996, United States ∥ Center for Nanophase Materials Sciences, Oak Ridge National Laboratory, One Bethel Valley Road, Oak Ridge, Tennessee 37831, United States S Supporting Information *

ABSTRACT: Relations between molecular design, chemical functionality, and stimulus-triggered response are important for a variety of applications of polymeric systems. Here, reactive amphiphilic block copolymers (BCPs) of poly(2-vinylpyridine)block-poly(2-vinyl-4,4-dimethylazlactone) (PVP-b-PVDMA) were synthesized and assembled into microgels capable of incorporating functional amines. The composition of the PVP-b-PVDMA BCPs was varied to control the number of reactive sites in the spherical aggregates created by self-assembly of PVP-b-PVDMA BCPs in a 2-propanol/THF (v:v = 19:1) solvent mixture, which is selective for PVP. PVDMA and PVP segments were selectively cross-linked by 1,4-diaminobutane (DAB) or 1,4-diiodobutane (DIB) to fabricate core- and corona-cross-linked azlactone-containing microgels, respectively. Non-cross-linked aggregates of PVP-b-PVDMA and DIB-cross-linked microgels dissociate when exposed to THF, which is a good solvent for both blocks. However, the DAB-cross-linked BCP microgels swell in THF, suggesting the formation of a stable, three-dimensional network structure. Because of their ability to be reactively modified in ways that allows their stability or disassembly characteristics to be tailored, these azlactone-containing BCP microgels provide an attractive platform for applications in a wide range of fields, including catalysis, imaging, molecule separation, and guest loading for targeted delivery.

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INTRODUCTION Reactive polymers1−4 have long been viewed as important materials and have found application in fields such as medicine,5 organic electronics,6 and biotechnology.7,8 The ability to address the reactive groups along the chain provides a facile way to impart stimuli responsiveness or specific selectivity, which are key for applications that take advantage of the reversible incorporation of guest molecules,9,10 stimuliresponsive deformations,11 selective separation of similarly sized biomolecules,12 or bioinspired self-healing of mechanical and structural properties.13−16 Microgels based on reactive polymers are of particular interest due to the ability to control their sizes on nanoscopic or microscopic length scales using microfluidic techniques,17−22 which enables their use as delivery vehicles in confined environments, such as blood vessels,23 for example. Furthermore, their cross-linked polymer network structure provides high stability and swelling-responsiveness in selective solvents.24,25 Most importantly, the reactive groups in the microgels allow them to be functionalized in a facile manner and actuated by external stimuli.11,26 Therefore, reactive polymeric microgels are regarded as a type of “small but smart” soft materials which are of interest for both fundamental research and practical application.27,28 © XXXX American Chemical Society

There have been several reports describing the preparation of reactive polymeric microgels. For example, the random copolymerization of N-isopropylacrylamide (NIPAM) and acrylic acid (AA) is a well-known process to synthesize reactive polymeric microgels that are multiresponsive.29 By using poly(NIPAM-co-AA) microgels, Lyon and co-workers demonstrated the pH- and ionic strength-controlled encapsulation of proteins in solution and the fabrication of thermoresponsive multilayer films.30,31 In another route, Wang et al. reported the formation of microgels by chemically cross-linking poly(allylamine hydrochloride) (PAH) and dextran.32 They showed that small dye molecules and larger metal nanoparticles (size ∼3.6 nm) could be loaded in the PAH−dextran microgel-based films and released slowly under physiological conditions.32−35 Supramolecular self-assembly is another versatile technique to construct well-defined core−corona particles from amphiphiles, including amphiphilic block copolymers (BCPs).36,37 Chemically cross-linking the core−corona BCP aggregates provides a way to increase the stability of the nanoaggregate, which leads Received: February 22, 2018 Revised: April 25, 2018

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DOI: 10.1021/acs.macromol.8b00405 Macromolecules XXXX, XXX, XXX−XXX

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Macromolecules to the formation of robust, functional microgels.25,37 Saito et al. first demonstrated the synthesis of flower type microgels by using 1,4-diiodobutane (DIB) to cross-link micelles formed by self-assembly of poly(2-vinylpyridine)-block-polystyrene-blockpoly(2-vinylpyridine) (PVP-b-PS-b-PVP) triblock copolymers in toluene and toluene/cyclohexane mixtures, which are selective for PS.38 The self-assembly and cross-linking of BCPs have also been used to design microgels with more complex structures. For example, Wooley and co-workers produced hollow polymeric microgels of poly(ε-caprolactone)block-poly(acrylic acid) (PCL-b-PAA) that contain a degradable PCL core and a 2,2′-(ethylenedioxy)bis(ethylamine)-crosslinked PAA corona.39 These reports merely highlight the broad interest in developing novel and versatile microgels that can be synthetically tailored and reactively modified to control their structure and properties. As demonstrated by early reports by Heilmann and coworkers, poly(2-vinyl-4,4-dimethylazlactone) (PVDMA) is a useful reactive polymer that can be chemically modified in a variety of ways.40−43 VDMA monomers or PVDMA polymers and copolymers react with primary amines (or alcohols) under mild conditions, producing a stable amide linkage without formation of byproducts, which makes these materials useful as functional materials. For instance, Lynn and co-workers reported the layer-by-layer assembly of PVDMA-based multilayers for DNA delivery, superhydrophobic surfaces, and selfhealing coatings.3,44−46 Fontaine and co-workers developed a class of stable, azlactone-functionalized thermoresponsive nanoparticles47 that have potential application as theranostics. Similarly, Lowe and co-workers synthesized a series of pHand/or thermoresponsive polymers by functionalization of PVDMA48,49 and also created multiresponsive hydrogels50 via postpolymerization modification of PVDMA homopolymers, both of which represent smart polymer platforms that may be useful in tissue engineering or as biomaterials. In our previous studies, we reactively modified PVDMA to alter interfacial properties, created bioconjugates for control of liver cell growth, and examined formation and structure of polymer brushes based on PVDMA homopolymers and dually reactive BCPs containing VDMA.51−58 However, to our knowledge, this is the first report detailing the fabrication of reactive microgels using azlactone-containing BCPs. In this study we report the synthesis of azlactone-based microgels by successive self-assembly of PVP-b-PVDMA BCPs in solution and then reactively modify the nanoassemblies by cross-linking in a selective fashion. PVP-b-PVDMA BCPs of different molecular weight and PVDMA content were synthesized in order to investigate self-assembly and guest loading characteristics. A diamine or a diiodide was used to cross-link either the PVDMA or the PVP blocks in the selfassembled aggregates. In addition to assessing the stability and swelling properties of the resulting microgels in THF, a series of functional primary amines were used to investigate the chemical reactivity of the covalently cross-linked PVP-bPVDMA BCP microgels.

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methoxy-2,4-dimethylvaleronitrile) (V-70) was purchased from Wako Specialty Chemicals and recrystallized from methanol before use. 2-Vinyl-4,4-dimethylazlactone (VDMA; Isochem North America, LLC) was fractionally distilled under reduced pressure and the middle fraction (∼70%) reserved for use. 1,4-Diaminobutane (DAB), 1aminopyrene, and anhydrous potassium carbonate were purchased from Acros. 1,4-Diiodobutane (DIB) and anhydrous magnesium sulfate were purchased from Alfa Aesar. 4-Aminoazobenzene and 4phenylazobenzoyl chloride were purchased from TCI. Dansylcadaverine (DC) was purchased from Anaspec, Inc. Synthesis of PVP MacroCTA. A poly(2-vinylpyridine) macrochain-transfer agent (PVP MacroCTA) was synthesized via reversible addition−fragmentation chain-transfer (RAFT) polymerization of 2vinylpyridine (VP). Reactions were formulated in a single-neck 100 mL Airfree round-bottom reaction flask equipped with a Teflon-coated magnetic stir bar. In a typical reaction, VP (4.94 g, 4.70 × 10−2 mol) was combined with CDB (168.28 mg, 6.18 × 10−4 mol; VP:CDB = 76) and AIBN (20.29 mg, 1.24 × 10−4 mol; molar ratio of CDB:AIBN = 5:1). The polymerization was conducted in bulk for 6 h at 65 °C. Polymerization was stopped by immersing the reaction vessel in liquid nitrogen and exposing it to air. The reaction mixture was diluted with THF and the PVP MacroCTA purified and isolated by precipitating into hexanes (3×), followed by drying overnight in vacuo. Synthesis of PVP-b-PVDMA BCPs. Reactions were formulated in a single-neck 50 mL Airfree round-bottom reaction flask equipped with a Teflon-coated magnetic stir bar. Two PVP-b-PVDMA BCPs with PVDMA blocks of different length were synthesized by chain extension of the PVP MacroCTA by RAFT polymerization: (i) For PVP-b-PVDMA BCPs with shorter PVDMA blocks, VDMA (2.78 g, 2.00 × 10−2 mol) was combined with the PVP MacroCTA (362.24 mg, 1.13 × 10−4 mol; VDMA:PVP MacroCTA = 177) and V-70 (11.44 mg, 3.71 × 10−5 mol; molar ratio of PVP MacroCTA:V70 = 3:1). (ii) For PVP-b-PVDMA BCPs with longer PVDMA blocks, VDMA (6.96 g, 5.00 × 10−2 mol) was combined with the PVP MacroCTA (342.53 mg, 1.07 × 10−4 mol; VDMA:PVP MacroCTA = 467) and V-70 (11.00 mg, 3.57 × 10−5 mol; molar ratio of PVP MacroCTA:V70 = 3:1). Polymerizations were conducted in benzene at a VDMA concentration of 1 M for 12 h at 30 °C. Polymerization was stopped by immersing the reaction vessel in liquid nitrogen and exposing it to air. The block copolymers were recovered and purified by precipitating the solution (in benzene) into hexanes (3×), followed by drying overnight in vacuo. Synthesis of Reactive Microgels. 2-Propanol and THF were dried over 5 Å molecular sieves before use. Self-assembled aggregates were formed via the dropwise addition of 2-propanol (4.75 mL) to a solution of the PVP-b-PVDMA BCP (2.5 mg) dissolved in THF (0.25 mL). The solution was then stirred at room temperature for 6 h before 5 mol % of DAB (relative to the number of VDMA repeat units) or 40 mol % of DIB (relative to the number of VP units) was added to the mixture. The reaction mixture was stirred at room temperature for an additional 24 h, resulting in the formation of reactive microgels. The microgel solutions were dialyzed against THF for 6 days using MWCO 6000−8000 dialysis tubing. Additional THF was added to the microgel solution until the final polymer concentration was ∼0.1 mg/mL. Reactive microgels cross-linked with DAB or DIB are noted as DABcross-linked microgel (DABCM) or DIB-cross-linked microgel (DIBCM), respectively. Synthesis of N-(4-Aminobutyl)azobenzenecarboxyamide (NAzo). 1,4-Diaminobutane (3.0 g), K2CO3 (0.3 g), and CH2Cl2 (25 mL) were added to a round-bottom flask, which then was sealed with a septum, placed under positive N2 pressure to create an inert atmosphere, and cooled to 0 °C. 4-Phenylazobenzoyl chloride (0.5 g) in CH2Cl2 (50 mL) was added dropwise using a nitrogen purged syringe, after which the temperature of the flask was raised slowly to room temperature. The reaction mixture was stirred for 24 h. The solution was extracted with deionized water (3×), and the organic layers were combined and dried over anhydrous magnesium sulfate. After filtration to remove solids, the solvent was removed by distillation at 60 °C, and residual 1,4-diaminobutane was completely removed in vacuo at 80 °C, leaving an orange solid. Subsequently, the

EXPERIMENTAL SECTION

Materials. 2-Vinylpyridine (VP), 2-phenyl-2-propyl benzodithioate (commonly referred to as cumyl dithiobenzoate, or CDB), 1,8diazabicyclo[5.4.0]-undec-7-ene (DBU), 1-pyrenemethylamine hydrochloride, and 2,2′-azobis(2-methylpropionitrile) (AIBN) were purchased from Sigma-Aldrich. AIBN was recrystallized from anhydrous methanol three times and dried under vacuum. 2,2′-Azobis(4B

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Macromolecules Scheme 1. Synthesis of PVP-b-PVDMA BCPs by RAFT Polymerization

orange solid was reconstituted in THF and precipitated into deionized water (3×). The precipitate was isolated by filtration and dried in vacuo at 60 °C to obtain a yellow solid in 75% yield. 1H NMR (300 MHz, CDCl3, 25 °C): δ = 2.78 (t, −CH2NH2), 3.50 (t, −NHCH2−), 7.52 (br d, aromatic H), 7.94 (br s, aromatic H). AccuTOF DART mass spectrometry (m/z): [M + H+] calculated 297.16; found 297.17. Self-Assembly on Surfaces. Silicon substrates obtained from Silicon Quest were cleaned via successive sonication in toluene, 2propanol, methanol, and deionized water (15 min in each solvent). The wafers were then immersed in piranha acid solution (1:3 (v/v) mixture of 30% H2O2 and 98% H2SO4) and heated until no bubbles were evolved. After removing the surfaces from the piranha acid solution and rinsing them with copious amounts of deionized water, the silicon substrates were dried with a stream of dry nitrogen. This treatment is known to create hydroxyl groups on the silicon substrate, which facilitates the self-assembly of PVP-b-PVDMA BCPs due to hydrogen bonding between hydroxyl and pyridine groups.59 Silicon substrates were immersed in the BCP solution (1 mg/mL in a 19:1 v/v mixture of 2-propanol/THF) overnight. After self-assembly, the substrate was rinsed with nonsolvent 2-propanol, then sonicated in 2-propanol for 5 min (2 × ) to remove loosely bound materials, and dried under ambient conditions. Functionalization of Reactive Microgels. 4 mL of a THF solution containing functional amines (c = 1.0 mg/mL) was added to 15 mL of a THF solution containing the reactive DABCMs (c = 0.1 mg/mL). The solution was stirred at room temperature for 24 h to facilitate the nucleophilic addition of the amines to the azlactone repeat units in the reactive DABCMs. Excess amine was removed by dialysis against THF for 6 days. The amine-modified DABCM solution was diluted with THF to a final volume of 20 mL, which results in a polymer concentration of ∼75 μg/mL. Characterization. Molecular weights and dispersities were determined by size exclusion chromatography (SEC) using a Waters Alliance 2695 separations module equipped with three Polymer Laboratories PLgel 5 μm mixed-C columns (300 × 7.5 mm) in series, a Waters Model 2414 refractive index detector (λ = 880 nm), a Waters Model 2996 photodiode array detector, a Wyatt Technology miniDAWN multiangle light scattering (MALS) detector (λ = 660 nm), and a Wyatt Technology ViscoStar viscometer. THF was used as the mobile phase at a flow rate of 1 mL/min. Refractive index increment, dn/dc, values were calculated based on 100% mass recovery using Astra V software. Dynamic light scattering (DLS) measurements were performed on a four-detector ALV goniometer according to methods described previously.60−62 Details of the DLS measurements and data analysis are available in the Supporting Information. Values of hydrodynamic radius, Rh, are considered apparent Rh by virtue of being measured at finite concentration. Transmission electron microscopy (TEM) images were acquired using a Zeiss Libra 200 MC transmission electron microscope that is equipped with a Gatan UltraScan US1000XP CCD camera. The polymer solution was drop-cast onto a carbon film grid. The sample was then stained by exposure to iodine vapor for 24 h. Atomic force microscopy (AFM) images were collected using a Veeco Instruments Nanoscope IIIa multimode atomic force microscope in tapping mode using silicon cantilevers from Applied NanoStructures, Inc. (Mountain View, CA). Samples drop-cast on silicon wafers were characterized by attenuated total reflectance−Fourier transform infrared (ATR-FTIR) spectroscopy using a Bruker Optics Vertex 70 spectrometer equipped

with a Harrick Scientific VariGATR accessory, which has a germanium hemisphere internal reflection element. Spectra were acquired using the narrow-band mercury−cadmium−telluride (MCT) detector, and at least 128 scans were collected. A blank silicon surface was used to acquire the background spectrum. Quaternization of the DIB-cross-linked BCP microgels was examined via X-ray photoelectron spectroscopy (XPS) using a Thermo-Fisher ESCALAB 250 X-ray photoelectron spectrometer, which uses a monochromated aluminum X-ray source. The binding energy scale was corrected to aliphatic carbon of 285.0 eV. Samples for XPS measurement were prepared by drop-casting the polymer solutions onto silicon substrates. Ultraviolet−visible (UV−vis) spectra were recorded using a Thermo Scientific Evolution 600 UV−vis spectrophotometer. Spectra were acquired using solutions having concentrations of 75 and 0.19 μg/mL over the wavelength range of 230−600 nm and corrected for the absorbance of the pure solvent. Steady-state fluorescence spectra were measured for solutions having a concentration of 0.19 μg/mL over the wavelength range of 350−650 nm using a PerkinElmer LS55 luminescence spectrometer using an excitation wavelength of λ = 335 nm. The 1H NMR spectrum was recorded on a Varian Mercury Vx 300 spectrometer in CDCl3 at ambient temperature with tetramethylsilane (TMS) as internal standard. Mass spectra were acquired using a JEOL (Peabody, MA) JMST100LC (AccuTOF) orthogonal time-of-flight (TOF) mass spectrometer equipped with an IonSense (Danvers, MA) Direct Analysis in Real Time (DART) source. Calibration was performed using DART with a mixed solution of 5 μL/mL PEG 200 and 10 μL/mL PEG 600 in a mixed solvent of methanol and methylene chloride (1:1 v/v) using [M + H+] ion series. The mass acquisition range was m/z = 50−600. Samples were dissolved in acetonitrile at a concentration of 1.0 mM for the measurement. The UV irradiation light source used to trigger photoisomerization was a pulse laser operating at 10 Hz (10 pulses/s; pulse fwhm = 6 ns) and λ = 355 nm with an energy per pulse of 4 mJ. The visible irradiation light source was a high-pressure mercury lamp with an optical fiber, and a band-pass filter (450 nm wavelength) was used.

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RESULTS AND DISCUSSION Synthesis and Self-Assembly of PVP-b-PVDMA BCPs. In a previous report we detailed the utility of reversible addition−fragmentation chain transfer (RAFT) polymerization to synthesize dually reactive BCPs of VDMA and poly(glycidyl methacrylate).57 Building on this previous work, we chainextended a PVP MacroCTA (Mw = 3.2 kg/mol; PDI 1.05) with VDMA and altered the VDMA block length to target different PVDMA:PVP ratios. Two PVP-b-PVDMA BCPs, noted as PVP30-b-PVDMA88 and PVP30-b-PVDMA144 with subscripts identifying the number-average degree-of-polymerization of each block, were synthesized and characterized. The synthetic route is shown in Scheme 1, and the molecular properties are listed in Table 1. When a solvent that is selective for PVP blocks is used, the PVP-b-PVDMA BCPs microphase segregate into well-defined nanostructures. As shown in Scheme 2, PVP-b-PVDMA BCPs were first dissolved in a small amount of THF, which is a good solvent for both blocks, followed by the dropwise addition of 2C

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Macromolecules Table 1. Molecular Properties of PVP MacroCTA and BCPs samplea

Mwb (kg/mol)

mol % PVDMAb

Đb

dn/dcc (mL/g)

PVP macroCTA PVP30-b-PVDMA88 PVP30-b-PVDMA144

3200 15500 23300

75 83

1.05 1.08 1.13

0.167 0.098 0.090

a

Subscripts indicate the degree of polymerization of each block. Determined by size exclusion chromatography−multiangle laser light scattering (SEC-MALS). cDetermined by Astra V software assuming 100% mass recovery. b

propanol, which induces self-assembly because it is selective for PVP. 2-Propanol was chosen because more nucleophilic solvents, such as acidic water, methanol, or ethanol, could lead to unwanted side reactions (ring-opening or nucleophilic addition53) of the repeat units of the PVDMA blocks. The selfassembled structure of PVP-b-PVDMA BCPs in a 2-propanol/ THF (v:v = 19:1) mixture at a concentration of 0.5 mg/mL was characterized by dynamic light scattering (DLS). The apparent hydrodynamic radius (Rh) distributions for PVP30-b-PVDMA88 and PVP30-b-PVDMA144 BCPs are unimodal and broad, as shown in Figure 1a. The Rh values calculated from the Stokes− Einstein equation for self-assembled aggregates of PVP30-bPVDMA88 and PVP30-b-PVDMA144 BCPs are ∼172 and ∼191 nm, respectively. As expected, the Rh for assemblies formed from PVP30-b-PVDMA88 BCPs is smaller than that of the aggregates of PVP30-b-PVDMA144 due to the former BCP having shorter PVDMA blocks and a lower molecular weight. Transmission electron microscopy (TEM) images of the aggregates indicate that self-assembly of PVP30-b-PVDMA88 BCPs leads to structures that are nearly spherical in shape with an average radius of ∼121 nm (Figure 2a). It should be noted that the size measured by DLS is larger than that deduced from the TEM images because translational diffusion (DLS) is sensitive to the swelling of the assembled BCPs and the presence of a solvation layer around the aggregates in solution.63 In combination these results demonstrate that selfassembly of PVP-b-PVDMA BCPs provide access to welldefined aggregates for the fabrication of reactive microgels by chemical or physical cross-linking. Cross-Linked PVP-b-PVDMA BCP Microgels. The PVDMA and PVP segments in the aggregates formed by self-

Figure 1. Distribution of hydrodynamic radii, Rh, of (a) self-assembled aggregates of PVP-b-PVDMA BCPs and (b) DAB- and DIB-crosslinked PVP30-b-PVDMA88 BCP microgels (DABCM and DIBCM) formed in a solvent mixture of 2-propanol/THF (v:v = 19:1) at a concentration of 0.5 mg/mL. The apparent Rh (measured at finite concentration) of the aggregates or microgels are given in each plot.

Figure 2. TEM images of (a) self-assembled aggregates, (b) DABCMs, and (c) DIBCMs made from PVP30-b-PVDMA88 BCPs. The samples were drop-cast from a solvent mixture of 2-propanol/THF (v:v = 19:1). The radii (R) of the spherical aggregates or microgels are indicated in each image.

Scheme 2. Fabrication of DAB- or DIB-Cross-Linked Amine-Reactive Microgels from Self-Assembled Aggregates of PVP-bPVDMA BCPsa

a

DIBCMs disassemble in a nonselective good solvent, but DABCMs do not dissolve and can be functionalized. Color is used to distinguish the different blocks and cross-linking agents as indicated in the drawing, with reactive azlactone groups schematically represented by light green spheres along the PVDMA blocks. D

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Macromolecules assembly of PVP-b-PVDMA BCPs can be selectively crosslinked by 1,4-diaminobutane (DAB) and 1,4-diiodobutane (DIB), respectively, which enables the formation of robust microgels. With the former, nucleophilic addition of the alkyl amine DAB to the pendant azlactone rings of the PVDMA blocks results in stable, covalent bonds.51 These DAB-crosslinked microgels are referred to here as DABCMs (see Scheme 2). The cross-linking reaction was monitored using attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, as seen in Figure 3. To support the contention of

Figure 4. XPS spectra of the nitrogen 1s region for PVP30-b-PVDMA88 BCPs, including non-cross-linked BCPs deposited on a silicon substrate by drop-casting from a 2-propanol/THF (v:v = 19:1) solvent mixture (trace a, black), and DIBCMs on silicon substrates drop-cast from a 2-propanol/THF (v:v = 19:1) solvent mixture (trace b, red) and from a THF solution (trace c, blue). The arrow indicates the peak assigned to quaternized pyridine.

suggests that the level of cross-linking is low. The quaternization persists even if the solvent is changed to THF (Figure 4, curve c), which is consistent with a previous report by Hayward et al.64 Results from DLS measurements indicate that both DABCMs and DIBCMs prepared from PVP30-bPVDMA88 have similar distributions of hydrodynamic size when compared to their non-cross-linked analogues (Figure 1b), and TEM images show that the particles retain their spherical shape upon cross-linking (Figures 2b and 2c). Distributions of hydrodynamic sizes of DABCMs and DIBCMs are broad and indicate that these microgels are composed of aggregates of different sizes, which is also observable in Figures 2b and 2c. In view of these results, it can be concluded that cross-linking reactions used to form the microgels does not affect their structure. This is consistent with results of van Oers et al., who showed that vesicles formed by self-assembly of poly(ethylene glycol)-b-poly(styrene-co-4-vinylbenzyl azide) diblock copolymers retain their shape when low cross-link densities are targeted.67 The stability of the self-assembled aggregates of non-crosslinked and cross-linked PVP-b-PVDMA BCPs upon absorption was investigated by immersing freshly cleaned silicon wafers into BCP solutions (2-propanol/THF, v:v = 19:1, as the solvent), allowing the aggregates to absorb on the substrate, and then imaging the resultant surface topography by atomic force microscopy (AFM). The self-assembled structure of PVPb-PVDMA BCPs before cross-linking also were examined, and as shown in Figure 5a, cylindrical microdomains oriented parallel to the substrate (described as wormlike structures) were observed. Given that TEM images indicate spherical aggregates, the difference in morphology is most likely caused by merging of spherical aggregates during the casting process and interactions of the BCP with the hydrophilic surface. We reported similar structural changes for the self-assembly of polystyrene-b-PVP BCP mixtures in a previous study.61 On the other hand, cross-linking of the self-assembled BCPs in solution can partly prevent reorganization of the nanoaggregates on the surface. For example, the topological image of films of DABCMs shows spherical nanostructures with a horizontal radius of ∼25 nm (Figure 5b), which is smaller than the sizes determined from DLS and TEM. These structures may correspond to the microdomains formed by DAB-cross-linking PVDMA blocks within the core of the DABCMs. The

Figure 3. Partial ATR-FTIR spectra obtained for (a) the PVP30-bPVDMA88 BCP, (b) the DABCM produced by reacting the PVP30-bPVDMA88 aggregate with DAB, and (c) the dansylcadaverine-modified DABCM of PVP30-b-PVDMA88. Modes associated with characteristic groups that change upon modification are labeled.

cross-linking of the self-asssembled BCP aggregates with DAB to create the DABCMs, the ratio of the peak areas assigned to the carbonyl of the azlactone ring at 1823 cm−1 (A1 = C Oazlactone) to the peak assigned to the imine stretch (which is transformed to an amide stretch due to nucleophlic addition) at 1672 cm−1 (A2 = C−Nazlactone) was calculated by fitting Gaussian curves to these peaks. The area ratio for PVP30-bPVDMA88 BCP aggregates (black curve in Figure 3) before cross-linking was A1/A2 = 1.8, and the ratio after cross-linking to produce the DABCM (red curve in Figure 3) was A1/A2 = 1.2. The relative changes implied by this decrease in A1/A2 is consistent with nucleophlic addition of amines to the azlactone rings, which cross-links the BCP aggregates (depicted in Scheme 2). In addition, Figure 3 shows that a peak at 1823 cm−1 remains present after cross-linking, indicating that azlactone groups are not completely consumed and therefore available for additional functionalization. Both Saito and co-workers and Kramer and co-workers have shown that DIB can be used to cross-link PVP blocks via the interactions between pyridine rings and alkyl iodides.38,64,65 These DIB-cross-linked microgels, referred to here as DIBCMs, are cross-linked through the corona blocks, as shown in Scheme 2. The cross-linking reaction to form DIBCMs results in quaternization of pyridine rings, which is confirmed by X-ray photoelectron spectroscopy (XPS) measurements. The XPS spectra for PVP30-b-PVDMA88 BCPs before and after crosslinking by DIB are presented in Figure 4. In the case of noncross-linked sample, a single peak is observed at a binding energy of 399.1 eV (Figure 4, curve a), which corresponds to the N 1s electrons from nitrogen heteroatoms of VDMA and 2vinylpyridine.65,66 Following cross-linking of the BCP nanoparticles using DIB, a second peak corresponding to quaternized pyridine is seen at a binding energy of 401.9 eV (Figure 4, curve b), indicating successful cross-linking of the BCP micelles by DIB. The intensity of the peak at 401.9 eV E

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Macromolecules

Figure 5. AFM height images of self-assembled structures created from PVP30-b-PVDMA88 BCPs that are absorbed on silicon substrates. Wormlike structures are observed for (a) non-cross-linked, self-assembled BCPs, but the (b) DABCMs, which are core-cross-linked, retain their spherical shape. Image (c) shows that films of the corona-cross-linked DIBCMs contain both spherical aggregates and wormlike structures.

topographical imaging of films composed of DIBCMs cast on silicon substrates indicates both wormlike structures and spherical aggregates with a horizontal radius of ∼30 nm (Figure 5c). We believe this mixed structure is consistent with less cross-linking by DIB of the PVP corona, which enables some morphological remodeling during absorption and/or drying. Additionally, the phase images for the various systems (shown as Figure S1) indicate that the aggregates formed from the PVP-b-PVDMA BCP robustly absorb and uniformly cover the silicon surface with no significant differences in material hardness. DLS was used to examine the stability of PVP-b-PVDMA DABCMs and DIBCMs in THF, which is a good solvent for both blocks. Self-assembled BCPs, DABCMs, and DIBCMs in 2-propanol/THF (v:v = 19:1) mixed solvents with an initial polymer concentration of 0.5 mg/mL were diluted with THF to a final polymer concentration of 75 μg/mL. The normalized light intensity autocorrelation function for each of these solutions is shown in Figure 6a, and the absence of a wellformed autocorrelation function for either the self-assembled BCPs or the DIBCMs indicates that these two systems disassemble in THF. As a result, they are not suitable candidates for guest loading. The disassembly of DIBCMs occurs in THF presumably because of the low level of crosslinking, which is demonstrated in the XPS spectra. In essence, either the short DIB chains are unable to bridge adjacent PVP chains that are swollen in solvent (Scheme 2) or the degree of cross-linking is simply too low. In comparison, PVDMA reacts robustly with amines, and because PVDMA is in the core of the self-assembled BCP micelles, it is easier to cross-link the aggregates with DAB than with DIB. Therefore, the DABCMs can maintain their three-dimensional structure when challenged with the good solvent THF. The Rh distribution for DABCMs in THF presented in Figure 6b shows a single, broad distribution with an Rh of 342 nm, which is much larger than their size in the 2-propanol/THF (v:v = 19:1) solvent mixture (Figure 1). PVP30-b-PVDMA88 DABCMs drop-cast from THF solution were imaged by TEM. As shown in the inset of Figure 6b, these microgels are nearly spherical in shape with an average radius of 195 ± 53 nm, confirming the swelling of these microgels in THF. Because of their stability and ability to swell/ deswell by changing solvent quality, these DABCMs are promising candidates for functional molecule immobilization and their ability to carry guest molecules. Functionalization of PVP-b-PVDMA DABCMs. Because of their excellent stability in THF at low concentration, the incorporation of functional amines into PVP-b-PVDMA

Figure 6. (a) Normalized light intensity autocorrelation functions for PVP30-b-PVDMA88 BCP self-assembled aggregates (black) and microgels indicate that DAB-cross-linked DABCMs (red) are stable upon dilution with THF, but DIB-cross-linked aggregates (DIBCMs) (blue) and un-cross-linked self-assembled BCPs are not. (b) Rh distribution for PVP 30 -b-PVDMA 88 DABCMs in THF at a concentration of 75 μg/mL indicates that DABCMs swell significantly. The inset TEM micrograph shows that PVP30-b-PVDMA88 DABCMs drop-cast from a THF are spherical in shape. The radii R of the DABCMs determined by TEM and DLS are inset in their respective images.

DABCMs was investigated. Dansylcadaverine (DC) (chemical structure inset in Figure 7) was chosen as a model compound because it has a single primary amine to enable nucleophilic addition to VDMA repeat units, and it also is fluorescent. The conjugation of DC with PVP-b-PVDMA BCP DABCM was monitored by ultraviolet−visible (UV−vis) absorption spectroscopy. Figure 7 (curve a) shows the broad absorption peak at ∼260 nm for the PVP30-b-PVDMA88 DABCM, which is attributed to overlapping π → π* and n → π* transitions of pyridine rings of the PVP segments.68 After dialysis against THF to remove unbound DC from the system, a new absorption peak at 335 nm is observed (curve b in Figure 7), indicating the successful modification of DABCMs with DC. F

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ments performed on DC-functionalized DABCMs made from PVP30-b-PVDMA88 show an Rh = 243 nm in THF, which is smaller than the size of unloaded DABCMs in THF. The decrease in Rh upon DC conjugation is likely the result of a decrease in solubility after addition of DC, which is possibly augmented by π−π stacking of naphthalenic subunits of DC, which makes the microgels more compact. In addition, the functionalization ratios of DC-modified PVP30-b-PVDMA144 unimers and DABCMs were also calculated, and the results are given in Table 2. The functionalization ratio of DC for PVP30-b-PVDMA144 unimers is 41.4%, which is similar to the value determined for PVP30-b-PVDMA88 unimers. However, the functionalization ratio of DC for PVP30-bPVDMA144 DABCMs calculated from the UV−vis absorption difference spectrum (shown as Figure S4) is higher than that of PVP30-b-PVDMA144 unimers or the smaller microgels because the UV−vis absorption spectra was significantly affected by the scattering from the turbid solution of DC-loaded PVP30-bPVDMA144 DABCMs. This is likely a direct result of the large particle size (Rh = 323 nm, determined by DLS). Therefore, the functionalization ratio (61.8%) of DC for reactive VDMA groups in the PVP30-b-PVDMA144 DABCM calculated from the UV−vis spectra is likely an overestimation. The modification of PVP-b-PVDMA DABCMs with DC is further confirmed by the ATR-FTIR. The FTIR spectrum of the DC-modified DABCM presented in Figure 3 (spectrum c) shows clearly shows a peak at 1643 cm−1 corresponding to amides resulting from ring-opening nucleophilic addition of primary amine to the azlactone rings of PVDMA. In addition, peaks at 1307 and 1143 cm−1 for sulfonamide and at 1452 and 790 cm−1 for the naphthalenic subunit are observed, which confirm successful attachment of DC to PVDMA blocks. In addition, a signal at 1823 cm−1 from the carbonyl of the azlactone ring remains after functionalization, which indicates that not all of the azlactone rings of the PVDMA blocks are reactively modified with DC. The presence of unreacted azlactone rings is also in qualitative agreement with functionalization ratios determined by UV−vis spectroscopy. The DC-conjugated PVP-b-PVDMA DABCMs are fluorescent due to the addition of DC to the azlactone rings. The photograph inset in Figure 8 shows the fluorescence of DCconjugated PVP30-b-PVDMA88 DABCMs in THF. The DCmodified DABCMs are bright cyan in color while the non DC-

Figure 7. UV−vis spectra of PVP30-b-PVDMA88 DABCMs at c = 75 μg/mL in THF (a) before and (b) after functionalization of DC. The curve labeled as (c) is the difference spectrum (of spectra a and b). The inset shows the chemical structure of DC.

The absorption due to the conjugation of DC in the microgels is determined from the difference between curves a and b in Figure 7, and this difference spectrum is represented by curve c. The functionalization ratio of DC in the microgels is determined by comparing the absorbance (at 335 nm) of the microgel to a calibration curve generated for DC in THF (Figure S2), and the results are presented in Table 2. The Table 2. Functionalization Ratio of PVP-b-PVDMA BCPs and DABCMs block copolymer PVP30-bPVDMA88 PVP30-bPVDMA88 PVP30-bPVDMA144 PVP30-bPVDMA144

aggregation state in THF

functionalization ratioa (%)

unimers

42.1

DABCMs

38.9

unimers

41.4

DABCMs

61.8

Rhb (nm)

243

323

a Calculated from UV−vis absorbance at 335 nm assuming 10 mol % of VDMA repeat units are consumed due to cross-linking of the aggregate with DAB. bObtained from DLS in THF at c = 75 μg/mL.

functionalization ratio for free VDMA groups in the microgels is found to be 38.9%. This is based on the assumption that 10% VDMA groups are consumed in the initial cross-linking reaction, which is based on the molar feed ratio of DAB to VDMA repeat units. As a control, a solution of non-cross-linked PVP30-b-PVDMA88 in THF (unimers) also were functionalized with DC followed by the removal of excess DC molecules by dialysis, and the UV−vis spectrum acquired after DC functionalization is shown in Figure S3. Table 2 shows that the functionalization ratio of DC for these PVP30-b-PVDMA88 unimers is 42.1%, which is similar to the value estimated for reactive modification VDMA repeat units in PVP30-b-PVDMA88 DABCMs. These results suggest that the azlactone groups are not exhaustively modified, either during the initial cross-linking with DAB or from the subsequent functionalization with DC. This result is consistent with recent studies by Aden et al., who demonstrated that PVDMA brushes were not completely functionalized when modified with a series of small molecule amines.54 While PVDMA is not completely modified at room temperature by small molecule amines, ostensibly due to steric hindrance, Lynn and co-workers demonstrated that exhaustive functionalization of PVDMA by primary amines could be realized at elevated reaction temperatures.69 DLS measure-

Figure 8. Fluorescence emission spectra of PVP30-b-PVDMA88 DABCMs (a) in THF at a concentration of 0.19 μg/mL and (b) in solution after functionalization with DC (the excitation wavelength is 335 nm). Inset photograph shows fluorescent behavior of PVP30-bPVDMA88 DABCMs in THF at a concentration of 75 μg/mL (left) and the solution of DC-functionalized DABCMs (right) when excited using 365 nm UV light. G

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Figure 9. (a) UV−vis spectra of PVP30-b-PVDMA88 DABCMs (curve a) in THF at a concentration of 0.19 μg/mL, (curve b) after functionalization with NAzo, and (curve c) the NAzo-grafted microgels after exposure to UV light at λ = 355 nm for 3 min. Inset shows the reversible photoisomerization of azobenzene derivatives, with colors corresponding to the relevant spectra. (b) Measured absorbance at a wavelength of 335 nm (black) and hydrodynamic radius (blue) of NAzo-functionalized PVP30-b-PVDMA88 DABCMs in THF after exposure to UV light at λ = 355 nm (odd cycle numbers) and after exposure to visible light at λ = 450 nm (even cycle numbers) in a repeated sequence.

created by the microgel is reversible by simply alternating the exposure (Figure 9b). In addition, the size of NAzofunctionalized, DAB-cross-linked microgels is generally constant during the trans-to-cis isomerization process (Figure 9b). We also found that 1-pyrenemethylamine could be conjugated to the PVP-b-PVDMA DABCMs (Figure S10), which suggests facile reactivity of the microgels with primary alkylamines.

conjugated microgel solution is nonfluorescent (left vial pictured in inset). The fluorescence emission spectrum of unfunctionalized PVP-b-PVDMA DABCMs presented in Figure 8 shows three small peaks located at 371, 407, and 431 nm (curve a). Except for the peak at 371 nm that is present in both the DC-functionalized and unfunctionalized DABCMs, the DC-conjugated DABCMs have a broad, featureless emission peak centered at 485 nm that swamps the peaks of DABCMs at 407 and 431 nm in the fluorescence spectrum (curve b). (A similar behavior is seen for DC-conjugated PVP30-b-PVDMA144 DABCMs, as shown in Figure S5.) This confirms the fluorescent behavior is derived from conjugation of DC in the PVP-b-PVDMA DABCMs. Two other functional amines, 1-aminopyrene and 4-aminoazobenzene, were used to expand the functionality of the PVPb-PVDMA DABCMs. However, neither of these compounds reacted with azlactone groups of the PVDMA-containing DABCMs at room temperature, even when the organic base, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),3,44 was used. While this is likely due to the relatively weaker nucleophilicity of aromatic amines compared to alkyl amines, the results suggest that PVP-b-PVDMA microgels offer selectivity toward incorporation of primary alkyl amines rather than aromatic amines. To test this hypothesis, we synthesized N-(4-aminobutyl)azobenzenecarboxyamide (NAzo), which is analogous to 4aminoazobenzene but contains a primary alkylamine. The synthetic scheme for NAzo involving acylation of phenylazobenzoyl chloride with DAB is shown in Figure S6, and the product was confirmed by 1H NMR and ATR-FTIR spectroscopies and by mass spectrometry (see Figures S7−S9). The conjugation of NAzo within PVP-b-PVDMA DABCMs and the photoisomerization of the NAzo-conjugated DABCMs in THF solution were monitored by UV−vis absorption spectroscopy. A strong absorbance peak centered at ∼340 nm is observed (c = 0.19 μg/mL) for the NAzo-conjugated microgels (Figure 9a, curve b), indicating successful grafting of trans-NAzo within the PVP-b-PVDMA DABCMs. To trigger the reversible trans-to-cis photoisomerization of the azobenzene derivative,70−72 the solution was irradiated at λ = 355 nm for 3 min. A UV−vis spectrum acquired immediately after this exposure (Figure 9a, curve c) shows that the absorbance peak at ∼340 nm is strongly diminished, and a peak at ∼450 nm appears. These changes in absorbance were tracked over multiple cycles, as shown in Figure 9b. The results indicate the photoinduced trans-to-cis isomerization of the azobenzene derivative in the confinement

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CONCLUSIONS A facile method involving self-assembly and cross-linking of azlactone-containing BCPs is established as a useful route to fabricate microgels containing reactive “handles” that can be selectively modified to incorporate additional functionality. In this approach, the surfactant-like nature of PVP-b-PVDMA BCPs enables the formation of nanoscale aggregates in solvents selective for PVP blocks, and the size of the self-assembled structure can be manipulated by tailoring block lengths of the BCPs. Moreover, the reactive azlactone rings in the selfassembled PVP-b-PVDMA BCP soft nanoparticles offer binding sites for covalent cross-linking to fabricate stable PVP-b-PVDMA microgels, which can be subsequently functionalized with guest molecules. These advantages derive from the inherent polymerizability of VDMA and efficacy of reactive modification without formation of byproducts, which make azlactone-containing BCPs useful as building blocks for functional self-assembled systems or soft scaffolds, which may make them applicable as nanoreactors or in sensing applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.8b00405. Experimental details of DLS measurements, AFM phase images, UV−vis calibration curve of DC in THF, UV−vis spectra and fluorescence spectrum of copolymers and DC-conjugated microgels, synthesis scheme and molecular characterization of NAzo, including 1H NMR and ATR-FTIR spectroscopies and mass spectrometry (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (S.M.K.). *E-mail: [email protected] (X.W.). H

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Xu Wang: 0000-0002-6646-0515 Bethany M. Aden: 0000-0003-1115-3830 Bradley S. Lokitz: 0000-0002-1229-6078 S. Michael Kilbey II: 0000-0002-9431-1138 Author Contributions

X.W. and J.L.D. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported in part by the National Science Foundation (Award Nos. 1131252 and 1512221) (S.M.K.II, B.A., J.D.), the Program of Qilu Young Scholars of Shandong University (X.W.), and National Natural Science Foundation of China (NSFC grant 21704057) (X.W.). A portion of this research was conducted at the Center for Nanophase Materials Sciences, which is a DOE Office of Science User Facility. Prof. Robert N. Compton, Dr. Mohammadreza Rezaee, and Dr. Weikun Li are gratefully acknowledged for assistance with photoisomerization studies, and Dr. Zhenqian Zhu is thanked for assistance with mass spectroscopy measurements. Prof. Brian Long is thanked for the helpful discussions, as are Prof. Wenshou Wang and Zongpeng Gao for their assistance with XPS studies.

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DOI: 10.1021/acs.macromol.8b00405 Macromolecules XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.macromol.8b00405 Macromolecules XXXX, XXX, XXX−XXX