Functionalized Stimuli-Responsive Nanocages from Photocleavable

Article pubs.acs.org/Macromolecules

Functionalized Stimuli-Responsive Nanocages from Photocleavable Block Copolymers Olivier Bertrand, Elio Poggi, Jean-François Gohy,* and Charles-André Fustin* Institute of Condensed Matter and Nanosciences (IMCN), Bio- and Soft matter (BSMA) division, Universite catholique de Louvain, Place L. Pasteur 1, Louvain-la-Neuve, Belgium S Supporting Information *

ABSTRACT: The light-induced formation of pH and temperature-responsive poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) nanocages is demonstrated here. The strategy is based on the self-assembly in aqueous solutions of a photocleavable poly(tert-butyl acrylate)-hv-poly[2(dimethylamino)ethyl methacrylate] block copolymer (PtBAhv-PDMAEMA, where -hv- is the photocleavable junction) into spherical micelles, followed by first PDMAEMA crosslinking by a bis-iodo compound and then by UV light irradiation. The exposure to light induces the cleavage of the junction between the PtBA core and the cross-linked PDMAEMA shell of the micelles. The PtBA block is then extracted to obtain the desired nanocages. The size change of the nanocages in response to pH and temperature is investigated by dynamic light scattering. Finally, the ability to functionalize the internal cavity of the nanocages is demonstrated.

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INTRODUCTION During the past decade, the formation of hollow nanocapsules (or nanocages) has attracted extensive attentions.1−3 Indeed, such type of structure with an empty core domain allows the encapsulation of a large amount or guest molecules which is valuable for drug delivery systems4,5 and nanoreactors.6,7 Moreover, hollow nanocapsules open the door to compartmentalization and nature mimics (e.g., cells and viruses).6,8 With such type of applications in mind, the development of methodologies to produce well-defined, functionalized and responsive, hollow nanocapsules is highly desired. Over the years, hollow polymeric nanocapsules were produced via several methodologies, which can be divided into two categories. The first one involves the direct polymerization of monomers, either in a scaffold9−11 or onto the surface of a sacrificial nanoparticle.12,13 The second category relies on the assembly of homopolymers or block copolymers. In this category, the layer-by-layer technique is one of the most attractive technique for the preparation of hollow nanocapsules because the process is environmentally friendly, inexpensive and versatile.3,14,15 However, the functionality of the internal walls of the nanocage is difficult to control by this technique. The self-assembly of block copolymers has been also used for the production of nanocages since the size of the nanocage membrane and cavity can be easily tuned by the copolymer composition, molecular weight and self-assembly conditions or even by external stimuli.16 Methodologies involving the self-assembly of copolymers into core−shell structures combined with the migration of the core forming chains into the corona were recently developed.17,18 Other © 2013 American Chemical Society

methodologies involve the self-assembly of copolymer precursors into a core−shell structure combined with the extraction19−22 or the degradation4,23−25 of the core forming polymer. Although this last method allows the production of responsive nanocages, the control of the nanocage internal wall functionality is not always trivial.20 To solve this issue and obtain nanocages with well-defined chemical functionalities, O’Reilly and co-workers developed a methodology based on supramolecular copolymers.20−22 They used polystyrene-blockpoly(acrylic acid) supramolecular copolymers (PS-b-PAA), in which the junction between the two blocks was a Ruthenium or Palladium containing metal−ligand complex. After the selfassembly of those supramolecular copolymers into micelles and the chemical cross-linking of the shell, the metal−ligand complexes were cleaved and the PS chains were extracted to form the nanocages. They thus obtained pH responsive PAA nanocages with the internal cavity decorated by ligands. Although, this methodology allows the production of functionalized and responsive nanocages, the further functionalization of the nanocages may be an issue since it requires the reformation of new metal−ligand complexes and is limited to molecules bearing a complementary ligand. In this contribution, we report on the formation of functionalized stimuli-responsive nanocages via the selfassembly into micelles, the shell cross-linking and the Received: November 7, 2013 Revised: December 18, 2013 Published: December 31, 2013 183

dx.doi.org/10.1021/ma402301w | Macromolecules 2014, 47, 183−190

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Figure 1. Schematic representation of the strategy toward nanocage formation. 2NB-BiB) was synthesized under the conditions described in ref 32. Sodium azide (NaN3, Acros, 99%), CuBr (Aldrich, 99.999%), CuCl (Aldrich, 99.999%), ethyl 2-bromoisobutyrate (EBiB, Acros, 98%), anisole (Acros, 99%), N,N,N′,N″,N″-pentamethyldiethylenetriamine (PMDETA, Aldrich, 98%), bis(2-iodoethoxy)ethane (BIEE, Aldrich, 96%), N,N′-dicyclohexylcarbodiimide (DCC, Acros, 99%), 1-aminopyrene (Aldrich, 97%), and all the other chemicals were used as received. Instrumentation. Proton and carbon nuclear magnetic resonance (NMR) spectra were acquired on a 500 MHz Bruker Avance II. Molar masses (Mn) and polydispersity indices (PDI) of the polymers were measured on an Agilent gel permeation chromatography (GPC) system equipped with an Agilent 1100/1200 pump (35 °C; eluent: DMF; flow rate of 1 mL/min), an Agilent differential refractometer and two PSS GRAM columns (beads 10 μm; porosity of column 1, 1000 Å; porosity of column 2, 100 Å). The calibration was performed using polystyrene standards. Irradiation was performed with 3 Rayonet photochemical reactor lamps (maximum emission wavelength at λ = 300 nm). The intensity received by the sample was equal to 41.6 mW/ cm2 and was measured with a radiometer RM12 from Dr Gröbel UVelectronik GmbH equipped with a UVB (280−315 nm) sensor. UV− vis spectra were recorded on a Varian Cary 50 Conc. spectrophotometer. Dynamic light scattering (DLS) measurements were performed at an angle of 90° on a Malvern CGS-3 apparatus equipped with a He−Ne laser with a wavelength of emission at λ = 632.8 nm. Atomic force microscopy was performed on a Digital Instruments Nanoscope V scanning force microscope in tapping mode using NCL cantilevers (Si, 48 N/m, 330kHz, Nanosenors). Fluorescence spectra were recorded on a Varian Cary Eclipse spectrophotometer. General Procedure for the Synthesis of PtBA−Br. Under argon, a round-bottom flask, with a stopcock, containing CuBr (26.1 mg; 0.182 mmol; 1 equiv) was filled with a solution containing tertbutyl acrylate (tBA; 7.14 mL; 36.4 mmol; 190 equiv), EBiB (35.5 mg; 0.182 mmol, 1 equiv), PMDETA (34.7 mg; 0.200 mmol; 1.1 equiv), and anisole (4.22 mL; 40 wt %) previously degassed by three freeze− pump−thaw cycles. The mixture was degassed by three freeze− pump−thaw cycles, filled with argon and stirred in an oil bath at 80 °C for 8h (tBA conversion = 40%). The polymerization was quenched by quickly cooling the tube in a water−ice bath and exposing the reaction mixture to air. The reaction mixture was diluted with CH2Cl2 and washed with an aqueous solution of EDTA (0.04 M). The organic phase was dried over MgSO4, filtered and the solvent was removed

photocleavage of a poly(tert-butyl acrylate)-hv-poly[2(dimethylamino)ethyl methacrylate] photocleavable copolymer (PtBA-hv-PDMAEMA), where -hv- is the photocleavable junction. This approach presents the advantage of producing well-defined carboxylic acid groups, a versatile moiety easily transformed into many other functions of interest, on the nanocage inner walls after photocleavage of the copolymer. Indeed, the copolymer junction is composed of an onitrobenzyl ester function, which splits into two parts (a carboxylic acid and a nitrosobenzaldehyde) under irradiation.26−31 The photocleavable junction is introduced between the PtBA and the PDMAEMA blocks via a copper(I) catalyzed azide−alkyne cycloaddition−atom transfer radical polymerization (CuAAC−ATRP) simultaneous one-pot process developed in our group.32 The formation of the PDMAEMA nanocages is realized in four steps (Figure 1). In the first step, the photocleavable PtBA-hv-PDMAEMA copolymer is selfassembled in water to obtain micelles with a PtBA core, a PDMAEMA shell and the photocleavable junction localized at the core−shell interface. The chemical cross-linking of the micellar shell is realized with bis(2-iodoethoxy)ethane in the second step. In the third step, the irradiation of the micellar solution is performed leading to the cleavage of the junction to obtain PDMAEMA nanocages loaded with PtBA chains. The last step is the removal of the PtBA chains in order to empty the cavity of the PDMAEMA nanocages. The stimuliresponsive behavior of the PDMAEMA nanocages is then studied by dynamic light scattering (DLS). Finally, the ability to functionalize the inner wall of the nanocages is demonstrated by the grafting of 1-aminopyrene onto the carboxylic acid groups, which are formed after photocleavage.

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EXPERIMENTAL SECTION

Materials. tert-Butyl acrylate (tBA, Acros, 99%), 2-(dimethylamino) ethyl methacrylate (DMAEMA, Aldrich, 98%) were passed through activated basic alumina (Acros) columns prior use. N,Ndimethylformamide (DMF, Aldrich, 99.8%) was distilled on CaH2 prior use. 5-Propargyl ether-2-nitrobenzyl bromoisobutyrate (5Pr184


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Scheme 1. Strategy for the Synthesis of PtBA-hv-PDMAEMA Copolymer

PDMAEMA, Et−N−(CH3)2), 2.10−1.45 (b, 636H; PDMAEMA backbone, CH2; PtBA backbone, CH2 + CH), 1.40 (s, 1062H; PtBA-tert-butyl, CH3), 1.20−0.8 (b, 423H; PDMAEMA backbone, CH3). Mn(NMR) = 40 100 g/mol. General Procedure for the Micellization of PtBA-hvPDMAEMA. PtBA118-hv-PDMAEMA141 was dissolved in DMF at a concentration of 12.5 g/L. A volume of water equal to 3 times the DMF volume was then added dropwise to induce aggregation of the insoluble PtBA blocks, followed by the addition of a volume of water equal to the DMF volume in one shot to “freeze” the micelles (Cfinal = 2.5 g/L). Subsequently, the DMF/water solution was dialyzed against water (pH = 6) for 24 h replacing the water at least three times (Spectra-Por dialysis bags, cutoff 14−16 kDa). The final concentration of the copolymer in water (pH = 6) was set to 2.5 g/L. General Procedure for the Cross-Linking of the Corona of the PtBA-hv-PDMAEMA Micelles. To a stirred solution of micelles (12.0 mL, 30.0 mg, 7.90 × 10−4 mmol (PtBA118-hv-PDMAEMA141), 0.11 mmol (amine residues)) in water (pH = 9) was added a solution of bis(2-iodoethoxy)ethane (20.7 μL, C = 100 mg/mL 5.60 × 10−3 mmol, 0.05 equiv of amine residues) in methanol. The resultant solution was allowed to stir for 16 h at 40 °C. Removal of low molar mass contaminants was achieved through dialysis through Spectra-Por dialysis bags (cutoff 14−16 kDa) against methanol. General Procedure for the Photocleavage (UV−Vis). A solution of PtBA118-hv-PDMAEMA141 micelles in water (C = 2.5 g/ L) was irradiated at 300 nm in a quartz cell. The photocleavage was followed by UV−vis spectroscopy. General Procedure for the PtBA Extraction. The aqueous solution of PDMAEMA nanocages containing the cleaved PtBA chains was first basified with NaOH to obtain uncharged PDMAEMA. Water was removed under vacuum and the yellow powder was redispersed in methanol. The solution was allowed to stir at 20 °C for 24 h; methanol was afterward removed under vacuum. Basic water (pH = 9) was added to the crude mixture of PDMAEMA nanocages and PtBA, the solution was stirred at 20 °C for 24 h. The stirring was stopped to let PtBA sediment. The separation of the nanocages from the precipitated PtBA was realized by filtration using syringe filters (1.2 μm) and water was removed under vacuum. The extraction cycle was afterward restarted. The extraction was monitored with 1H NMR. General Procedure for the Grafting of 1-Aminopyrene on the PDMAEMA Nanocages. To a solution of PDMAEMA nanocages (1.00 mL, C = 1.48 g/L, 6.5 × 10−5 mmol (carboxylic acid residues)) in dichloromethane (DCM), a solution of 1aminopyrene (28.2 μL, C = 5 g/L, 6.5 × 10−4 mmol, 10 equiv

under reduced pressure. The residue was precipitated in MeOH:H2O (50:50), filtered and dried in vacuo at 35 °C for 24 h, affording a white solid (2.2 g, 73%). PtBA118−Br: Mn(GPC) = 14 200 g/mol; PDI(GPC) = 1.12. 1H NMR (500 MHz, CDCl3): δ (ppm) 4.1 (m, 3H, CH−Br + CH2−OCO), 2.3−2.0 (b, 117H, PtBA backbone: CH), 1.9−1.3 (s, 1298H; PtBA backbone, CH2; tert-butyl, CH3). Mn(NMR) = 15 200 g/mol. General Procedure for the Synthesis of PtBA−N3. To a solution of PtBA118−Br (2.00 g, 0.131 mmol, 1 equiv) in dry DMF (6.53 mL, 0.02 M) was added sodium azide (170 mg, 2.61 mmol, 20 equiv). The solution was stirred under argon at 25 °C for 24 h. Distilled water (50 mL) was added to the mixture inducing the precipitation of the polymer, which was extracted with CH2Cl2. The organic phases were combined, dried over Na2SO4, filtered and the solvent was removed under reduced pressure. The residue was precipitated in water, filtered and dried one night in vacuo at 35 °C, affording a white powder (1.62, 81%). PtBA118−N3: Mn(GPC) = 14 200 g/mol; PDI(GPC) = 1.22. 1H NMR (500 MHz, CDCl3): δ (ppm) 4.1 (m, 2H, CH2−OCO), 2.3−2.0 (b, 118H; PtBA backbone, CH), 1.9−1.3 (s, 1298H, PDMAEMA backbone, CH2; tert-butyl, CH3). General Procedure for the Synthesis of PtBA-hv-PDMAEMA. Under argon, a Schlenk tube containing CuCl (3.80 mg; 0.038 mmol; 1 equiv) and PMDETA (13.3 mg; 0.077 mmol; 2 equiv) was filled with previously degassed 1,4-dioxane (300 μL). The mixture was degassed by one freeze−pump−thaw cycle and allowed to stir for 30 min. To the Schlenk tube, a solution containing 2-(dimethylamino)ethyl methacrylate (DMAEMA; 1.49 g; 9.51 mmol; 250 equiv), 5Pr2NB-BiB (137 μL (C = 100 g/L); 0.038 mmol; 1 equiv), PtBA118−N3 (586 mg; 0.038 mmol, 1 equiv), and 1,4-dioxane (1.87 mL; 60 wt %) previously degassed by three freeze−pump−thaw cycles. The mixture was degassed by three freeze−pump−thaw cycles, filled with argon and stirred in an oil bath at 70 °C for 1 h 30 (DMAEMA conversion = 54%). The polymerization was quenched by quickly cooling the tube in a water−ice bath. The reaction mixture was filtered on neutral Al2O3 (eluent: CH2Cl2). The solvent was removed under reduced pressure. The residue was precipitated twice in hexane, filtered and dried in vacuo at 35 °C for 24 h, affording a white solid (0.798 g, 60%). PtBA118-hv-PDMAEMA141: Mn(GPC) = 26 500 g/mol; PDI(GPC) = 1.1; ec = >99%. 1H NMR (500 MHz, CDCl3): δ (ppm) 8.13 (d, 1H, junction: HAr in ortho of the NO2), 7.82 (s, 1H; junction, CH triazole), 7.08 (s, 1H, junction, HAr), 7.03 (m, 1H; junction, HAr), 5.44 (m, 2H; junction, CH2−OCO), 5.23 (b, 3H; junction, O−CH2− triazole; PtBA, CH−triazole), 4.03 (b, 242H; PDMAEMA, COO− CH2), 2.54 (b, 242H; PDMAEMA, CH2−N−(Me)2) 2.25 (b, 846H; 185


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(carboxylic acid residues)) in DCM was added. The solution was allowed to stir for 1h at 25 °C. A solution of N,N′-dicyclohexylcarbodiimide (5.4 μL, C = 5 g/L, 1.3 × 10−4 mmol, 2 equiv (carboxylic acid residues)) was then added to the mixture. The solution was stirred at 25 °C for 24 h. The grafted nanocages were purified by dialysis against methanol for 3 days replacing methanol at least three times (Spectra-Por dialysis bags, cutoff 14−16 kDa).

PDMAEMA141 copolymer was realized by the cosolvent method. The copolymer was first dissolved in a nonselective solvent (dimethylformamide, DMF, C = 12.5 g/L), the selective solvent (H2O at pH = 6) was then added dropwise in the solution to induce the micellization by insolubilization of the PtBA block. DMF was finally removed from the solution by dialysis against Milli-Q water. The micelles were then analyzed by dynamic light scattering (DLS) and atomic force microscopy (AFM). Figure 3a displays the CONTIN size distribution

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RESULTS AND DISCUSSION Copolymer Synthesis. The general strategy for the synthesis of the linear photocleavable block copolymer via a CuAAC−ATRP is presented in Scheme 1. The first step is the synthesis by ATRP of a PtBA homopolymer. The chain end of the PtBA is then substituted by an azide group (step 2). Finally, the CuAAC−ATRP is carried out with 5-propargyl ether-2nitrobenzyl bromoisobutyrate (5Pr-2NB-BiB) and 2(dimethylamino)ethyl methacrylate (DMAEMA) to obtain the desired PtBA-hv-PDMAEMA copolymer. The key step of the synthesis is the one-pot CuAAC−ATRP process, in which the PtBA-N3 is coupled to the photocleavable initiator (5Pr-2NB-BiB) on one side by a CuAAC click reaction and the PDMAEMA block is synthesized by ATRP on the other side. This reaction was carried out in 1,4-dioxane (60 wt %) at 70 °C with PtBA118-N3/5Pr-2NB-BiB/CuCl/PMDETA/ DMAEMA molar ratios equal to 1:1:1:2:250. Under these conditions, a well-defined PtBA118-hv-PDMAEMA141 copolymer was obtained with a molar mass of 40100 g/mol, as determined by proton nuclear magnetic resonance (1H NMR), and a polydispersity index (PDI) of 1.13 as determined by gel permeation chromatography (GPC). The molar mass of the block copolymer and the CuAAC coupling efficiency (ec) were determined by 1H NMR by comparing the signals of the aromatic protons of the junction, the CH2−COO protons of the PDMAEMA block and the PtBA protons (Figure S1, Supporting Information). A quantitative coupling of the PtBA block to the photocleavable junction was obtained under these conditions. The quantitative coupling was confirmed by GPC. Indeed, a clear shift toward higher molar masses is observed between the PtBA118 and the PtBA118-hv-GPC traces. Moreover, the copolymer trace is characterized by a symmetrical monomodal distribution (Figure 2). Nanocage Formation. The first step for the formation of nanocages is the self-assembly of the photocleavable block copolymer in water. The micellization of the PtBA118-hv-

Figure 3. Characterization of the PtBA118-hv-PDMAEMA141 micelles in water (pH = 6) by DLS and AFM: (a) CONTIN size distribution diagram and (b) AFM images.

diagram of the obtained micelles in water (pH = 6, C = 0.1 g/ L). A single population (Rh,app = 28 nm, PDI from cumulant analysis: 0.153) is observed in agreement with the formation of rather well-defined spherical micelles, as further confirmed by AFM imaging (Figure 3b). The height of the objects measured by AFM was 12 ± 0.7 nm and the spread diameter was 63 ± 2.3 nm. These results are in good agreement with the DLS results considering that imaging is performed in the dry state, and that the measured height is essentially proportional to the micellar core with a small contribution from the collapsed corona. The second step for the formation of nanocages is the crosslinking of the PDMAEMA micellar shell. The cross-linking was realized following experimental conditions developed by Armes and co-workers using bis(2-iodoethoxy)ethane (BIEE).33,34 This bifunctional reagent selectively quaternizes the tertiary amine groups of the PDMAEMA block via a nucleophilic substitution of the iodine groups by the amine functions of the polymer. The cross-linking degree of the shell was maintained low to facilitate PtBA extraction in the next step.19 The crosslinking was carried out by the addition of a solution of BIEE (0.05 equiv of amine functions, targeted cross-linking degree = 10%) in methanol to a solution of the PtBA 118 -hvPDMAEMA141 micelles in water at pH = 9. The solution was allowed to stir for 24 h at 40 °C. The micelles were purified by

Figure 2. GPC chromatograms of the PtBA precursor and of the PtBA-hv-PDMAEMA copolymer. 186


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decorated with carboxylic acid groups and filled with cleaved PtBA chains bearing a nitrosobenzaldehyde chain-end (Figure 5). The photocleavage was carried out in methanol at λ = 300 nm for 4 min (C = 2.5 g/L) and was followed by ultraviolet− visible (UV−vis) spectroscopy (Figure 5). In the UV−vis spectrum, the typical behavior of o-nitrobenzyl esters under irradiation is observed: the decrease of the band centered at λmax = 280 nm corresponds to the cleavage of the o-nitrobenzyl ester functions whereas the rise of the band centered at λmax = 345 nm with irradiation time is due to the appearance of nitrosobenzaldehyde functions in solution.32,35 Following the successful photocleavage of the block copolymer junction, the extraction of the core forming PtBA chains was realized. The extraction was carried out by selective/ nonselective solvent cycles (MeOH/H2O) and the extraction process was monitored by 1H NMR. After six extraction cycles, more than 97% of the PtBA chains were extracted out of the nanocages (See Figure S3). The extracted nanocages were finally analyzed by DLS and AFM. Figure 6a presents the CONTIN size distribution diagram of PtBA118-hv-PDMAEMA141 cross-linked micelles and of PDMAEMA nanocages in water (pH = 6, C = 0.1 g/L). The nanocages are characterized by a larger apparent hydrodynamic radius (48 nm) compared to the cross-linked micelles (28 nm). This could be explained by the fact that PDMAEMA chains are no longer tethered by the PtBA core in the nanocages and could swell further in water. Figure 6b presents the AFM image of the PDMAEMA141 nanocages. Spherical objects with a height of 4.5 ± 1.1 nm and a spread diameter of 35 ± 5.5 nm are observed. The size of the nanocages measured by AFM is much smaller in comparison to the PtBA118-hv-PDMAEMA141 micelles (Figure 6c). The difference observed between micelles and nanocages by AFM demonstrates, in addition to the spectroscopic proofs, the hollow structure of the nanocages. Indeed, micelles have a solid PtBA core that prevents the complete collapse of the objects upon drying. On the other hand, the empty core of the nanocages leads to a complete collapse and a smaller size is thus observed in AFM. Since PDMAEMA well-known pH and temperature responsive polymer,36−39 those stimuli can be used to tune the size of the nanocages in water. The size of the nanocages

3 days of dialysis against methanol to remove small molar mass byproducts. 1H NMR analysis of the cross-linked micelles revealed that the cross-linking reaction was quantitative (see Figure S2). In order to demonstrate that 10% of cross-linking will be sufficient to maintain the integrity of the nanocages formed after irradiation, the shell-cross-linked micelles were analyzed by DLS in MeOH, a nonselective solvent for the PtBA118-hv-PDMAEMA141 copolymer. The CONTIN analysis of the cross-linked micelles in methanol demonstrates that the cross-linking was successful since only a single population was observed in the size distribution diagram (Rh,app = 43 nm, Figure 4). The increase of the apparent hydrodynamic radius of

Figure 4. CONTIN size distribution diagram of the PtBA118-hvPDMAEMA141 cross-linked micelles in water and methanol (C = 0.1 g/L), and of the starting micelles in water as comparison.

the shell-cross-linked micelles in methanol may be explained by the swelling of the PtBA core, which induces the expansion of the whole micelle. On the other hand, the size of the micelles and of the shell cross-linked micelles is similar in water since the cross-linking degree is low. The third step for the formation of nanocages is the photocleavage of the copolymer junction. The irradiation of the micellar solution leads to the cleavage of the block copolymer junction to yield a PDMAEMA nanocage with its inner cavity

Figure 5. Photocleavage of the copolymer junction: (left) photocleavage reaction and (right) evolution of the UV−vis spectra with irradiation time (C = 2.5 g/L, λ = 300 nm). 187


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Figure 6. Characterization of the PDMAEMA141 nanocages: (a) CONTIN size distribution diagram of PtBA118-hv-PDMAEMA141 cross-linked micelles and PDMAEMA nanocages in water (pH = 6, C = 0.1 g/L), (b) AFM images of the nanocages, and (c) comparison of the PtBA118-hvPDMAEMA141 micelles and PDMAEMA141 nanocages AFM profile.

response at pH = 3 was expected since the large quantity of positive charges on the nanocages shifts the lower critical solution temperature (LCST) of PDMAEMA to temperatures higher than 90 °C.37 At pH = 9, the collapse of the system is observed around 62 °C with the decrease of Rh,app from 43 to 27 nm. The cloud point observed here is higher than the LCST of PDMAEMA free chains at this pH, which is located around 45−50 °C.37 The increase of the cloud point for our system in comparison to linear PDMAEMA can be explained by the presence of permanent positive charges resulting from the cross-linking of the PDMAEMA by BIEE. At pH = 6, the behavior of the nanocages differs from the behavior of PDMAEMA free chains. Indeed, PDMAEMA homopolymers do not exhibit a LCST at pH = 6 whereas our system presents a cloud point around 72 °C, where the Rh,app decreases from 48 to 30 nm. The collapse of the nanocages observed here is attributed to the reduction of the mobility of the PDMAEMA chains due to the cross-linking. The decrease of the LCST of thermo-responsive polymers upon a reduction of chain mobility has already indeed been previously observed for poly(oligoethylene glycolmethacrylate) (POEGMA) brushes on silica surfaces40 and for POEGMA and poly(Nisopropylacrylamide) (PNIPAm) brushes grafted on polystyrene latex particles.41,42 In order to confirm this hypothesis, the thermo-responsive behavior of PtBA118-hv-PDMAEMA141 micelles was monitored at pH = 6. The decrease of the micelle size was also observed around 52 °C (Figure S4). The difference between the cloud points of the PtBA118-hvPDMAEMA141 micelles and of the PDMAEMA nanocages is attributed to the increase of the hydrophilic character of the nanocages compared to the micelles due to the disappearance of the hydrophobic PtBA block and the creation of permanent charges during the cross-linking reaction. It should be noted that no macroscopic precipitation of the collapsed nanocages

was monitored by DLS as a function of pH and temperature. Figure 7 plots the evolution of the apparent hydrodynamic

Figure 7. Evolution of the PDMAEMA nanocage apparent hydrodynamic radius with pH and temperature.

radius (Rh,app) of the PDMAEMA nanocages at pH = 3, 6, and 9 versus temperature. At 25 °C, the effect of pH on the nanocage size is observed. The Rh,app are respectively equal to 76, 48, and 43 nm for the PDMAEMA nanocages at pH = 3, 6, and 9. The expansion of the nanocages upon acidification is the consequence of the increase of electrostatic repulsions among positively charged DMAEMA units. As far as the temperature dependence of the nanocage size is concerned, two cases can be distinguished. At pH = 3, the PDMAEMA nanocages do not respond to the increase of temperature, whereas the sizes of the nanocages at pH = 6 and 9 decrease when the solution is heated. The absence of 188


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Figure 8. Functionalization of the nanocages: (a) functionalization conditions, (b) normalized fluorescence emission spectra (λex = 328 nm) of PtBA-hv-PDMAEMA shell cross-linked micelles (dashed curve), of PDMAEMA nanocages reacted with 1-aminopyrene in methanol with DCC (full curve) and without DCC (dotted curve).

realized in four steps: the self-assembly of the copolymer in water, the chemical cross-linking of the PDMAEMA corona, the photocleavage of the copolymer junction and the extraction of the core forming PtBA chains. Moreover, the size of the nanocages can be tuned by changing pH or temperature thanks to the stimuli responsive nature of PDMAEMA, as shown by DLS. Finally, the ability to functionalize the internal cavity of the nanocages was demonstrated by fluorescence spectroscopy. The major advantages of the photocleavage approach to create functional nanocages are the mild conditions employed for the cleavage of the copolymer junction, which does not require the addition of reagents in the medium, and the easy tuning of the irradiation parameters (wavelength, intensity and duration) to fit the system. Moreover a wide range of functionalities may be obtained after photocleavage depending on the photocleavable initiator used during the synthesis.44,45 Such functional stimuliresponsive nanocages have potential as nanoreactors in which catalysts could be grafted into the hollowed nanocapsules. The stimuli responsive behavior of the nanocages may be used to encapsulate and release guest molecules. Moreover, the ability to easily functionalize the internal cavity of the nanocages is a major advantage for applications were the immobilization of guest molecules is needed.

was observed above their cloud points, confirming the beneficial role of the permanent positive charges, resulting from the cross-linking reaction, on the colloidal stability of the system. Nanocage Functionalization. Following the successful formation of the PDMAEMA nanocages, an interesting feature of the photocleavable approach should be highlighted: the ability to functionalize the inner wall of the nanocages. Indeed, the photocleavable approach allows, as opposed to degradation methods,24,25,43 the formation of nanocages with an internal cavity decorated by well-defined functions, here carboxylic acid groups. The availability of the internal carboxylic acids of the PDMAEMA nanocages was evidenced by fluorescence spectroscopy. The selective functionalization of the nanocages was carried out by grafting an amine bearing chromophore (1aminopyrene) via carbodiimide coupling. Figure 8a presents the functionalization conditions. A solution containing 1-aminopyrene and N,N′-dicyclohexylcarbodiimide (DCC) was added to a solution of the PDMAEMA nanocages. The mixture was allowed to stir for 24 h at 20 °C. The grafted nanocages were purified by dialysis against methanol to remove all traces of unreacted 1-aminopyrene. Finally, the fluorescence emission spectrum of the solution was recorded (λex = 328 nm). Figure 8b presents the fluorescence emission spectrum of the pyrene functionalized PDMAEMA nanocages (full curve). To ensure that the recorded fluorescence emission spectrum is specifically due to the aminopyrene that has reacted with the carboxylic acids of the nanocage inner wall, two reference experiments were performed. The same grafting experiment was realized with shell cross-linked micelles (dashed curve) and with the PDMAEMA nanocages in absence of DCC (dotted curve). The experiment on the shell cross-linked micelles demonstrates that the dye cannot be grafted when the carboxylic acid functions are masked. The experiment carried out without DCC demonstrates that there is no encapsulation of 1-aminopyrene into the nanocage cavity and that any free 1aminopyrene is efficiently removed from the nanocages by dialysis.

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ASSOCIATED CONTENT

S Supporting Information *

1

H NMR spectra of the pure polymer, shell cross-linked micelles, and of the nanocages and a plot of the evolution of the micelles hydrodynamic radius. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: (C.-A.F.) [email protected]. *E-mail: (J.-F.G.) [email protected]. Notes

The authors declare no competing financial interest.

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CONCLUSIONS In this paper, the formation of nanocages by light was presented. The CuAAC−ATRP process enables the synthesis of a well-defined photocleavable PtBA-hv-PDMAEMA block copolymer. The formation of the nanocages was successfully

ACKNOWLEDGMENTS

The authors thank the Communauté française de Belgique for financial support in the frame of ARC SUPRATUNE and to the P2M Programme from the ESF. C.A.F. is Research Associate of the FRS-FNRS. E.P. thanks FRIA for financial support. 189


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