University of Birmingham CO2/pH-responsive particles with built-in fluorescence read-out

A novel ﬂ uorescent monomer was synthesized to probe the state of CO 2 -responsive cross-linked polymeric particles. The ﬂ uorescent emission of this aminobromomaleimide-bearing monomer, being sensitive to protic environments, can provide information on the core hydrophilicity of the particles and therefore indicates the swollen state and size of the particles. The particles ’ core, synthesized from DEAEMA ( N , N -diethylaminoethyl methacrylate), is responsive to CO 2 through protonation of the tertiary amines of DEAEMA. The response is reversible and the ﬂ uorescence emission can be recovered by simply bubbling nitrogen into the particle solution. Alternate purges of CO 2 and N 2 into the particles ’ solution allow several ON/OFF ﬂ uorescence emission cycles and simultaneous particle swelling/shrinking cycles.


Introduction
Stimuli-responsive polymers have received great interest in recent years and have been developed for different applications such as nanotechnology, bio-materials and drug delivery. [1][2][3][4] To trigger a response and/or change in properties, external stimuli such as temperature, pH, CO 2 or light can be utilized. [5][6][7][8][9] The recent interest in CO 2 as a stimulus for responsive materials is due to the natural abundance of this gas and its bio-compatibility. The usage of CO 2 -responsive polymers is widely reported to trigger and control self-assembly morphology transitions. [10][11][12][13] These responsive polymers are usually synthesized with monomers containing tertiary amine, amidine, guanidine or imidazole functional groups. [14][15][16] These functional groups can all be protonated upon a decrease in pH which in turn can be induced by CO 2 . Tertiary amine-containing monomers such as DMAEMA (N,N-dimethylaminoethyl methacrylate), DEAEMA and DPAEMA (N,N-diisopropylaminoethyl methacrylate) exhibit a pH-responsive behavior, as they can react with strong and weak acids. 17,18 These pHresponsive monomers are also responsive to CO 2 in water, as CO 2 partially dissolves in water to form an equilibrium with carbonic acid, which is a weak acid that can dissociate into HCO 3 − , CO 3 2− and H + , allowing protonation of the amine monomers. The CO 2 response is a reversible process where reversal is carried out by simply bubbling nitrogen or argon in the solution, which displaces carbonic acid and shifts the equilibrium toward the initial pH. 19 CO 2 -responsive polymer assemblies can also be obtained via encapsulation of aminebearing small molecules into non-responsive systems such as micelles. 20 Further examples of CO 2 -responsive materials include the use of CO 2 /pH-responsive latexes as Pickering emulsifiers, reported by Morse and co-workers. 21 The crosslinked latexes were synthesized from DEAEMA, DVB (divinylbenzene) and PEGMA ( poly(ethylene glycol methacrylate)) and showed a reversible diameter increase from 230 nm to 590 nm using HCl/KOH or CO 2 /N 2 gas purges to change the pH. The protonation of the DEAEMA cross-linked core, using HCl or CO 2 , increases the core hydrophilicity, which induces a swelling effect. The deprotonation of DEAEMA via N 2 purging or with KOH decreases the hydrophilicity of the core and results in a reduction of the particle diameter back to its original size. Recently, Chen and co-workers also reported the preparation of CO 2 -responsive polymeric microgels which are composed of a DEAEMA core covalently stabilized with PEGA (poly(ethylene glycol acrylate)), cross-linked with EGDMA (ethylene glycol dimethacrylate) or BIS (methylene bis(acrylamide)). 22 The different microgels obtained showed a reversible size increase upon CO 2 and argon bubbling. Depending on the CO 2 concentration, these microgels can reversibly swell, or swell then collapse. Fluorescent dyes are of key interest owing to their potential use in drug delivery systems. [23][24][25] Liang and co-workers presented the advantages of a responsive and fluorescent combination for bio-medical applications with the preparation of polymeric nanoparticles that are fluorescent, pH-responsive and biocompatible for intracellular imaging and drug † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c6py01254j a delivery. 26 We previously reported the synthesis of fluorescently labelled proteins and polymers with a dithiomaleimide moiety; a small functional group that does not affect the polymer or protein scaffold. 27 This fluorescent functional group was also incorporated into an amphiphilic block copolymer which self-assembled into spherical micelles and can be used in nanomedicine. 28 We also recently reported the one-pot synthesis of fluorescent nanogels that were covalently dyed using a dithiomaleimide methacrylate monomer. 29,30 It was also demonstrated that these particles do not exhibit self-quenching at high concentration unlike commonly used fluorophores such as phloxine B. Dithiomaleimides have been previously demonstrated to be highly fluorescent when the maleimide unit is conjugated to an alkyl thiol, 27,31 although dithiomaleimides in the presence of an excess of thiol can undergo substitution, which may result in loss of fluorescent properties if substituted with an aromatic thiol. 32 To counter this substitution effect and thus the loss of fluorescence, we have recently developed a new class of highly emissive fluorophores, the aminobromomaleimides (ABM). 33 Their fluorescence properties are environment dependent; in protic solvents a loss of fluorescence can be observed. Herein, we report the first synthesis of fluorescent CO 2 -responsive polymeric particles by emulsion polymerization. A novel ABM functional fluorescent monomer, present in the particle core, was utilized as a probe of the core hydrophobicity. By simple CO 2 bubbling, the particles become swollen and, as a consequence of the increased hydrophilicity of the particles, their fluorescence drastically decreases. This swelling is reversible by purging the solution with nitrogen and ON/OFF cycles of fluorescence are reproducible with successive CO 2 /N 2 purges. PDEAEMA ( poly(N,N-diethylaminoethyl methacrylate)) was used as the CO 2responsive core-forming segment, with OEGMA (oligoethylene glycol methacrylate) as the hydrophilic shell-forming block.

Experimental
Materials Dry solvents were obtained by passing over a column of activated alumina using an Innovative Technologies solvent purification system. DEAEMA was filtered through a plug of alumina prior to use and stored at 4°C. All other chemicals were purchased from Aldrich, Fluka or Acros and used as received.

Synthetic procedures
Synthesis of the dibromomaleimide methacrylate monomer (DBMMA). To an oven-dried round-bottom flask under an inert nitrogen atmosphere was added triphenyl phosphine (1.03 g, 1 equiv.) and dry THF (35 mL). The mixture was cooled to −78°C before the dropwise addition of diisopropyl azodicarboxylate (DIAD) (0.769 mL, 1 equiv.). The mixture was stirred for 5 min before adding 2-hydroxyethyl methacrylate (0.475 mL, 1 equiv.), stirred for a further 5 min before adding 2,2-dimethylpropan-1-ol (0.170 g, 0.5 equiv.), and stirred a further 5 min before adding 2,3-dibromomaleimide (1.00 g, 1 equiv.). The reaction was allowed to warm to room temperature while stirring for 18 h. The solvent was removed in vacuo, and the crude mixture purified by column chromatography on silica gel using a 1 : 1 mixture of petroleum ether 40-60°C and dichloromethane, to give the product as a white solid (1.037 g, 73%). The monomer was characterized by 1 H NMR spectroscopy in CDCl 3 , 13 C NMR spectroscopy and mass spectrometry, see ESI. † Synthesis of aminobromomaleimide methacrylate (ABMMA). DBMMA (1) (1.00 g, 1 equiv.) was dissolved in THF (50 mL). To the solution, sodium carbonate (0.720 g, 2.5 equiv.) was added and stirred. Isopropylamine (0.250 mL, 1.05 equiv.) was added dropwise to the solution, whereby an immediate color change of the solution to yellow and the formation of a white precipitate was observed. Upon complete addition of isopropylamine, the solution was left to stir for 2 h at room temperature. The solvent was removed in vacuo, the residue taken up in CH 2 Cl 2 (150 mL), washed with water (2 × 150 mL) and dried with MgSO 4 . The organic layer was concentrated in vacuo and the product purified by column chromatography on silica gel using a 10 : 1 mixture of petroleum ether 40-60°C and ethyl acetate to yield the product as a yellow-orange crystalline solid (0.88 g, 94%). The product was characterized by 1 H NMR spectroscopy in CDCl 3 , 13 C NMR spectroscopy, mass spectrometry and fluorescence spectroscopy, see ESI. † General procedure for the synthesis of fluorescent particles. OEGMA (0.11 mmol) was first dissolved in 44 mL of deionised water and then EGDMA (0.126 mmol), and ABMMA (2) (0.145 mmol) were dissolved in DEAEMA (13.5 mmol) and added dropwise to the solution. The mixture, whilst stirred, was degassed with nitrogen for 30 min and further heated at 65°C for 30 min. The initiator, potassium persulfate (KPS) (0.093 mmol), was dissolved in water (1 mL) and degassed with nitrogen before being added to the reaction mixture. The reaction was stirred at 65°C for 16 h under a nitrogen atmosphere. The particles were purified by exhaustive dialysis (MWCO 3.5 kDa) against deionised water.

Synthesis and characterization of the fluorescent monomer
The fluorescent monomer containing the ABM functionality was synthesized in two steps, see Scheme 1. First, 2,3-dibromomaleimide methacrylate (DBMMA, 1) was synthesized by alkyl-ation of 2-hydroxyethyl 2-methylprop-2-enoate with 2,3-dibromomaleimide using a modified Mitsunobu reaction procedure reported by Walker. 34 The DBMMA monomer (1) was then functionalized via mono-substitution of the bromine with isopropylamine to obtain an aminobromomaleimide methacrylate (ABMMA, 2), following a procedure similar to that used for the synthesis of a library of aminomaleimides, previously reported by our group. 33 The monomer presents excitation maxima at 247 nm and 372 nm corresponding to an emission maximum at 482 nm in 1,4-dioxane.

Synthesis and characterization of fluorescent PDEAEMA particles
Fluorescent CO 2 /pH-responsive particles were designed with a tertiary amine-bearing monomer (DEAEMA) and the fluorescent ABMMA monomer (1), the former allows a pH-response while the latter monomer allows a fluorescence read-out of the particles' state. Particles were synthesized via emulsion polymerization in water and the polymerization was initiated by using potassium persulfate (KPS) as the initiator, see Scheme 2. The emulsion polymerization procedure consists of emulsifying an insoluble monomer phase in water in the presence of a stabilizing amphiphilic compound. The amphiphilic compound in this case is OEGMA, which copolymerizes with DEAEMA to form a covalently linked hydrophilic shell. The hydrophobic core is composed of PDEAEMA cross-linked with 1 wt% of EGDMA and 2 wt% of the fluorescent ABMMA monomer (2). The tertiary amines of the core-forming block allow a CO 2 -responsive behavior while the presence of the ABM allows a built-in fluorescence read-out. Several batches of particles were synthesized. The influence of the OEGMA molecular weight (from 360 Da to 2000 Da) on the particles' size and responsive character was studied. As a control experiment, particles were also synthesized without the fluorescent monomer in order to confirm it does not affect the particles' response to CO 2 . The hydrodynamic diameter of the different particles in solution in deionized water was measured by dynamic light scattering (DLS) and it was found that the particles are all in the same size range ca. 200 nm, see Table 1 and Fig. 1b, d for the DLS of particles 4 and 8, see ESI † for particles 3, 5, 6 and 7. The variation of the OEGMA molecular weight or the incorporation of ABMMA did not affect the particles' morphology and size range. The different OEGMA utilized changed the shell's density as the degree of polymerization of OEGMA was kept the same whilst the monomer increased in size. The size of fluorescent particles with different shells (4 and 8) was also confirmed by transmission electron microscopy (TEM), see Fig. 1a and c. The particles' spherical morphology was confirmed and the average diameter was measured to be 257 nm for particles 4 and 330 nm for particles 8. The difference between the Scheme 2 Synthesis of particles (8) via emulsion polymerization in water at 65°C and the reversible particle response upon CO 2 and N 2 bubbling.  Particle size monitoring DLS was utilized to monitor changes in the particle diameter in response to bubbling with CO 2 . When CO 2 is bubbled in the particle solution, the amines of PDEAEMA present in the core of the particles become protonated and therefore hydrophilic. Owing to their cross-linked structure, the particles swell and increase in diameter instead of disassembling. A size increase upon CO 2 bubbling was observed for all the particles, see Fig. 2b for particles 8 and ESI † for particles 3, 4, 5, 6 and 7.
For example, the hydrodynamic diameter of particles 3 increased from 185 nm to 390 nm and particles 7 present a diameter increase from 200 nm to 435 nm. The size increase of the non-fluorescent particles with the small molecular weight OEGMA shell (3) was also monitored by small angle X-ray light scattering (SAXS) with an in situ CO 2 purge, see Fig. 2c. The SAXS data shows an increase of the radius of gyration within minutes. Analysis of the SAXS curves over a period of 21 min indicates an increase of particle size as well as an increase of the dispersity. The initial particles could be analyzed as spherical micelles with really low core dispersity with some polymer coils accounting for the outer hydrated shell. 36,37 After bubbling CO 2 the dispersity of the core slightly increased (as evidenced by the loss of oscillations in the raw SAXS profiles) and a more pronounced core-shell spherical morphology was observed with an increase of core radius. An initial diameter of 180 nm is observed, and an increase of the diameter is observed over time: after 2 min of bubbling, 255 nm and after 21 min, 280 nm.

Fluorescence emission monitoring
As previously demonstrated by our group, the ABM functional group is sensitive to polar protic environments. 33 Thus, the ABM-bearing monomer should be able to probe the protona-tion of its environment. Therefore, the increasing hydrophilicity of the core upon CO 2 bubbling should quench the fluorescence emission of the particles. Fluorescence emission of the particles 8 was measured before and after CO 2 bubbling, a drastic decrease of the intensity was observed at 487 nm from 810 a.u. to 50 a.u. (see Fig. 2a). The decrease in fluorescence emission upon CO 2 bubbling was observed under a UV lamp (λ = 345 nm), see video in ESI. †

Reversibility of the system: CO 2 /N 2 purge cycles
To test the reversibility of the system, the particle solution was repeatedly purged with successive cycles of CO 2 and N 2 bubbling. Purge cycles were monitored by fluorescence spectroscopy and DLS. The purging time was kept constant for the entire experiment, CO 2 was bubbled for 15 min and N 2 was bubbled for 30 min. Cycle experiments were performed on fluorescent particles with the denser OEGMA shell (8). The hydrodynamic diameter and the fluorescence emission at 487 nm of the particles (8) were measured after each CO 2 or N 2 purge. As shown in Fig. 3, the size of the particles alternatively increases and decreases six times. Similarly the emission intensity is reversibly quenched eight times (Fig. 4). Although  particles with a less dense shell (3,4,5,6) can swell with CO 2 , they are unable to collapse upon N 2 bubbling. For example, particles 5 exhibit a diameter of 235 nm before bubbling, 430 nm after CO 2 bubbling and 1000 nm after N 2 purge. This irreversible swelling occurs as a consequence of the density of the shell that cannot counter the effect of core chains that irreversibly penetrate the shell during the first protonation. These hydrophobic chains get trapped in the shell when the particles shrink and their presence promotes inter-particle interactions, which leads to the formation of uncontrolled large aggregates. The formation of aggregates is significantly reduced with a denser shell made of OEGMA with a molecular weight of 2000 Da.

Conclusions
In conclusion, we have presented the synthesis of fluorescent CO 2 -responsive cross-linked polymeric particles. The fluorescent dye was incorporated in the core in the form of a novel fluorescent monomer containing an ABM functional group while the CO 2 -responsive character was introduced by the presence of tertiary amines. These particles swell as the core is made hydrophilic by bubbling CO 2 into the solution. This change of local hydrophobic character then induces a decrease in ABM fluorescence intensity. Thus, the fluorescence intensity can be used to probe the particle core hydrophobicity and particle size.