pH-Dependent Morphology and Photoresponse of Azopyridine-Terminated Poly(N-isopropylacrylamide) Nanoparticles in Water

A series of azopyridine-terminated poly(N-isopropylacrylamide)s (PNIPAM) (C12-PN-AzPy) (∼5000 < Mw < 20 000 g mol–1, polydispersity index 1.25 or less) were prepared by reversible addition–fragmentation chain-transfer polymerization of NIPAM in the presence of a chain-transfer agent that contains an AzPy group and an n-dodecyl chain. In cold water, the polymers form nanoparticles (5.9 nm < Rh < 10.9 nm) that were characterized by light scattering (LS), 1H NMR diffusion experiments, and high-resolution transmission electron microscopy. We monitored the pH-dependent photoisomerization of C12-PN-AzPy nanoparticles by steady-state and time-resolved UV–vis absorption spectroscopy. Azopyridine is known to undergo a very fast cis-to-trans thermal relaxation when the azopyridine nitrogen is quaternized or bound to a hydrogen bond donor. The cis-to-trans thermal relaxation of the AzPy chromophore in an acidic nanoparticle suspension is very fast with a half-life τ = 2.3 ms at pH 3.0. It slows down slightly for nanoparticles in neutral water (τ = 0.96 s, pH 7.0), and it is very slow for AzPy-PNIPAM particles in alkaline medium (τ > 3600 s, pH 10). The pH-dependent dynamics of the cis-to-trans dark relaxation, supported by Fourier transform infrared spectroscopy, 1H NMR spectroscopy, and LS analysis, suggest that in acidic medium, the nanoparticles consist of a core of assembled C12 chains surrounded by a shell of hydrated PNIPAM chains with the AzPy+ end groups preferentially located near the particle/water interface. In neutral medium, the shell surrounding the core contains AzPy groups H-bonded to the amide hydrogen of the PNIPAM chain repeat units. At pH 10.0, the amide hydrogen binds preferentially to the hydroxide anions. The AzPy groups reside preferentially in the vicinity of the C12 core of the nanoparticles. The morphology of the nanoparticles results from the competition between the segregation of the hydrophobic and hydrophilic components and weak attractive interactions, such as H-bonds between the AzPy groups and the amide hydrogen of the PNIPAM repeat units.


Introduction
Among the various stimuli for responsive polymer-based devices, light possesses several advantages: it is directional, tunable in terms of energy, and it can be turned on and off rapidly. [1][2][3] Azobenzene, which undergoes reversible trans to cis photoisomerization, is commonly used for such applications. The trans-to-cis isomerization requires UV light irradiation. The cis to trans back conversion occurs upon irradiation with visible light and also by thermal relaxation in the dark. 4,5 The thermal cis-to-trans relaxation of azobenzene is very slow. It takes several hours for completion.
Disubstituted azobenzenes, such as 4-N,N-dimethylamino-4'-nitro-azobenzene, undergo thermal cis to trans isomerization much faster, with relaxation times on the order 10-100 s. 6,7 The fast response results from the "push-pull" electronic distribution imposed by the electron donor and the electron withdrawing substituents linked, respectively, to the 4 and 4' positions. This speed is critical for applications that require real time information without the use of a second photostimulation to regenerate the trans form, an important thrust in current material science.
Replacement of one phenyl ring of azobenzene with a pyridinium ring has a similar effect on the -electron distribution of azo chromophores, as exemplified by azopyridinium methyl iodide for which the cis-to-trans thermal relaxation time is on the order of 10 to 100 s. 6 The pyridine ring nitrogen is a powerful H-bond acceptor 8 , a property employed extensively in the construction of supermolecular assemblies, such as liquid crystals [9][10][11] , metal-organic frameworks 12,13 , fibers 14 , films 15,16 , and gels [17][18][19] . It turns out that binding of the AzPy nitrogen to common H-bond donors, such as phenols, accelerates the cis-to trans thermal relaxation rate of neutral AzPy due to the redistribution of the AzPy -electron imposed by the H-bond formation. This effect was exploited recently by Gelebart et al. who succeeded in generating continuous, macroscopic mechanical waves by continuous irradiation of films containing AzPy H-bonded to benzoic acid moieties. 16 In spite of such spectacular achievements, the design of simple, easily controllable, and fastrelaxing azo systems remains challenging, particularly in the case of water-borne polymeric materials. 20,21 Amphiphilic copolymers containing AzPy have been reported and evaluated in solutions, micro-, and nanoparticles in suspensions, or as hydrogels, 22,23 For instance, Zhang et al. reported AzPy-containing PNIPAM block copolymers in water form of giant vesicles, which undergo photo-controlled swelling and shrinking. [17][18][19] Supramolecular hydrogels formed by coassembly of phenylalanine-based amphiphiles and AzPy moieties were shown to undergo macroscopic gel−sol transition in response to light, and also through changes in temperature, or pH. 24 The dynamics of the thermal cis-trans relaxation were not assessed in these studies.
We report here the preparation of amphiphilic poly(N-isopropylacrylamides) (PNIPAM) bearing an AzPy moiety on one chain end and an n-dodecyl group on the other end, as simple models to evaluate the dynamics of neutral and charged AzPy in aqueous environments. A series of polymers (C12-PN-AzPy) of well-defined molar mass were synthesized by RAFT polymerization using a chain transfer agent bearing AzPy and n-dodecyl groups (Scheme 1). Light scattering measurements, 1 H NMR spectroscopy diffusion and fluorescence probe studies indicate that the polymers self-assemble in neutral water to form colloidally-stable nanoparticles. The photophysical properties of the colloidal dispersions were monitored as a function of the dispersions pH.
Transient absorption spectroscopy measurements indicated that the cis to trans thermal relaxation of AzPy is sluggish in alkaline dispersions of C12-PN-AzPy, but extremely fast in acidic and, unexpectedly, in neutral dispersions of the polymer. The fast dynamics of the cis-to-trans dark relaxation exhibited by neutral C12-PN-AzPy suggest that the AzPy end-groups form H-bonds with amide hydrogens of the PNIPAM repeat units in neutral conditions. This hypothesis was confirmed by several control measurements. It led us to conclude that C12-PN-AzPy chains do not form typical core-shell flower micelles, but adopt a more complex morphology that varies depending on the solution pH. This result is of interest in the context of polymer self-assembly and also from the practical view point as an entry to fast responsive light-driven systems.
The solid was removed by filtration. The filtrate was evaporated to dryness. The solid residue was purified by chromatography over a silica column eluted with hexane/ethyl acetate (4/1, v/v) as eluent.

General procedure for the synthesis of end-functionalized PNIPAMs
The polymers were prepared by RAFT polymerization of NIPAM in the presence of either CTA-AzPy or CTA-Azo. The following procedure leading to C12-PN-AzPy 12K is typical. In a 50 mL flask, NIPAM (1.13 g, 10 mmol), the CTA-AzPy (0.589 g, 1.0 mmol) and 4,4′-azobis(4-cyanovaleric acid) (ACPA, 0.056g, 0.2 mmol) were dissolved in 1,4 dioxane (10 mL). 1,3,5-Trioxane (0.02 g) was added to the solution as an internal reference for 1 H NMR measurements monitoring the progress of the polymerization. The solution was degassed with nitrogen for 30 min at room temperature. The flask was placed in a preheated oil bath set at 80 °C and kept at this temperature for 6 hours. The polymerization mixture was cooled to room temperature, and the polymer was purified by three consecutive precipitations into hexane. The sample was further purified by dialysis against water for 3 days and isolated by freeze-drying. See Figures S5 and S6 for the 1 H NMR spectra of C12-PN-AzPy and C12-PN-Azo.

Quaternization of the azopyridium end group of AzPy-terminated PNIPAM
Iodoethane (0.5 mL, 6.25 mmol) was added to a solution of C12-PN-AzPy 12K (0.45 g) in CH2Cl2 (10 mL). The reaction mixture was refluxed at 40 °C for five days, while monitoring the degree of advancement of the reaction by 1 H NMR spectroscopy. After completion of the reaction, the solvent was removed by evaporation. The polymer was dissolved in methanol, dialyzed against water for 3 days and isolated by freeze-drying (0.56 g) as an orange powder. See Figures S7 for the 1 H NMR spectra of the region part of C12-PN-AzPy 12K and C12-PN-AzPyC2H5 + 12K.

Characterization
Instrumentation 1 H NMR spectra were recorded on a Bruker AMX-400 (400 MHz) spectrometer. NMR diffusion experiments were performed with a Bruker Avance III (500 MHz) at 10 °C. Molecular weights and molecular weight distributions were determined with an Agilent 1100 gel permeation chromatography (GPC) system fitted with a TSK-gel R-M column (particle size 13 µm, exclusion limit 1 × 10 7 Da for polystyrene in DMF) and a TSKgel R-3000 column (particle size 7 µm, exclusion limit 1 × 10 5 Da for polystyrene in DMF) (Tosoh Biosep; DMF containing 0.4 wt % LiBr was used as eluent and the flow rate was set at 0.5 mL/min; the column temperature was set at 40 °C. Mass spectra were acquired on an Agilent 6224 Accurate-Mass TOF LC/MS. Fourier Transform Infrared Spectra (FT-IR) were recorded on a Nicolet 8700 FTIR spectrometer. Critical assembly concentration (CAC) values determined by a fluorescence probe were conducted with a Varian fluorimeter (Agilent Technologies).

Determination of the polymers molar mass from UV-Vis absorption data
The molecular weight was determined according to Eq 1: where w is the weight of polymer in the solution (in g), cCTA is the amount of RAFT agent residues (end-groups of the polymer) in solution (in mol), determined experimentally by application of Beer's law. Gaussian functions were used to separate the overlapping absorbances of the trithiocarbonate, azopyridine and azobenzene chromophores (fitting from 270 nm to 450 nm).

Solution preparation.
For

Light scattering (LS) Measurements
Dynamic light scattering (DLS) and Static light scattering (SLS) studies were carried out with a light scattering system equipped with a CGS-3 goniometer (ALV GmbH) fitted with an ALV/LSE-5003 multiple correlator (ALV GmbH) and a C25P temperature controller (Thermo Haake). The light source was a He-Ne laser (632 nm). Polymer solutions in water were refrigerated overnight and filtered through a 0.2 μm Millex Millipore PVDF filter prior to analysis.
In SLS experiments, the scattering intensity was measured at several angles from 30-150° against a toluene standard. The time averaged excess scattered intensity at angle θ, also known as the Rayleigh ratio Rvv(q), was related to the weight-averaged molar mass Mw, the Z-averaged root mean square radius Rg, the second virial coefficient A2 and the scattering vector (q), where K = 4π 2 n 2 (dn/dc) 2 /( NAλ0 4 ) and q = (4πn/λ0)sin(θ/2), with NA, n , (dn/dc) and λ0 being Avogadro's constant, the refractive index of the solvent, the specific refractive index increment of the solution, and the wavelength of light in vacuum, respectively. The dn/dc value of PNIPAM in water was assumed to be independent of temperature and equal to 0.167 mL/g. 28 The partial Zimm plot and the aggregation number, Nagg for aggregates were obtained from equations 2 and 3: where k is the fitting constant; β the stretched exponential parameter; τ the half-life of cisazopyridine; A0, A(t), and A∞ are the absorbances at 355 nm before UV irradiation, after UV irradiation at time t, and in the photostationary state, respectively.

Transient absorption measurements
The relaxation rate of the N-ethyl cis-azopyridinium moiety in C12-PN-AzPyC2H5 + (12K) was measured at room temperature with a laser flash photolysis system (LP 920, Edinburgh Instruments).
The laser wavelength was 355 nm. The pulse time was10 ns with an energy of 10 mJ / pulse. The detection wavelength was 365 nm. The data was fit with the exponential function defined in Eq 7: where ∆OD0 is the initial optical density, τ is the relaxation time; ∆OD is the time dependent optical density.

Transmission electron microscopy (TEM) measurements.
TEM images were recorded with a Tecnai G2 F20 at 200KV (FEI, USA), equipped with a 4k CCD camera. An aqueous solution of C12-PN-AzPy 20K (0.5 mg mL -1 ) was kept at 5 °C for at least 24 hours. One drop of the cold solution was deposited on a carbon-coated copper grid placed on a filter paper. The prepared TEM grid was dried at r.t. under vacuum before measurement.
The polymers were prepared by RAFT polymerization of NIPAM in dioxane using the chain transfer agents CTA-AzPy or CTA-Azo, the latter CTA leading to azobenzene-modified PNIPAM used in control experiments described below (Scheme 1). The structure of the two CTAs was confirmed by their 1 H NMR, 13 C NMR, and 2D-HMQC NMR spectra, shown in Figure   Mn values ( see Figure S9 in the case of C12-PN-AzPy, 12K) agree well the GPC-derived values (Table 1).

Solution properties of the polymers in water (10 o C)
Light scattering is a powerful technique to characterize the size and structure of self-assembled amphiphilic PNIPAMs. 31 Table 2.  Figure S10 and Figure S11) decrease with increasing molar mass of the polymers. The structure parameter, ρ = Rg/Rh, which reflects the mass distribution of the scattering object, is useful to assess the morphology of self-assembled nanoparticles. 28 (Figure 2c, d). The higher contrast of the particles core, visualized by high resolution TEM, suggests that the core contains closely-packed hydrophobic end groups (Figure 2d). The critical aggregation concentration (CAC) of the C12-PN-AzPy samples was evaluated by fluorescence spectroscopy using Nile Red (NR). This probe was selected since its absorption window (max= 520 nm) does not overlap with the absorption spectra of the AzPy and trithiocarbonate chromophores (see Figure S9) 35 Table 2 also presents the size of nanoparticles obtained for C12-PN-AzPyC2H5 + 12K. Both Rg and Rh are significantly larger than the corresponding neutral polymer nanoparticles, but their ratio is not affected, implying that the overall core-shell morphology of the particles is preserved. The Nagg of the C12-PN-AzPyC2H5 + 12K micelles decreases slightly, compared to C12-PN-AzPy12K.

pH-dependent photophysical properties of C12-PN-AzPy nanoparticles in
water (15 o C). Since the pKa of AzPy is ~ 4.53, 39 spectra recorded for solutions of pH 7 and 10 correspond to the chromophore in its neutral form, while the spectrum measured at pH 3 is characteristic of the azopyridinium protonated form. (Figure 3a). 40,41 The UV spectra of solutions of C12-PN-AzPy 7K of pH 7 and 10 present a band at 354 nm, characteristic of the π-π* transition of the transazopyridine group. This band undergoes a red shift from 354 nm to 385 nm upon protonation of the pyridine group at pH 3. The red shift results from the strong push-pull electronic redistribution from the oxygen atom of the alkoxy substituent to the positively charged nitrogen atom of the azopyridium group. Similarly, the UV-vis spectra of aqueous C12-PN-AzPyC2H5 + solutions, presented in Figure 3b, have a band at 390 nm, independently of the solution pH, as expected since the N-ethyl-pyridinium group is not pH-responsive . 6,42 The UV-Vis spectra of all samples ( Figure   3a) have an additional band centered at 310 nm attributed to the π-π* transition of the thiocarbonyl group linked to the -chain end. 43 It will not be included in the following discussions since the thiocarbonyl chromophore is inert at all pH values under the irradiation conditions used here. We confirmed by 1 H NMR spectroscopy that the ester that links the azopyridine group to the polymer main chain is not hydrolysed when the polymers are kept at pH 3 or 10 for up to 5 days at room temperature and ~ 1 day when heated to 70 °C. (Figure S13, S14.)  consists of an n-dodecyl core surrounded by AzPy groups confined in a restricted environment through H-bond to PNIPAM chains, possibly close to the core in view of their hydrophobicity and the limited hydration of the PNIPAM chains confined close to the core. 49 The UV-VIS absorption spectrum of C12-PN-AzPy 7K in a pH 10 aqueous environment undergoes significant changes upon irradiation of the sample: the band at 350 nm attributed to the π-π* transition decreases rapidly, while the band at 430 nm attributed to n-π* transition grows in. The thermal cis-to-trans isomerization takes over 2 hours (Figure 4b, pH=10), which is typical of neutral azopyridine in which the nitrogen electronic doublet is retained. Indeed, the electron-rich azopyridine nitrogen is unlikely to form hydrogen bonds with hydroxyl anions known to be strong H-bonds acceptor 52,53 and can interact with the amide hydrogen of the PNIPAM repeat unit. We measured the cis-to-trans thermal relaxation time of C12-PN-Azobenzene nanoparticles in water of pH 10. This polymer is a good model of C12-PN-AzPy in terms of self-assembly characteristics but the azobenzene end group is unable to form H-bounds. Its relaxation time in water is similar to that of C12-PN-AzPy in an alkaline environment ( Figure S16).
These photophysical properties, together with the LS results described above, lead us to conclude that C12-PN-AzPy micelles in an pH 10 medium also adopt a core shell morphology.
The precise morphology of the micelles cannot be ascertained from the data obtained so far.
Further characterization by SANS or SAXS is needed to determine if the AzPy groups cluster within the micelle core or clustered in the vicinity of the core, as shown in Scheme 2 (right).

Scheme 2.
Schematic representation of C12-PN-AzPy nanoparticles dispersed in water of pH 3, 7, and 10 based on data from LS, FTIR and 1 H NMR measurements and on the kinetics of the cis-to-trans thermal relaxation of azopyridine.

Conclusion.
When an amphiphilic copolymer is placed in contact with water, the hydrophobic moieties try to minimize contact with water and self-assemble. The segregation of the hydrophobic and hydrophilic components can be mitigated by attractive interactions between the two components through dipoledipole interactions or hydrogen-bonds. 50 The balance of the opposite effects determines the morphology of the copolymer micelles. Routine physico-chemical micelles characterization methods often fail to detect inter-components complexation. The phenomenon can reveal itself through the emergence of unique macroscopic properties, as reported recently in a study of polylactide-b-poly(-2-isopropyl-2-oxazoline) micelles in water. In the course of this study of azopyridine-end modified poly(N-isopropylacrylamides) dispersed in neutral aqueous media, we recorded an exceptionally fast cis-to-trans thermal relaxation of the chromophore attributed to the formation of H-bonds between AzPy and PNIPAM chains. The "flower micelle" morphology typical of ,-hydrophobically-modified PNIPAMs cannot account for this observation. We postulate a micelle morphology consisting of a core of segregated hydrophobes linked to one chain end. The other type of hydrophobes are distributed throughout the micelle PNIPAM shell. Their location within the shell depends on the pH of the aqueous environment. This unique morphology is responsible for the fast photo-response of the chromophore and its sensitivity to the environment pH.