The correlation between thermally induced precipitate‐to‐coacervate transition and glass transition in a polyelectrolyte‐bolaamphiphile complex

The precipitate and the coacervate are two aggregated states in the polyelectrolyte complexes (PECs). The precipitate‐to‐coacervate transition and glass transition in PECs have been widely reported in the past. In many cases, the two phenomena are studied independently, although both of them are apparently affected by water and small ions. Here, utilizing a PEC system consisting of poly(acrylic acid) (PAA) and a cationic bolaamphiphile (DBON), we explore the states of PECs as a function of salt, temperature, and the molecular weight of PAAs. By a combination of microscopic observation, time‐resolved fluorescence measurements, and differential scanning calorimetry, we identify salt/temperature driven precipitate‐to‐coacervate transitions of the complexes. The thermally induced morphology transformation from the precipitate to coacervate occurs around the glass transition temperature, indicating a strong correlation between the two processes. As the molecular weight of the PAA increases, the thermal transition temperature becomes higher. This finding offers new insights on the mechanistic interactions that dictate the aggregated states of PECs. Based on the photothermal effect of DBON, we also develop a UV light‐induced strategy to mediate the precipitate‐to‐coacervate transition, providing a fantastic platform to create functional PEC materials.


INTRODUCTION
When one polyelectrolyte (PE) solution is mixed with another oppositely charged species (surfactant, PE, nanoparticle, etc.), a PE complex (PEC) is formed via electrostatic interactions.[3][4] The water contents of a coacervate and a precipitate are usually different.With a water content high up to 70%, the coacervate behaves like a viscoelastic fluid with low viscosity and interfacial tension, [5] showing fantastic applications in protein encapsulation, [6] drug delivery, [7] and underwater adhesion. [8][11][12] In contrast, the solid-like precipitate, containing much less water, is a kinetically trapped polymer-rich phase.The irreversible precipitation of PECs was problematic in many practical applications.[16] Recent observations on the protein aggregation in vivo and in vitro strongly suggest that the macromolecular aggregates share the common morphology of the precipitates formed by synthetic PEs. [17]They may emerge from metastable coacervates and appear to be toxic to cells. [10]The physical pictures of the aggregated states in synthetic systems should be insightful for the understanding of the aggregation/condensation in cells.However, mechanistic interactions that dictate the formation of the precipitate/coacervate and the transformation between them have remained poorly understood for years.
A breakthrough was reported by Schlenoff's group on "saloplastic" PECs, [18,19] where the electrostatic interaction between polyions was largely reduced by salt ions.They observed the PE complex/coacervate continuum in a ternary polymer/salt/water system.With increased salt concentration, the solid-like complex underwent a solid-liquid-solution transition, leading to the PE complex/coacervate continuum. [20][23] Salt-driven precipitate-tocoacervate transition is a universal phenomenon in PEC systems.The origin has been discussed through rheological measurements by several groups, such as Schlenoff, [20] Perry, [24] and Tirrell. [25]Based on the idea of ion-doping in the polyion network, it is believed that the associated salt ions paired with polyions break the intrinsic polyion pairs, facilitate chain rearrangement, and lower the activation energy barrier, or the friction for such motions. [26,27]he underlying mechanism of salt-driven transition has inspired new ideas to modulate the morphologies of PECs and the transition pathways through manipulating external conditions, such as temperature, [28][29][30] pH, [31] light, [32][33][34] enzyme, [35] or changing the chemical structures of charged components. [36]However, only a few studies cover the precipitate-to-coacervate transition in PECs under such stimuli.Tirrell found that DNA hybridization induced a liquid-to-solid transition in a DNA-peptide coacervate, and the complex formed by a short DNA and the peptide displayed a precipitate-to-coacervate transition at ∼50 • C due to the melting of DNA. [37]Mann's group observed a light-induced morphological transformation from irregularly shaped particles to droplets in a PE-surfactant complex via substrate-mediated azobenzene photomechanics. [38]The transformations observed in these examples are specific, because the phase states were strongly influenced by the variation of physiochemical properties of one charged component (usually the small molecules in PECs) in response to temperature or light.
The glass transition in solid-like PECs and multilayers, generally in a hydrated state, has been emerging as an important dynamic event in PE community. [27,39]As with neutral polymers, the glass transition temperature (T g ) of PEC can be determined by DSC and dynamic mechanical analysis. [20,40][42][43][44] Lutkenhaus observed a monotonic decrease in T g with increasing water content in PECs. [41]ater weakens intrinsic ion pairing and also facilitates the sliding motion and relaxation of PEs. [43]Additionally, the salt can break the interactions between polyions, bring in more water, and thus lower the T g . [19]he structure and properties of PEC materials are influenced by the glass transition in many aspects.For instance, the thickness, stiffness, and surface smoothness of PE multilayers can be affected by thermal annealing of PECs above the T g . [45,46]The kinetically arrested nonequilibrium morphology of solid-like PEC could also be varied as the temperature rises.In 2010, Tirrell reported a thermally induced decrease in the turbidity and the size of the flocculated precipitate in poly(acrylic acid)/poly(allylamine hydrochloride) complex equilibrated with the supernatant. [47]t was suspected that a partial transformation of precipitate to coacervate occurred at 70 • C. Unfortunately, the pronounced temperature effect observed in this early work and its exact nature did not gain as much attention as the salt effect on the states of PECs.An intriguing question is whether the thermally induced precipitate-to-coacervate transition is related to the glass transition of PECs.If so, PECs may undergo a morphology transformation at a transition temperature possibly related to T g .
Here we report a PEC system consisting of poly(acrylic acid) (PAA) and a tetraphenylethene (TPE)-based cationic bolaamphiphile (DBON, chemical structure shown in Figure 1A).[50][51][52] The fluorescent emission of DBON in water is rather weak, but it can be significantly enhanced upon the association with negatively charged PE. [53] Thus, it offers inherent fluorescence labeling of PECs and facilitates the characterization of the complex by fluorescence microscopy. [54]By mixing PAA and DBON in aqueous solution, solid-like precipitates or liquidlike coacervates are obtained, depending on the molecular weight of PAAs.For the solid-like complex equilibrated with the supernatant, the precipitate-to-coacervate transition can be achieved not only by adding salt but also by heating.It is demonstrated that the thermally induced precipitateto-coacervate transformation is strongly correlated with the glass transition of the complex.Moreover, based on the photothermal effect of DBON, we develop a light-induced strategy to mediate the morphology transformation.

Solution properties of DBON
As a derivative of TPE, DBON has two conformers, cis and trans, [55] which coexist (1/1) as synthesized without further purification in this work.With two triethylammonium head groups on the ends of the hydrophobic TPE unit, DBON is a bolaamphiphile which may form micelles/aggregates at a certain concentration.The self-assembly behavior of DBON in water solution was studied by fluorescence spectra.At lower concentration (<1 mM), DBON has a maximum emission wavelength at 488 nm when excited at 330 nm (Figure 1B).With increased concentration, the fluorescence of DBON increases, reaches a maximum at 2.0 mM, and then decreases at higher concentrations (Figure S1).The fluorescence response versus the concentration of DBON indicates that the molecules form micelles/aggregates at 2.0 mM, which is the critical micelle concentration (CMC) of DBON in water.In this work, we kept the concentration of DBON below the CMC to avoid the self-aggregation.

Preparation of PAA60k/DBON complex
For the preparation of the complex, the pH was set at 10 for both of PAA (5 mM) and DBON (1 mM, below the CMC) solutions, so that we deal with a fully charged system.PAA with a molecular weight (M w ) of 60 kDa was used first, abbreviated as PAA60k.The mixing charge ratio, z (+/-), is defined by the ratio of the molar charges of DBON to those of PAA.With the addition of PAA into DBON solution at room temperature (RT), the turbidity of the mixture increased with z and plateaued at z = 1.0 (Figure S2), suggesting that the maximum complex formation and phase separation occurs most likely at the 1:1 stoichiometry.Indeed, flakelike precipitates can be observed for PAA60k/DBON at z ≥ 1 (Figure 1C,D).The precipitates are strongly emissive whereas the background is "dark", producing a high contrast under fluorescence microscopy (Figure 1E,F).
To determine the water content and chemical composition of the complex, the precipitate prepared at z = 1.2 was isolated from the supernatant by centrifugation.The water content in PAA60k/DBON was 18.4% by TGA, suggesting that most of the water molecules are repelled out of the complex.Surprisingly, the effective charge ratio of the complex was 0.80 (Figure S3), indicating an excess of negative charges of the complex.The mismatching of the charge densities of PAA and DBON may cause the deviation of z from the apparent mixing ratio.With a higher charge density of PAA, some of the charges on the polymer chains cannot be neutralized by the opposite charges.These complexes are kinetically trapped and eventually precipitate from the solution when the strong hydrophobic interaction overwhelms the electrostatic repulsion interaction.The solid-like precipitate has a storage modulus of ∼1 MPa at RT at 0.1 Hz (Figure S4).

Salt driven precipitate-to-coacervate transition
Small ions can screen the electrostatic interaction among PEs, rendering a precipitate-coacervate-solution continuum.When a small amount of NaCl was introduced, PAA60k/DBON complexes remained as precipitates (5-20 mM, Figure 2A).The transition from the precipitate to the coacervate occurred at c(NaCl) = 25 mM, as droplets were found under the microscope (25-200 mM, Figure 2A).The turbidity of the mixture remained high during the transition (Figure 2B,C).As more salt was added, a coacervate-to-solution transition occurred at c(NaCl) = 240 mM, the so-called critical salt concentration for PAA60k/DBON.When c(NaCl) was higher than this critical concentration, the turbidity decreased dramatically and a homogeneous solution was obtained (Figure 2A, 250 mM).
We also performed time-resolved fluorescence spectra of the complexes to obtain the fluorescence lifetime, an intrinsic property of the fluorophore sensitive to the aggregation state and local environment. [52,54]For AIE-gens in the aggregated state, they exhibit longer lifetimes than those in the solution, since the motion/rotation of the molecules are largely restricted when they are close to each other.The amplitude-averaged lifetime (τ) of PAA60k/DBON (equilibrated with the supernatant) was monitored during the salt-driven precipitate-to-coacervate transition (Figure 2C and Figure S5).Upon complexation with PAA60k, τ of DBON in the solid-like complex was 1.75 ns and remained nearly unchanged while c(NaCl) ≤ 20 mM.At c(NaCl) = 25 mM, τ declined to 1.67 ns.In the coacervate regime, τ of DBON decreased with increasing c(NaCl).When the complex was dissolved at c(NaCl) = 250 mM, a significant decrease of the lifetime down to 1.02 ns was observed.
As one might expect, the solid-like complex should cause more severe restriction on the rotation of phenyl rings in DBON than the liquid-like one, resulting in a considerable lifetime variation between the precipitate and the coacervate.Here, we only observed a subtle decrease of the lifetime (∼5%) across the precipitate-to-coacervate transition, suggesting that the electrostatic interaction network consisting of polyions and DBON remains compact across the transition.That is, the polyion interactions between PAA/DBON pairs are close, and the network is not strongly swollen by adding small ions.The evolution of the lifetime is consistent with Schlenoff's prior scattering experiments [56] on a PE complex doped with KBr.They observed that the radius of gyration (R g ) of the PE in the complex remained surprisingly constant up to 1.4 M KBr, close to the transition between solidlike complex and coacervate.Thereafter, R g decreased with increasing KBr.They proposed that a nonaffine swelling of the polyion network occurs when the precipitate transforms into the coacervate.Following this model, the conformations of polyions are retained during the transition, and water and salt ions fill the extra space, resulting in a microphase separated area in the coacervate.Similarly, DBON molecules should be undisturbed across the precipitate-to-coacervate transition, so the lifetime of DBON is nearly unaffected.After the transition, the doped ions significantly reduce the interaction of ion pairs, leading to a shorter fluorescence lifetime of DBON in the coacervate and dissolved states.Since the fluorescence lifetime of DBON is extremely sensitive to local constraint, the fluorescence method thus provides an accurate detection of the microstructure of the PECs.

Thermally induced precipitate-to-coacervate transition
We noted that the regime of solid-like PAA60k/DBON is quite narrow in the presence of salt (0-20 mM), indicating that the solid is somehow "soft".A slight degree of external ion doping already makes the interaction strength weak enough to get a coacervate.We wondered whether the transition could be triggered by the temperature.In order to study the temperature effect on the morphology of PAA60k/DBON, two pathways were used to prepare the complex (Scheme 1).In pathway 1, when the freshly prepared PAA60k/DBON mixture was heated to 50-55 • C, flake-like complexes split into small pieces, slowly "melted", and transformed into droplets (Figure 3A,B, and Figure S6).As the heating temperature further increased, the transition speeds up.Spherical droplets were observed when heating at 57 • C for 3 min (Figure 3C).The droplets were stable even at 80 • C (Figure S6).The water content of PAA60k/DBON coacervate was 78.3%.In pathway 2, preheated PAA60k and DBON stock solutions were mixed at 50 • C. A lot of tiny particles formed immediately after the mixing (Figure 3D).As keeping at 50 • C for 30 min, they merged into larger droplets (Figure 3E), suggesting that the coacervate is a relative stable state at high temperature.On the other hand, when these fresh particles prepared at 50

S C H E M E 2
Sample preparation procedure of poly(acrylic acid) (PAA)/DBON complexes."Water immersed" polyelectrolyte complexes (PECs) are PECs equilibrated with the supernatant.They were used for microscope observation."Water immersed" PAA60k/DBON was also used for DSC measurement.However, the DSC tests on "water immersed" samples were quite challenging.Therefore, we used "isolated" PECs to get their glass transition temperatures."Isolated" PECs refer to the isolated samples dried overnight at 60 • C under reduced pressure.The water contents in "isolated" PECs are comparable with those in the "water-immersed" samples.appeared again (Figure 3F), although the transition took some time.
The thermally induced precipitate-to-coacervate transition was confirmed by DSC experiment.The precipitates as prepared at RT were left to settle for a few minutes (Figure 1C) and transferred into the pan with a little supernatant for DSC measurement.This "water-immersed" PEC (Scheme 2, water content of 18.4%) exhibits an endothermic transition around 50 • C on the heating curve, and the profile looks like a glass transition (Figure 4A, black curve)!Unfortunately, the DSC signal of "water-immersed" solid-like complex was very weak and could not be repeated every time, so we did DSC test on the "isolated" PEC.The "isolated" PEC was prepared according to Scheme 2 and had a water content of 16.6% as shown Table 1.The glass transitions occur at 49.1 and 53.2 • C in the first and second heating ramps, respectively (Figure 4A, blue and red curves).The coincidence of the transitions in "water-immersed" and "isolated" PECs strongly suggests that there should be some relevance between the precipitate-to-coacervate transition and the glass transition: the precipitate transforms into the coacervate at T > T g .Since T g relies on the water content and thermal history, the transi- tion temperatures of "water-immersed" and "isolated" PECs are not exactly the same: T g of "water-immersed" PEC is slightly lower than that of "isolated" PEC.
As pointed out by Huglin [57] , the solid-like polyion complex is a "frozen liquid", and the state above the T g can be defined as a "liquid with physically fixed structure".In such a structure, both the electrostatic ion interaction and the chain entanglement serve as physical crosslinking points holding the network.Considering that DBON is a small molecule, the chain entanglement effect in PAA60k/DBON can be reduced, leading to a lower storage modulus of PAA60k/DBON (1 MPa at RT in Figure S4) than those glassy PECs reported in the literature (typically 100 MPa).The low modulus could also result from the porous nature of the complex. [39]We measured the linear viscoelastic response of PAA60k/DBON at varied temperatures (Figure S4).As the  temperature increases, the modulii of the complex show significant decrease when it goes through the glass transition.
The peak in tan (δ) curve indicates a glass transition at 45 • C, a bit lower than the value determined by DSC.Indeed, at T < T g , the storage modulus of the complex is higher than its loss modulus, indicating solid-like behavior; at T > T g , the loss modulus becomes higher than the storage modulus, suggesting a liquid-like behavior of the complex at higher temperature.

The influence of the molecular weight of PAA
The molecular weight of the PE may affect the phase state [1] and glass transition [39] of PECs.Here, another three PAAs with the M w of 2000, 8000, and 250,000 Da were used to prepare the complexes, namely PAA2k/DBON, PAA8k/DBON, and PAA250k/DBON.The turbidity plot versus the mixing charge ratio in three PAA/DBON systems follows the same trend as that of PAA60k/DBON (Figure S2).However, the morphology of the PECs varies with the M w : flake-like precipitates were observed in PAA8k/DBON, PAA60k/DBON, and PAA250k/DBON, whereas micro-sized droplets formed in PAA2k/DBON (Figures S7-S9).The distinctive difference between liquid/solid complexes lies in the water content (Table 1), which is 85.6 and 18.4% for PAA2k/DBON and PAA60k/DBON, respectively.
For simplicity, we define the thermally induced precipitateto-coacervate transition temperature (T tr ) by the temperature where the solid-like complexes are fully transformed into droplets upon heating (for 10 min) under microscopy observation (Figures S8 and S9).A molecular weight dependence of T tr is shown in Table 1.PAA8k/DBON has a similar T tr to PAA60k/DBON, whereas PAA250k/DBON displays a higher T tr up to 63 • C. The thermal transition was also recorded by DSC measurement using the "isolated" PEC (Figure 4B,C), which has a comparable water content with the "water-immersed" PEC.With increasing M w of PAAs from 2 to 250 kDa, the T g of "isolated" PEC increased from 27.4 to 63.2 • C (T g read from the seconding ramp in Figure 4).Most significantly, the T tr of each solid-like complex is consistent with the T g of "isolated" PEC.This supports our idea that the precipitate-to-coacervate transition in PAA/DBON is indeed dictated by the glass transition.Since the complex formed with longer PAA chains has more intrinsic ion pairs, PAA250k/DBON has the highest T g and T tr .In contrast, the T g of PAA2k/DBON is around RT, so a liquid-like coacervate is directly obtained when mixed with DBON.

2.6
The mechanism of the thermally induced precipitate-to-coacervate transition Here, we report the precipitate-to-coacervate transition driven by temperature: the irregular solid-like complex transforms into a liquid-like coacervate around the T g of the complex in the presence of water.Water plays a key role during the thermal transition as salt does in the salt-driven transition.As illustrated by Figure 5, the solid-like complexes are kinetically trapped when long PAA chains are mixed with DBON in the aqueous solution.A few water molecules (free or bound) exist in the network as plasticizers, whereas most water molecules are expelled out of the complex.As the temperature rises around T g , the relaxation processes (chain rearrangement, water diffusion, etc.) inside the PECs are activated.Since PECs are immersed in the supernatant phase, a large amount of water enters the assembly and reduces the energy barrier of the thermal transition, leading to a fluidic liquid state eventually.As shown in Table 1, the water contents of PAA/DBON increases significantly after the transition.For instance, the water contents of PAA60k/DBON increase from 18.4% in the precipitate to 78.3% in the coacervate.Both the water content and water dynamics, such as the diffusion and the bound state of the water, change significantly across the glass transition, leading to the precipitate-to-coacervate transition in PAA/DBON complex.

Other factors affecting the transition
The thermal transition is strongly affected by the ionic strength in the PECs. [26]For PAA60k/DBON complexes, they form the precipitates (Figure 2) when c(NaCl) ≤ 20 mM at RT.At this concentration regime, the T g of the complex should decrease with increasing c(NaCl). [43]Correspondingly, the T tr also decreases with c(NaCl) (Figure S10).A T tr of 42 • C is found for PAA60k/DBON at c(NaCl) = 20 mM.Since the salt ions can break intrinsic ion pairs of PAA/DBON, the glass transition of the complex is activated at a lower temperature, and a reduced T tr is observed with increasing c(NaCl).Moreover, the chemical structures (the kind of polyions, the flexibility and aromaticity of polymer chains, etc.) also have an effect on T g .For example, DBON can form a solidlike complex with poly(sodium 4-styrenesulfonate) (PSS), a polyanion bearing aromatic groups in the repeating units.The compact precipitate (Figure S11) remains stable over the entire working range between 0 and 100 • C. Neither a thermally induced precipitate-to-coacervate transition nor a glass transition was observed in this temperature window.

Light-triggered precipitate-to-coacervate transition
Besides the radiative-decay-induced emission of DBON, its nonradiative decay pathway is not completely suppressed.The nonradiative decay of AIE-gens is responsible for the energy dissipation in the form of heat.The photothermal effect offers tremendous opportunity for their applications in the fields including thermal imaging and photothermal therapy. [58,59]The photothermal conversion of DBON solution (0.8 mM) was characterized by infrared camera when it was irradiated by an UV LED (365 nm). Figure 6A shows the thermographic experimental setup when the UV LED was turned on.The camera recorded the temperature of the liquid surface (Figure 6B).From the temperaturetime plot in Figure 6C, it was found that the temperature of DBON solution increased and plateaued at 60 • C in 10 min, whereas the temperature of pure water or PAA60k solution increased only slightly.When the light was off, the temperature of DBON solution decreased immediately.With a maximum temperature variation of 35 • C, the efficiency of photothermal conversion is about 45.8% (Figure S12).
The photothermal effect of DBON maintains in PAA/DBON complex (Figure 6C).The temperature of PAA60k/DBON has risen by 30 • C under the same condition.It is noted that the rising of the temperature was rather fast till 47 • C and then slowed down.Considering the thermally induced transition of the complex, we suspect that a precipitate-to-coacervate transition occurs above 47 • C during the UV irritation.Indeed, all precipitates transformed into droplets after the exposure of UV light.The decomposition of DBON in the photothermal experiment was negligible (less than 1%), as indicated by the 1 H NMR spectra of the complex before and after the UV irradiation (Figure S13).Interestingly, the transition can be in situ visualized under a fluorescence microscope (Figure 6D-G, Figure S14 and the Video in SI).When the sample was imaged in "DAPI" channel (excited with UV light), the irregular aggregates quickly "melted" in several seconds (depending on the light power) and fluorescent droplets were found in the sight, whereas those out of sight kept their original shapes.Thanks to the photo-thermal effect of DBON, PAA60k/DBON is locally heated after absorbing UV light, and the complexes transform into droplets when the temperature approaches the T g of the complex.The advantage of light triggered precipitate-to-coacervate transition lies in that the morphology of the complex can be precisely modulated in a small area (∼µm 2 ) by an accurate control of the light source.

CONCLUSION
In summary, the state of a PE complex, solid or liquid, depends on many factors such as salt, water, chain length, and temperature.In the PE-bolaamphiphile complex system, we observed a thermally induced precipitate-to-coacervate transition of PECs around the T g .The morphologies of PECs immersed in water are directly correlated with the T g : a solid below the T g and a liquid above the T g .Whether this correlation applies to PEC systems consisting of two PEs needs to be verified in future.The thermal effect on the morphology of PECs may provide an implication on the condensation of some heat-sensitive proteins. [5]This finding may be especially insightful to the practical application of PE multilayers, since their structure and properties are strongly affected by thermal annealing.Finally, the advantages of

Characterization of DBON
1H NMR (400 MHz) spectra were recorded on a Bruker AscendTM spectrometer at RT using deuterated solvents such as CDCl 3 and D 2 O and tetramethylsilane as the internal standard.Mass spectra were recorded on a Thermo Scientific Q Exactive Mass spectrometer.

Preparation of the complex
Stock solutions of PAA (5 mM) and DBON (1 mM) were prepared in Milli-Q water.The pH was controlled at pH 10 through addition of 1M NaOH and was measured using a FE28 pH meter (Mettler Toledo) with an LE438 pH electrode.
The complex was prepared at RT by mixing PAA and DBON of predetermined volumes with a pipette in a centrifuge tube (2.5 mL).The mixing charge ratio, z (+/−), is defined by the ratio of the molar charges of DBON to those of PAA.The molar charge of DBON is twice of the molecular amount, and the molar charge of PAA is equal to the molar amount of the repeat units.

Preparation of the complex with NaCl
The amount of sodium chloride required for a given salt concentration was measured and added to PAA and DBON stock solutions.Then the stock solutions were mixed at z (+/−) = 1.0.The freshly prepared complexes equilibrated with the supernatant were characterized by optical microscope and fluorescence techniques.

Thermally induced precipitate-to-coacervate transition
To study the temperature effect on the morphology of PAA60k/DBON, there are to two pathways to prepare the complex (z = 1.0).As shown in Scheme 1, Pathway 1 involves mixing PAA60k and DBON at RT and heating the complex at a certain temperature (T > RT) in a water bath.In Pathway 2, the two stock solutions are warmed separately at T, followed by a mixing at T to get the complex.After the preparation for several minutes, a few µL of the mixture was sampled by pipette for the optical observation.

Optical microscope observation
For the morphology observation, the complex, freshly prepared and equilibrated with the supernatant ("water immersed" PEC in Scheme 2), was placed on a standard 35 mm dish (Nest) with a glass coverslip bottom (#1.5, 0.17 mm).The optical images of the complexes were acquired on a Leica DMI6000B fluorescence microscope at 63× magnification equipped with an oil immersion objective.Confocal microscopy was performed on a NikonA1R confocal microscope at 100× magnification equipped with an oil immersion objective.The fluorescent imaging was performed using a 405-nm excitation laser and a 450-nm emission filter.The video of the light-triggered precipitate-to-coacervate transition was recorded on a Nikon Ti-U fluorescence microscope at 40× magnification.

Thermal analysis of the complex
The complex (concentrated phase) was separated from the aqueous supernatant by centrifugation at 5000 rpm for 10 min.The excess water on the surface of the complex was dabbed dry by a Kim Wipe.Then the sample was tested on a TGA instrument to determine the water content in the "water immersed" PECs.The water content of the concentrated phase was analyzed on a Discovery TGA (TA) instrument with a heating rate of 1 • C/min, with the maximum temperature being 120 • C. DSC measurement was conducted on a Discovery DSC (TA) instrument.To determine the thermal transition temperature of "water immersed" complex, the concentrated phase equilibrated with a small amount of aqueous supernatant was carefully transferred to the pan for liquid samples.The heating/cooling rate of was 2 • C/min.In the repeated experiments, the faint transition was not detectable every time because unavoidable evaporation of water may disturb the baseline at higher temperature.Therefore, we measured the T g of "isolated" PEC, which refers to the complex isolated from the supernatant and dried at 60 • C overnight under reduced pressure, as shown in Scheme 2.

Spectroscopy characterization
Turbidity measurements were collected in a 1.0 cm path length cell with Agilent Cary 60 UV-vis spectrophotometer.
The turbidity (T%) was recorded by monitoring the transmission at the wavelength of 600 nm and calculated as 100 -T%.
The steady state and time-resolved fluorescence spectroscopy were performed on an Edinburgh FLS1000 spectrofluorometer.A picosecond pulsed diode laser (EPL-375) was used for time-resolved measurements.The excitation wavelength was 375 nm, and the emission was collected in the region of 400 ∼ 800 nm.The lifetime was analyzed based on the Marquardt-Levenberg algorithm using a two exponential decay function provided by Fluoracle.

Dynamic mechanical testing
The linear viscoelastic properties (storage modulus G′, loss modulus G″, and the loss tangent tan (δ)) of PAA60k/DBON at varied temperatures were examined using a straincontrolled MARS 40 rheometer (HAAKE).The rheometer had an 8-mm parallel plate geometry equipped with Peltier temperature control to ± 0.1 • C. For the sample preparation, the precipitate of PAA60k/DBON was isolated from the supernatant without further drying.The bulk sample was molded into an 8-mm diameter 1.4-mm thick disk at RT.A solvent reservoir with a cap was used to enclose the bottom geometry.The upper geometry was lowered onto the sample to apply a 0.2 N axial force.Fresh water was added to the reservoir to keep the sample in contact with water.Strain sweep experiments were performed at 2% strain.Temperature sweep experiments were performed from 25 to 75 • C at 0.1 Hz and with a heating rate of 2 • C / min.

Thermography testing
The photothermal effects of DBON and PAA/DBON complex were monitored on an ImageIR 8300 IR camera (INFRATEC, Germany).As shown in Figure 6A, the solution (or the mixture) (0.85 mL) was placed in a quartz cell and irradiated with a PCB mounted LED (Thorlabs, Inc.) from one side.The UV LED (365 nm, model M365D2) was equipped with a custom-made heat sink and controlled by a T-Cube LED driver (LEDD1B, Thorlabs, Inc.) with adjustable power.The input setting of the T-Cube LED driver was kept at the five-sixths of the maximum drive current, which produces a light power of 1150 mW according to the manufacturer.The IR camera was fixed on the top of the cell to record the temperature on the liquid surface.The thermal images were analyzed using IRBIS3 Professional software.The temperature-time profiles were extracted and used for the calculation of photothermal conversion efficiency.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interests.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

F
I G U R E 1 (A) Chemical structures of poly(acrylic acid) (PAA) and DBON.The cartoon shows the complexation of PAA and DBON in the aqueous solution.Blue and red circles represent positive and negative charges, respectively.(B) Fluorescence spectra of DBON (0.4 mM) and PAA60k/DBON (prepared at 0.4 mM and z = 1.0).(C) The flake-like precipitates in the vials.(D) Bright field microscopic images of PAA60k/DBON.(E) Fluorescence (DIPI channel) images of PAA60k/DBON.Scale bar = 20 µm.(F) The fluorescence of the sample in (C) observed under a handheld UV lamp (365 nm).

F
I G U R E 2 (A) Morphologies poly(acrylic acid) (PAA)60k/DBON with NaCl observed at room temperature under the microscope.Upper, bright filed; lower, DAPI channel.c(NaCl) = 5, 10, 20, 25, 200, and 250 mM from left to right.Scale bar = 10 µm.(B) Precipitate-coacervate-solution continuum of PAA60k/DBON observed under visible (upper) and UV (lower, 365 nm) lights.The values on the vials indicate the salt concentrations in the unit of mM.(C) The turbidity (black circles) and fluorescence lifetime (blue squares) of PAA60k/DBON during the salt-driven precipitate-to-coacervate transition.The magenta dash lines indicate the regimes of precipitates, coacervates, and solutions with increased c(NaCl).
S C H E M E 1Two pathways to prepare poly(acrylic acid) (PAA)/DBON complexes for the study of the temperature effect on the states of polyelectrolyte complexes (PECs).
• C were cooled to RT (by removing the sample vial from the water bath), irregular aggregates F I G U R E 3 Bright field (upper pannel) and fluorescence (lower pannel) images of poly(acrylic acid) (PAA)60k/DBON during thermally induced precipitate-to-coacervate transition.(A-C) The complexes prepared by pathway 1 were heated to 50, 55, and 57 • C, respectively.The heating time was 3 min.(D) The complex was prepared by pathway 2 and the mixing temperature was 50 • C. (E) The complex in (D) was kept at 50 • C for 30 min.(F) The complex in (D) was cooled for 15 min.The fluorescence images in (B and D) do not match exactly with the bright field ones due to the fast movement of the particles.Scale bar = 10 µm.

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I G U R E 4 DSC heating curves of "isolated" polyelectrolyte complexes (PECs) (blue and red for the first and second heating ramps).(A) poly(acrylic acid) (PAA)60k/DBON; (B) PAA8k/DBON; (C) PAA250k/DBON; (d) PAA2k/DBON.The arrow indicates T g in the second heating ramp.The DSC curve of the "water-immersed" PAA60k/DBON is also shown in black line in (A).

4 Abbreviations:
Abbreviations: PAA, poly(acrylic acid); PECs, polyelectrolyte complexes.a As prepared at room temperature without salt.b Determined by TGA.For the measurement, the complexes were isolated from the supernatants.The excess water on the surface of the complex was dabbed dry by a Kim Wipe.c Observed by the microscope.d Determined by TGA. e Determined from the second heating ramp of DSC experiment.fThe coacervate complexes at 65 • C were isolated from the supernatants for TGA measurement.g Not determined.

F I G U R E 5
Schematic presentation of thermally induced precipitateto-coacervate transition in poly(acrylic acid) (PAA)/DBON complex.The charges on PAA and DBON molecules are not shown for clarity.

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I G U R E 6 (A) Thermal imaging setup of poly(acrylic acid) (PAA)60k/DBON in the cuvette.(B) Thermograph image of PAA60k/DBON during the UV irradiation.(C) Thermograph plots of the complexes.The plots of DBON, PAA60k, PAA2k/DBON, and water are shown for comparison.(D and F) Bright field images of PAA60k/DBON before and after the UV irradiation for 10 s. (E and G) Acquired in DAPI channel.Scale bar = 20 µm.
TA B L E 1 T g , T tr , and the water contents of PAA/DBON complexes.
DBON, an AIE-active molecule, should be emphasized at last.It has three roles: it is a charged component in the PEC, an intrinsic fluorescent label, and a photothermal agent to trigger precipitate-to-coacervate transition by UV light.Ongoing research involves probing the viscosity nature of PECs in varied states and designing other light stable AIE-gens to study the dynamics of the PECs.
This work is supported by the State Key Research Development Programme of China (grant number: 2021YFB3800702), the National Natural Science Foundation of China (grant number: 21902073), Shenzhen Science and Technology Innovation Committee (grant number: JSGG20210629144802007), and Post-Doctoral Later-Stage Foundation Project of Shenzhen Polytechnic (grant number: 6021271003K).The authors acknowledge the assistance of SUSTech Core Research Facilities.Xiaoqing Liu.thank Prof. Yi Liu (Shenzhen University) for helpful discussions.