Thermogelling of Amphiphilic Block Copolymers in Water: ABA Type

Article Cite This: Macromolecules XXXX, XXX, XXX−XXX

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Thermogelling of Amphiphilic Block Copolymers in Water: ABA Type versus AB or BAB Type Shuquan Cui, Lin Yu, and Jiandong Ding* State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular Science, Fudan University, Shanghai 200438, China

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S Supporting Information *

ABSTRACT: Some amphiphilic block copolymers exhibit sol−gel transition in water upon heating, which affords a promising injectable thermogel in clinic and an interesting soft matter system. While ABA-, BAB-, and AB-type block copolymers have all been reported, little is known about the comparative study of supermolecular structures of these polymer types in the physical hydrogels, which hinders the understanding of the universal mechanism of structural changes during thermogelling. Herein, a thermogellable aqueous system of ABA triblock copolymer poly(D,L-lactide-co-glycolide)-b-poly(ethylene glycol)-b-poly(D,Llactide-co-glycolide) was investigated by both experiments and computer simulations, with the corresponding AB diblock copolymer and BAB triblock copolymer as controls. The copolymers were synthesized via ring-opening polymerization, and their thermogelling behaviors in water were analyzed with transmission electron microscopy, three-dimensional dynamic light scattering, and so forth. Fluorescence resonance energy transfer, temperature-dependent 13C NMR, and rheological measurements were also carried out to investigate the internal structures and their evolutions during the sol−gel transition. A dynamic Monte Carlo simulation was operated to analyze the thermogelation further. We found two states with different structures in the thermogel window of the ABA block copolymer. The formation of a hydrophobic channel evolved from the semibald micelle was revealed as the key universal cue triggering the physical gelation for all types of thermogellable copolymers. Based on our structural studies, the molecular design principles for the thermogellable copolymers have been established.

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INTRODUCTION Hydrogels have been paid much attention over decades as a soft matter similar to human tissues.1−4 In the hydrogel family, an injectable physical hydrogel with a sol−gel transition is very promising in clinic owing to its convenient operation and minimal invasion and so forth.5−9 The thermogel is particularly interesting.10−13 The reversed sol−gel transition upon heating makes the system maintain a sol state at room temperature and thus can be mixed with drugs or cells conveniently but transform into a gel after being injected into the body. The physical thermogelation avoids the byproducts from the chemical reaction and suppresses the possible resulting inflammatory responses. Concentrated aqueous systems of some amphiphilic block copolymers composed of poly(ethylene glycol) (PEG) and some biodegradable polyesters such as poly(D,L-lactide-coglycolide) (PLGA) exhibit a reversible sol−gel transition upon heating. This thermogel is degradable and can maintain its integrity from 1 week to even 3 months when being put into a large amount of water after formation of the physical hydrogel. The maintaining duration can be adjusted by the molecular parameters.10 These properties endow this injectable thermogel with high prospects in many medical fields, such as drug delivery,14,15 tissue engineering,16,17 prevention of postoperative adhesion,18,19 and some other applications.20 © XXXX American Chemical Society

For a thermogellable copolymer composed of PEG and polyester, there are a series of different molecular structures including AB diblock, ABA triblock, BAB triblock, multiblock, and graft architectures.10,21 In this study, A and B represent hydrophobic block and hydrophilic block, respectively. Thermogels of ABA triblock copolymers with stable performance, high modulus, and a wide adjustable range of degradation periods have been the research focus22 and show great potential of industrialization. PEG/polyester thermogels have been studied for about 2 decades since the pioneering work by Jeong and Kim et al. in 1997.23 Control of the thermogelling behavior is very important in any clinical application. It has been reported that the susceptible thermogel properties are influenced by many internal or external factors, such as the block length,22 the molecular weight distribution,24,25 the modification of the end group,26 some additional agents,27,28 and so forth.29 Nevertheless, the universal guidelines for the system design are still needed, which should be based on a comprehensive illustration of the internal structure and mechanism of the thermogelling. Received: March 16, 2019 Revised: April 26, 2019

A

DOI: 10.1021/acs.macromol.9b00534 Macromolecules XXXX, XXX, XXX−XXX

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Figure 1. Schematic of the thermogelling process of AB or BAB copolymer (A) and ABA copolymer (B) in water. The orange and blue lines represent the hydrophobic and hydrophilic blocks, respectively. The red line and the mazarine line in the state boxes represent aggregates linked by the hydrophobic channel and the hydrophilic bridge, respectively.

Figure 2. (A) Synthesized routes of the PEG/PLGA block copolymers. (B) Chemical structures of the synthesized block copolymers and their 1H NMR spectra. (C) GPC profiles of the block copolymers and the corresponding initiators. (D) Molecular parameters of the synthesized copolymers.

Structural studies about soft matter have been challenging issues in the fundamental research studies.30−34 It is comprehensible that the amphiphilic block copolymers of PEG and polyester self-assemble into micelles at the sol state; but for this system without any strong ionic or hydrogen bond interaction, the mechanism of the further thermogelling and the structure of the thermogel are mysterious.

Some unique properties of PEG/polyester thermogels cannot be illustrated by the current theoretical models, such as the micelle jamming model, which has been confirmed as the internal structure of the thermogel of a commercial surfactant Pluronic composed of poly(ethylene oxide)-bpoly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPOPEO).35−37 The jamming without any real connection B

DOI: 10.1021/acs.macromol.9b00534 Macromolecules XXXX, XXX, XXX−XXX

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Figure 3. (A) Typical images of the Tri2 aqueous system (25 wt %) at different temperatures (25, 37, 60 °C). (B) Schematic of the relationship of block copolymers with different designed molecular weights (left) and their state diagrams in water (right). (C) Storage moduli (G′) and loss moduli (G″) of Di and Tri2 aqueous systems (25 wt %) as a function of temperature. (D) Complex moduli (G*) of different copolymer aqueous systems (25 wt %) as a function of temperature.

among different micelles explains why the duration of the Pluronic thermogel after being put into a large amount of water is only overnight. Compared with the Pluronic thermogel, the obvious longer duration of even 3 months of PEG/polyester thermogel indicates some strong connection among the micelles instead of micelle jamming. For the ABA triblock copolymer in water, once two hydrophobic end blocks of a copolymer are inserted into two micellar cores, the middle hydrophilic block is called a bridge.38,39 Lee’s group speculated that the formation of the bridge among different micelles was the key to the thermogelation of the PEG/polyester block copolymer.22 This speculation is reasonable, yet inapplicable to other thermogellable copolymers without the bridge such as BAB triblock copolymer or AB diblock copolymer. In our recent work, a semibald micelle and the corresponding percolated micelle network with a hydrophobic channel were proposed to interpret the thermogelling of the diblock copolymer.40 At low temperatures, the amphiphilic diblock copolymers self-assemble into crew-cut micelles in water.41,42 Then, as the temperature increases, the micellar corona consisting of the reversed thermosensitive PEG blocks collapses,43 leading to the formation of the semibald micelle with the micellar core partially exposed. To reduce the total exposed hydrophobic area, the exposed core areas of different semibald micelles come in contact and the hydrophobic channel forms at the core-to-core location. Finally, a percolated micelle network with hydrophobic channels as the crosslinking points forms, resulting in a gel at the macroscopic level.

In this mechanism model, the formation of the semibald micelle is the key to the thermogelling. The purpose of choosing a diblock copolymer as a model in our previous work40 is to avoid the disturbance of other complex factors, such as the bridge, and to obtain a universal understanding on the thermogelling. The BAB triblock copolymer shares a similar structure with the AB diblock copolymer without the bridge, thus we believe that it has a similar self-assembly process in water. The present work is focused on the structural studies of the thermogel of the ABA copolymer with the bridge structure, not only because it is more promising in clinic but also because it has richer physics. PLGA-PEG-PLGA triblock copolymer was selected as the model ABA copolymer in this work. Our basic idea of the thermogelling of the ABA copolymer is, in such a comparative study with AB- or BAB-type copolymers, schematically described in Figure 1. For ABA copolymers in water at low temperatures, the crewcut micelles form, and different micelles may be associated via the hydrophilic bridge. According to the dominant type of the cross-linking point, there might be two thermogel states in the ABA copolymer aqueous system. Nevertheless, the key evolution triggering thermogelling of the ABA copolymer (for both of these two states) is still the formation of the hydrophobic channel evolved from the semibald micelle, which is consistent with the thermogelling of the AB or BAB copolymer system. The present work aims to report, combining experiments and simulations, the existence of the two states of the C

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Macromolecules thermogel of ABA copolymers for the first time and meanwhile to reveal the universal mechanism of the structural evolution in thermogelation of all types of copolymers examined by us (ABA, BAB, and AB). The prerequisites of the molecular design for a thermogellable copolymer will finally be discussed.

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From the molecular parameters listed in Figure 2D, the relationship among the synthesized PEG/PLGA copolymers is graphically presented in Figure 3B and described as follows: the AB diblock copolymer Di is equivalent to half of the ABA triblock copolymer Tri2; the Tri1 and Tri3 copolymers correspond to shortening and elongating of the Tri2 copolymer, respectively. By comparing Di and Tri2 systems, the effect of the bridge structure of the ABA copolymer on thermogelling can be well studied; by comparing Tri1, Tri2, and Tri3 systems, the effect of the molecular weight on thermogelling can be investigated. Via a test-tube inversion method, state diagrams were obtained with three macroscopic states (sol, gel, and precipitate), as presented in Figure 3B. Once the copolymer concentration was higher than the critical gel concentration (CGC), sol−gel transition and gel−precipitate transition occurred upon heating, leading to the lower transition temperature (gelation temperature, Tgel) and the upper transition temperature (precipitate temperature, Tprecipitate), respectively. The Tgel decreased and the Tprecipitate increased with the increase of copolymer concentration. For the three triblock copolymer systems, with an increase in the molecular weight, the thermogel window moved to higher temperatures, while the basic shape remained similar. From Tri1 to Tri3, the CGC of these systems was almost at the same concentration, but the Tgel and Tprecipitate increased obviously. For the Tri3 aqueous system, besides a reversed thermogel state at high temperatures, a normal gel state at low temperatures was also observed. Compared with the Tri2 system, the CGC of the Di aqueous system was much higher, as well as the Tgel and the Tprecipitate. To characterize the thermogelling behavior quantitatively, storage moduli (G′) and loss moduli (G″) of Di and Tri2 aqueous systems (25 wt %) as a function of temperature were measured in dynamic temperature sweep experiments, respectively. The results are presented in Figure 3C. At low temperatures, the moduli of the system remained at low values and gradually decreased. The G″ was larger than the G′, indicating a sol state with good mobility. Then, around the Tgel, the moduli dramatically increased and the G′ exceeded the G″, indicating the formation of the thermogel without liquidity. In contrast to the Di aqueous system, the G′ and G″ of the Tri2 aqueous system were higher at low temperatures. In addition, upon heating, the divergent point of the moduli of the Tri2 system was at a lower temperature. Besides, the maximum values of G′ and G″ of the Tri2 system in the detected temperature range were much greater than those of the Di system. In order to characterize the strength of the copolymer aqueous system, the complex modulus G* was calculated from the G′ and G″ as follows

RESULTS

Synthesis and Characterization of the PEG/PLGA Block Copolymers. A series of PEG/PLGA block copolymers were synthesized by ring-opening polymerization with D,L-lactide (LA) and glycolide (GA) as monomers and PEG (for PLGA-PEG-PLGA) or methoxy PEG (mPEG for mPEGPLGA) as initiators. The synthesized routes are schematically presented in Figure 2A and described in the Supporting Information. The copolymers synthesized by us were characterized with 1 H NMR. The corresponding spectra are presented in Figure 2B. For the triblock copolymer PLGA-PEG-PLGA, the number-average molecular weight (Mn) was calculated from the peaks with chemical shifts at 3.60 ppm (CH2 of PEG), 4.80 ppm (CH2 of GA), and 5.25 ppm (CH of LA) and the known Mn of PEG. Similarly, for diblock copolymer mPEG-PLGA, the Mn was calculated from the peaks with chemical shifts at 3.40 ppm (CH3O of mPEG), 4.80 ppm (CH2 of GA), and 5.25 ppm (CH of LA) and the known Mn of mPEG. The molar ratio of LA and GA in a block copolymer was calculated by the areas of the peaks of CH of LA and CH2 of GA. The molar-mass dispersity Đ M of the PEG/PLGA copolymers and the corresponding initiators PEG or mPEG were measured by gel permeation chromatography (GPC) (Figure 2C). The relatively narrow GPC profiles without shoulder peaks indicate that the products are basically pure without obvious oligomers. Molecular parameters of the synthesized PEG/PLGA block copolymers are listed in Figure 2D. There are three ABA triblock copolymers PLGA-PEG-PLGA (named Tri1, Tri2, and Tri3) and one AB diblock copolymer mPEG-PLGA (named Di) with different molecular weights in this work. Except molecular weight, other molecular parameters of these copolymers were controlled in a similar manner in order to eliminate their disturbance on thermogelling. It should be noted that the BAB triblock copolymer PEGPLGA-PEG was not synthesized in our work. The reason lies in that the middle coupling agent linking two PEG-PLGA prepolymers in the PEG-PLGA-PEG copolymer influences the thermogelling process significantly,29,44 which will disturb our structural studies. Additionally, the BAB-type copolymer in water cannot form bridges between micelles (similar to the ABtype copolymer and different from the ABA-type copolymer). Therefore, it is not only relatively difficult to be synthesized but also less promising as a material. While we just prepared ABA and AB types of copolymers in our experiments, all of the three types were modeled in our dynamic Monte Carlo (MC) simulations. Sol−Gel Transition of the PEG/PLGA Block Copolymer Aqueous Systems upon Heating. The amphiphilic copolymer aqueous solutions were prepared by dissolving the synthesized block copolymers in water at low temperatures. Upon heating, the solution transformed into a physical gel; then at very high temperatures, the copolymer and water dissociated, and a precipitate was formed. The global views of the different states are presented in Figure 3A.

G* =

G′ + G″

(1)

The G* values of Tri1, Tri2, Tri3, and Di systems as a function of temperature are presented in Figure 3D. For G*, a gradual decay at the low-temperature range and an obvious enhancement around the sol−gel transition were observed. For triblock copolymers, the G* values at low temperatures increased with the molecular weight, so did the divergent temperature of the G*. In contrast to the Tri2 system, the G* of the Di system at a low temperature was much lower and the divergent point of G* appeared at a higher temperature. The D

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Figure 4. (A) CMC determinations of Di and Tri2 copolymers in water at 25 °C. The schemes inserted represent the morphologies of these two copolymers at different concentration regions. (B) Apparent hydrodynamic radii (Rh,app) of Di and Tri2 copolymers in water as a function of concentration measured by 3D DLS at 25 °C. (C) Schematic of the morphology evolutions of AB or ABA copolymer in water with an increase of concentration.

We first investigate the self-assembly morphologies of the amphiphilic block copolymers in water at low temperatures before thermogelling. For the injectability of the injectable thermogels, the properties of the sol before the thermogelling are critical. The sol state is the prestate of the thermogel, and its properties have a significant influence on the thermogelation. The critical micelle concentration (CMC or cmc) of the Tri2 copolymer in water was determined by a hydrophobic dye 1,6-diphenyl-1,3,5-hexatriene (DPH) as the probe. The cmc of the Di copolymer was also measured as a control. The ultraviolet (UV) absorption of DPH is sensitive to the polarity of the microenvironment, and it is enhanced once the hydrophobic probe DPH is encapsulated into the self-assembly core. Thus, the formation of the micelle leads to an uprush of the UV absorption of the DPH molecule. By the turning point of the UV absorption against the logarithm concentration, the cmc can be determined. From Figure 4A, the cmc values of Tri2 and Di copolymers in water were similar, and the UV absorptions of DPH at the same copolymer concentration of these two systems were very close. The UV absorption of the hydrophobic DPH molecule enriched in the micellar core was dependent on the corresponding microenvironment, and thus, the similar results of the Tri2 and Di systems indicated the similar core structures of these two systems. The aggregate size of the aqueous copolymer system was characterized by three-dimensional dynamic light scattering (3D DLS). While the traditional light scattering can only detect a dilute solution with just single scattering, the 3D DLS can be extended to a concentrated system by eliminating the interference of multiple scattering based on a special experimental design and data treatment.47 From the crosscorrelation function of the intensity of the scattered light, the hydrodynamic radius (Rh) can be calculated via the Stokes−

rheological measurements are consistent with the observations in the test-tube inversion experiments. The rheological results, especially from the dynamic frequency sweep experiments, can be utilized to analyze the internal structure of the hydrogel. As a control, a traditional amphiphilic block copolymer Pluronic F127 thermogel was measured. For different systems, the detection temperatures were set to guarantee that the systems were at gel states. Prior to the dynamic frequency sweep, the dynamic strain sweep was operated to determine the linear range of the moduli against strain (Figures S1 and S2). Then, a strain value in the linear range was determined in the subsequent dynamic frequency sweep experiments. Figure S3 shows the dynamic frequency sweep result of the Tri2 thermogel system. In the detected frequency range, the G′ and G″ increased with frequency as G′ ∼ f υ′ ;

G″ ∼ f υ″

(2)

Here, f is the frequency and υ′ and υ″ are the scaling exponents of G′ and G″ against f, respectively, and can be obtained from the data fitting. The values of υ′ and υ″ are related to the internal structure of the system to some extent.45,46 The G′ and G″ of different hydrogels against frequency and the corresponding scaling exponents are presented in Figure S4. The similar scaling exponents (both υ′ and υ″) of thermogels of Tri1, Tri2, Tri3, and Di indicate a similar structure of these reversed thermogels. However, for the scaling exponents of the normal gel (Tri3 system at 5 °C), the values were significantly smaller, indicating that the normal gel has a different structure from thermogels. For Pluronic F127, the exponents close to zero correspond to a gel of a known jamming structure. The difference between the exponents of F127 and PEG/PLGA thermogels indicates that the structure of the PEG/PLGA thermogel is far from a micelle jamming. Characterization of the Sol State To Investigate the Self-Assembly Morphologies Prior to Thermogelling. E

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Figure 5. (A) TEM images of the copolymer aqueous system (Tri2, 1 wt %) at the indicated temperatures. (B) Cross-correlation functions of the copolymer aqueous system (Tri2, 20 wt %) at the indicated temperatures (left) and the Rh,app of systems with different copolymer concentrations as a function of temperature (right). (C) Temperature-dependent 13C NMR spectra of a thermogellable copolymer aqueous system (Tri2, 25 wt %). A spectrum of copolymer in CDCl3 (25 wt %) at 25 °C was measured as a control. The right spectra are the magnified results of the left spectra in the box with the same color. The Tgel of this copolymer aqueous system was around 37 °C.

the Tri2 system might be ascribed to an early aggregation caused by the bridge association among different micelles. In the experiments, higher concentrated systems were not available because the copolymers were difficult to dissolve. Combining the results of the cmc and the aggregate size, the morphology evolutions of AB (Di) and ABA (Tri2) copolymers in water are schematically described in Figure 4C. In water, AB copolymers self-assemble into dispersed micelles. The number of the isolated micelles increases against copolymer concentration, whereas there are almost no interactions among different micelles. However, for the ABA copolymer aqueous system, different micelles may be associated by the bridge structure, indicating that the total aggregate size of the system is larger than that of the AB copolymer system at the same concentration, particularly at the high concentration region. Here, the micellar core of the ABA copolymer system is still isolated and similar to that of the AB copolymer system. Morphology Evolution of the ABA Copolymer Aqueous System during the Sol−Gel Transition. The

Einstein equation. Notably, the data processing approximates the aggregate as a perfect sphere, which may be different from the real structure. However, the evolution of the detected Rh can still reflect the evolution process of the aggregate in the system. In order to distinguish the detected Rh with the real Rh, the detected Rh was sometimes called apparent hydrodynamic radius Rh,app.48 The Rh,app values of Tri2 and Di copolymer systems with different concentrations were calculated from the corresponding cross-correlation functions shown in Figure S5. Some typical results of 3D DLS are presented in Figure 4B. For both Tri2 and Di copolymer aqueous solutions, at low concentrations (Tprecipitate), the peaks of the corresponding groups of the PLGA block became sharp, implying the destruction of the core−corona structure. In the good solvent CDCl3, both the PLGA and PEG blocks had good mobility, and thus, all typical peak signals of the copolymer were sharp. From the results about the morphology analysis in this section, the micelle network was confirmed as the internal structure of the thermogel. FRET Experiments To Investigate the Connections among Different Micelles of the Thermogel of the ABA Copolymer. In soft matter science, fluorescence resonance energy transfer (FRET) is usually employed to detect the nanoscale distance and its change among molecules, clusters, and so forth.49−52 In our study, FRET experiments were designed to detect the connections of different micelles in the thermogel. The mechanism of the FRET is schematically shown in Figure 6A, with two fluorescent molecules as donor and acceptor. If the distance r between donor and acceptor is short (r < 5 nm53), the excited fluorescent donor transfers energy to the fluorescent acceptor in the ground state via a long-range dipole−dipole interaction. Such a process is called FRET. The FRET efficiency (η) is related to r as η = R 0 6/(R 0 6 + r 6)

gel transition temperature (Tgel) of the system, which was consistent with the results of our previous work.40 When the FRET experiments were carried out in the ABA copolymer aqueous systems, an interesting phenomenon was observed. From Figure 6C, the TFRET was higher than Tgel. As the molecular weight increased, the difference between the values of TFRET and Tgel was even more pronounced. From an amplified figure (Figure S6), although the TFRET was higher than Tgel, a weak increase of FRET between the TFRET and Tgel was still observed, indicating a weak aggregation in this temperature range. By combining signals at the sol state with signals between the Tgel and TFRET, the other divergent temperature point of FRET (TFRET ′ ) was obtained. The TFRET ′ was similar to Tgel. Considering further that the TFRET ′ was not conspicuous, this parameter was not further analyzed. The FRET experiments were also carried out in other ABA systems with different concentrations (Figure S7). Except some low concentrated systems with always weak FRET signals and thus difficult to analyze, systems with relatively strong FRET signals exhibited an obvious difference between the values of TFRET and Tgel, which illustrated the universality of the phenomenon in the ABA copolymer aqueous system. The existence of the difference implied that there might be two different states in the thermogel of the ABA copolymer in water. Aimed at further investigating the internal structure of the thermogel, we designed a dynamic FRET experiment based on our analysis of the structure evolution. As schematically shown in Figure 6D, there are two possibilities when the aggregation among different micelles occurs. In the first case, a hydrophobic channel forms from the semibald micelles, and the fluorescent molecules can exchange between micellar cores via the channel, so after recooling the system to a sol, the FRET effect is still maintained. In the second case without any hydrophobic channel, the donor and acceptor are always isolated in different micellar cores, so the FRET effect might disappear once recooling. In our dynamic FRET experiment, the copolymer aqueous system containing donor and acceptor underwent a heating and recooling cycle. After equilibrating for a sufficiently long time, the still obvious FRET signal of the AB copolymer aqueous system confirmed the formation of the hydrophobic channel in the thermogelling process. For the ABA copolymer aqueous system, when the heating temperature was between the Tgel and the TFRET, the little FRET implied insignificant hydrophobic channel; when the heating temperature was higher than TFRET, the obvious FRET signal indicated the significant hydrophobic channel. On the basis of the FRET experiments and the following dynamic FRET experiments, we speculated that there were two states (gel-1 and gel-2) in the thermogel of the ABA block copolymer. In gel-1, the hydrophobic channel was insignificant. Considering the existence of the bridge structure in the ABA copolymer aqueous system, we suggested that the hydrophilic bridge acted as the main cross-linking point in this state. In gel2, by the evidence from the dynamic FRET experiments, the significant hydrophobic channel served as the main physically cross-linking point. Rheological Studies under Heating−Cooling Cycles To Confirm the Transition between Gel-1 and Gel-2. The FRET experiments implied two states in the thermogel of ABA copolymers. To further confirm the existence of gel-1 and

(3)

Here, R0 is the distance between donor and acceptor at which the FRET efficiency is 50%. In this work, phenanthrene (Phe, donor) and anthracene (An, acceptor) were chosen as the FRET pair. The FRET efficiency (η) was quantified by I402/I366, where I402 and I366 represent the intensities of the characteristic emission fluorescence peaks at 402 and 366 nm for donor and acceptor, respectively. The excitation wavelength was 294 nm. We first dispersed the donor or acceptor into the two separate aqueous solutions of PEG/PLGA copolymers at low temperatures before thermogelling. The fluorescent molecules were encapsulated into the micellar core owing to their strong hydrophobicity. Then these two systems with either donor or acceptor were mixed together. No FRET signal could be observed if the donor and acceptor are encapsulated into different micelles with a long distance. Once the aggregation occurs among different micelles, the shortened distance between the donor and acceptor leads to an enhancement of the FRET. Notably, the exchange of the donor and acceptor among different micelles through water can be ignored, which is ascribed to the strong hydrophobicity of the fluorescent molecules and is proved by an almost unchanged FRET signal of a copolymer aqueous solution with both donor and acceptor in a four-day tracking. A homogeneous system is a premise of the accurate measurement of the fluorescence signals, thus the FRET experiments were operated only at the sol and gel states and were stopped before the gel−precipitate transition. The FRET experiment was first carried out in an AB diblock (Di) aqueous system. As shown in Figure 6B, the weak and stable FRET signal at low temperatures implied many isolated micelles in the sol; the increased FRET signal with temperature indicated the aggregation of different micelles. The divergent temperature point of the FRET (TFRET) was close to the sol− H

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Figure 7. (A,B) G* under eight heating−cooling cycles with the indicated temperature ranges of the Tri2 copolymer aqueous system (25 wt %) as a function of time. (C) FRET signal of the Tri2 system (25 wt %) as a function of temperature. The blue and red dashed lines correspond to the Tgel and TFRET, respectively. The orange shadow corresponds to the transition range determined by the rheological measurements. The inserted schemes are the possible cross-linking points in gel-1 and gel-2.

ature range was first set from 37 to 47 °C in the thermogel state. For each cycle, the G* in the cooling process was much lower than that in the heating process. The maximum G* of a cycle decayed with the increase of the cycle number, resulting from the hysteresis in the whole process, which might be caused by a state transition. In order to determine the possible transition range, we did the rheological measurements in more narrow temperature gaps, as presented in Figure 7B. For temperature ranging from 37 to 40 °C, the hysteresis of the G* disappeared, and with the increase of the cycle number, the maximum G* of a cycle remained almost unchanged. The similar phenomenon was observed in the temperature range from 44 to 47 °C. In contrast, for an intermediate temperature range from 40.5 to 43.5 °C, both an obvious hysteresis and a decay of G* were observed, implying a state transition in this temperature range. From the rheological measurements of the ABA copolymer aqueous system, we found, besides a sol−gel transition, a new transition in the thermogel state. The transition temperature was located in the temperature range from 40.5 to 43.5 °C, consistent with the TFRET of about 42 °C obtained from the FRET experiment of the same system (Figure 7C). The existence of two states with different main cross-linking points of the thermogel of the ABA copolymer was thus confirmed. Computer Simulations To Mimic the Thermogelling Behavior of the Amphiphilic Copolymer Aqueous System. The above experiments illustrated the existence of two states of the thermogel of the ABA copolymer. The internal structures of these two states were revealed by dynamic FRET experiments indirectly. Next, we employed

gel-2 and the transition between these two states, a series of rheological studies were designed and carried out. First, rheological measurements under a heating−cooling cycle were operated on the Tri2 and Di copolymer aqueous systems. The Pluronic F127 aqueous system was also measured as a control. From Figure S8, for both Tri2 and Di copolymer aqueous systems, an obvious hysteresis of the moduli was observed when the temperature dropped below Tgel in the cooling process. The moduli of the cooling process were much higher than those of the heating process. However, for the F127 aqueous system, the moduli of the heating and cooling cycle overlapped very well. The hysteresis of the moduli of the PEG/PLGA aqueous system below the Tgel indicated the formation of strong physical interactions among different micelles during the sol−gel transition, far from micelle jamming without strong interactions like thermogelling of the Pluronic F127 aqueous system. In the thermogel state above Tgel of the ABA copolymer system, the moduli in the cooling process were lower than those in the heating process, whereas in the thermogel state of the AB copolymer system, the moduli in the cooling process and the heating process were overlapped. The other hysteresis of the moduli above Tgel of the ABA copolymer system indicated that there might be a transition in the thermogel state. In order to analyze this interesting phenomenon, rheological measurements under several heating−cooling cycles in the temperature range of the thermogel state were employed. The complex modulus G* of the Tri2 system under eight heating−cooling cycles is shown in Figure 7A. The temperI

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Figure 8. (A) Schematic of the coarse-grained models in simulations. (B) Energy parameters in our simulations. The values of a and b in the relationship between ε*BV and T were a = 2.27 and b = 745 K, respectively. The red circles in the right curve correspond to the temperature points simulated in this work.

Here, kB is the Boltzmann constant and T is the Kelvin temperature. Between i and j beads, εij* > 0 indicates repulsion, while εij* < 0 indicates attraction. Thus, the reduced energy between a hydrophobic bead and a vacancy εAV * was set positive. According to a good compatibility between PLGA and PEG,63,64 the reduced energy between a hydrophilic bead and a hydrophobic bead εAB * was set negative. On account of the thermosensitivity of the PEG block, an appropriate relation of the reduced energy between a hydrophilic bead and a vacancy ε*BV versus temperature could help us simulate the systems with different temperatures. The determination of this quantitative relationship is described in Supporting Information Methods. The default energy parameters are presented in Figure 8B. In a dynamic MC simulation, the number of MC step (MCS) is linearly related to the real physical time.54 One MCS indicates the trial that every bead in the system has been randomly selected once on average. Therefore, the time t is measured in units of MCS in our dynamic MC simulations. Simulations at Low Temperatures To Analyze the Self-Assembly Morphologies of a Sol. For the sake of operability, a series of physical quantities were defined in our simulations. A micellar core was defined as follows: if any contact (nearest-neighbor or next-nearest-neighbor) occurred between any two hydrophobic beads of two hydrophobic A blocks, these two A blocks belonged to one core. If two end A blocks of a ABA model chain are inserted into different micellar cores, the middle B block was a hydrophilic bridge. If two micellar cores constituted of A blocks contacted, these two micellar cores might merge into one and the hydrophobic channel formed at the core-to-core location. According to the definition of the bridge in our simulations, the previous hydrophilic bridge between two micelles disappeared after fusion of these two cores. We introduced aggregate-total and aggregate-core as two crucial terms of the aggregate in our simulations, as

computer simulations of thermogelling to observe the structure directly and further reveal the mechanism of this process. The simulation program was made by ourselves. We carried out dynamic MC simulations54−58 to model the thermogelling of amphiphilic block copolymers in water. Selfavoiding chains were simulated in two-dimensional lattices with the periodical boundary condition at both directions (x, y). The Larson fluctuation model59 was selected as the main microrelaxation mode, and the partial reptation algorithm60,61 was employed to enhance the simulated efficiency. Metropolis sampling62 was used as the importance sampling approach. The corresponding simulation details are described in the Supporting Information Methods. In our simulations, an amphiphilic block copolymer was coarse-grained to a model chain comprising a series of beads, as schematically shown in Figure 8A. One bead was a collection of a few repeating units, similar to a Kuhn segment. In order to mimic the experiments, the default AB diblock model chain was half of the default ABA triblock model chain. Besides, the BAB copolymer with the same length as the default ABA copolymer was also investigated in the simulations. For the default AB diblock copolymer, BAB triblock copolymer, and ABA triblock copolymer, their chain models were represented as AxBy, ByA2xBy, and AxB2yAx, respectively, where A and B represent hydrophobic and hydrophilic beads. Subscripts represent the number of beads in the corresponding blocks. The default values of x and y were x = 24 and y = 8, respectively. A coarse-grained solvent in the simulation system was considered as a vacancy and represented by V. Both nearest-neighbor and next-nearest-neighbor pairwise interactions were considered in our simulations. The energy parameter determined directly was reduced energy ε*ij , where the subscripts i and j represent the species in the system. The εij* is related to the energy εij as εij* = εij /kBT

(4) J

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Figure 9. (A) Schematic of the aggregate-total and the aggregate-core (left) and the Mw(aggregate) of the A24B16A24 system as a function of φ (right). The dashed lines represent the transition positions between indicated states. (B) Schematic of the B block states in self-assembly morphologies of the ABA triblock copolymer in water (left) and the fractions of different states of the hydrophilic B blocks as a function of φ (right).

similar to that of the Rh,app against concentration in the ABA copolymer aqueous solutions (Figure 4B). The gel network at high concentrations was unavailable in experiments because the copolymer could not well dissolve under such a circumstance. The network at high φ might correspond to the normal gel observed in the Tri3 copolymer aqueous system at low temperatures. The low FRET signal of the normal gel implied that the micellar cores were dispersed (Figure 6C), consistent with the structure of gel(bridge) in our simulations. The molecular weight effect on the self-assembly morphologies at low temperatures was also examined, with results shown in Figure S11. In contrast to the A24B16A24 triblock copolymer, the elongated model copolymer A 96 B 64 A 96 exhibited an earlier aggregation against φ, which might be ascribed to the fact that a longer hydrophilic chain was favorable for the formation of the bridge. This result explained why the normal gel was only observed in the Tri3 copolymer aqueous system instead of other systems of copolymers with lower molecular weights in our experiments (Figure 3B). Besides, either an extreme short hydrophobic block (A8B16A8) or an extreme long hydrophilic block (A24B208A24) was unfavorable for the aggregation. For both systems, the low volume fraction of hydrophobic blocks resulted in an extreme long distance among micellar cores, hindering the formation of the bridge. In simulations, the fractions of the states of B blocks can be calculated conveniently, which is unavailable in experiments. There are four states of B blocks in the self-assembled structures of the ABA copolymer, as schematically shown in Figure 9B. If two end A blocks of a copolymer are inserted into two micellar cores, the middle B block forms a bridge; if two end A blocks of a copolymer are inserted into a micellar core, the middle B block forms a loop; if an end A block is inserted into a micellar core but the other is isolated, the B block forms

schematically shown in Figures 9A and S10. The aggregatetotal was linked by a hydrophobic channel or a hydrophilic bridge, corresponding to the total aggregate structure; whereas the aggregate-core was linked only by a hydrophobic channel, corresponding to the core structure. For the AB or BAB copolymer system without any bridge, these two terms were equivalent, but for the ABA copolymer system, the aggregatetotal had a wider range. The aggregate size was characterized by the weight-average molecular weight of aggregate Mw(aggregate). n

M w (aggregate) =

∑i = 1 Mi 2 n

∑i = 1 Mi

(5)

Here, Mi represents the number of beads in the ith aggregate and n represents the number of aggregates in the simulation system. For aggregate-total and aggregate-core, this parameter could be called Mw(aggregate-total) and Mw(aggregate-core), respectively. The evolution process of the aggregate size against concentration or volume fraction φ of the ABA copolymer aqueous system at 25 °C is presented in Figure 9A. An always low Mw(aggregate-core) indicated that the micellar cores were dispersed and did not significantly aggregate even at high φ. For Mw(aggregate-total), there were three φ regions with different evolutionary behaviors. When φ was below 0.18, the value was low and stable, and then the value steadily increased in the φ range from 0.18 to about 0.32; when φ was above 0.32, the value grew slowly and was almost proportional to φ, indicating the formation of a whole network. In the simulated φ range, there was always a difference between the Mw(aggregate-total) and the Mw(aggregate-core), which was caused by the hydrophilic bridge. On the basis of the Mw(aggregate-total), we named three regions: less-bridged micelle, bridged micelle, and gel(bridge). The evolutionary trend of Mw(aggregate-total) against φ was K

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Figure 10. (A) Mw(aggregate) of the A24B16A24 system as a function of temperature. (B) Fractions of different states of hydrophilic B blocks as a function of temperature. (C) Schematic of the evolution process of the ABA copolymer aqueous system upon heating.

and M w (aggregate-core) were called T gel‑1 and T gel‑2, respectively. In order to analyze the internal structures of the system, fractions of B block states were counted (Figure 10B). The fractions of bridge, isolated chain, and dangled chain decreased with the increase of temperature, while the evolution tendency of the fraction of loop was just the opposite. Around Tgel‑2, the changes of these fractions of states were evidently acute. Taking both results in Figure 10A,B into consideration, the morphology evolution of the ABA copolymer aqueous system upon heating is schematically shown in Figure 10C. At low temperatures, there are many bridged micelles linked by the bridges dispersed in the system. With an increase in temperature, the collapsed bridges of the reversed thermosensitive B block are disadvantaged for the association among the distant micellar cores. In some areas, some aggregate-totals dissociate, corresponding to the decrease of the Mw(aggregatetotal) and the fraction of bridge. According to the almost unchanged Mw(aggregate-core), the micellar cores are barely affected. The decreased aggregate size and almost unchanged micellar cores in simulations were consistent with the results from the 3D DLS (Figure 5B) and FRET experiments (Figure 6C) at low temperatures. As the B block further collapses with an increase of temperature, the hydrophobic channel is formed, and the bridge decreases correspondingly. The formation of the hydrophobic channel is revealed by a slight increase of M w (aggregate-core) and leads to an increase of the Mw(aggregate-total). When the temperature is above Tgel‑1, a small number of hydrophobic channels and a relatively large number of hydrophilic bridges are combined to constitute the network, corresponding to the gel-1 state. Then at higher temperatures above Tgel‑2, the hydrophobic channel becomes dominant, while the bridge becomes even less, corresponding to the gel-2 state. The driving evolution for both gel-1 and gel2 is the formation of the hydrophobic channel, which is evolved from the semibald micelles confirmed in our previous work.40 From the analysis of the internal structures, gel-1 and

a dangled chain; and if the ABA copolymer is isolated, the B block forms an isolated chain. From Figure 9B, with the increase of φ and the maturing of the self-assembled structures, the fractions of the bridge and the loop increased, the fraction of the dangled chain first increased and then decreased, and the fraction of the isolated chain always decreased. The obvious increase of the fraction of the bridge explained the increase of the Mw(aggregate-total) very well. According to our simulations, the existence of numerous bridges in the ABA block copolymer aqueous systems before thermogelling was confirmed, which was consistent with our speculations based on experiments. Simulations of Systems at Different Temperatures To Investigate the Thermogelling Behavior. On the basis of the relationship between ε*BV and T, the systems at different temperatures can be modeled. Thus, the thermogelling process was analyzed in our simulations. Mw(aggregate-total) and Mw(aggregate-core) were calculated to characterize the evolution of the aggregate with increase of temperature in the ABA copolymer aqueous system (Figure 10A). At low temperatures, the decreased Mw(aggregate-total) indicated the decrease of the aggregate size; with an increase in temperature, an enlarged Mw(aggregate-total) indicated the further aggregation. The low Mw(aggregate-core) at low temperatures implied that the micellar cores were isolated, whereas the increased Mw(aggregate-core) at high temperatures indicated the aggregation of the cores and the formation of the hydrophobic channel. According to our previous work and some reports from others, 40,65 the divergent temperature point of the Mw(aggregate)-temperature curve was defined as the sol−gel transition temperature (Tgel). From Figure 10A, the divergent temperature point of Mw(aggregate-total) was obviously lower than that of the Mw(aggregate-core), implying two transitions. These two transition temperatures from Mw(aggregate-total) L

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Figure 11. (A) State diagrams of B8A48B8, A24B8, and A48B16 systems. (B) Typical snapshots of the A24B8 system. The snapshots correspond to positions indicated by the sunlike symbols in the state diagram in (A). For each snapshot, the corresponding scheme is inserted into the bottom right.

Figure 12. (A) State diagram of the A24B16A24 system. (B) Typical snapshots of the A24B16A24 system. The snapshots correspond to the positions indicated by the symbols linked by dashed lines with respect to the four paths in the state diagram in (A). For each snapshot, the corresponding scheme is inserted into the bottom right.

As presented in Figure S12, the molecular weight effect on the thermogelling was investigated. When the model chain was elongated from A24B16A24 to A96B64A96, the aggregation

gel-2 in our simulations correspond well to gel-1 and gel-2 in our experiments, respectively. M

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besides the two thermogel states, the ABA copolymer system had the lower CGC, the lower Tgel, and the lower Tprecipitate, which arose from the bridge effect. The CGCs of AB and BAB block copolymers were similar, while both the Tgel and the Tprecipitate of the BAB copolymer system were lower. For the AB copolymer systems with an increase in the chain length, the CGC remained unchanged, but both the Tgel and the Tprecipitate moved to higher temperatures. Bridge is a feature of the ABA copolymer system. To further analyze the effect of the bridge, the aggregate sizes of the A24B16A24 system and A24B8 (equivalent to half of the A24B16A24) system were calculated. The corresponding statistics was first conducted at the sol state (Figure S18). For these two systems, the similar Mw(aggregate-core) at the same φ indicated a similar core structure. In contrast to the low Mw(aggregate-total) of the AB copolymer system, the high Mw(aggregate-total) of the ABA copolymer system resulted from the bridge structure. These results are consistent with those of the cmc measurements and the 3D DLS measurements for diblock and ABA triblock copolymer aqueous systems (Figure 4A,B). The effect of the bridge structure on thermogelling was also investigated. Figure S19A shows that the aggregation of the ABA block copolymer occurred earlier than that of the AB block copolymer when φ was 0.25; while with the increase of φ to 0.5, the contrary case was observed. The aggregation of the ABA copolymer at relatively low φ was attributed to the constraint of the bridge. However, at high φ, the constraint of the concentration was dominant; thus, the constraint of the bridge was obscured. Considering that the aggregation was driven by the formation of the semibald micelles that are mainly affected by the micellar corona, we also investigated the thickness of the micellar corona in the AB and ABA copolymer systems. In the simulated ABA system, the thickness of the micellar corona corresponded to half the length of bridge or loop, whereas in the AB copolymer system, the thickness corresponded to the length of B block. From Figure S19B, both for a system with φ of either 0.25 or 0.5, the micellar corona of the ABA copolymer system was thicker than that of the AB copolymer system. The thicker corona of the ABA copolymer system might inhibit the formation of the semibald micelles and further aggregation. At high φ, when the constraint of bridge was obscured, the inhibition became dominant in the ABA copolymer system. Figure S19B shows mean-squared end-to-end distances of half of the bridge and half of the loop in the A24B16A24 system and the B block in the A24B8 system as a function of temperature, and in the ABA copolymer system, half of the bridge was longer than half of the loop, indicating a more inhibition in the area rich in bridges for the formation of semibald micelles. Herein, the hydrophobic channel first forms in areas without the bridge. Only at higher temperatures with more collapse of the hydrophilic blocks, the hydrophobic channel forms in the bridge area. The uneven formation of the hydrophobic channel gives a relatively clear boundary of gel-1 and gel-2.

occurred at a higher temperature. Besides, shortening the hydrophobic block and elongating the hydrophilic block were disadvantageous for the aggregation. These results are comprehensible from the formation of the semibald micelle. Either absolutely long or relatively long hydrophilic blocks lead to a thick corona, not supporting the formation of the semibald micelle with a partially exposed core. State Diagrams of Aqueous Systems of AB and BAB Block Copolymers Obtained by Simulations. To understand the thermogelling intuitively and comprehensively, we determined the state diagram from our computer simulations based on the corresponding parameters and some definitions. The state diagrams of the systems of AB and BAB block copolymers without any bridge were first calculated. The criteria of different states are described in Figures S13 and S14. The state diagrams of AB and BAB block copolymer systems are presented in Figure 11A. Each system exhibited three statesmicelle, gel, and precipitate. Once φ was larger than CGC, a gel window with a lower transition temperature (Tgel) and an upper transition temperature (Tprecipitate) appeared. With an increase in φ, the change tendencies of Tgel and Tprecipitate are consistent with those in the state diagram obtained from experiments (Figure 2B). The typical snapshots of the AB system are shown in Figure 11B. At the micelle state, many isolated micelles led to a sol with fluidity; then these micelles aggregated together by the hydrophobic channel and formed the percolated micelle network at the gel state; at the precipitate state, all of the beads aggregated and were dissociated with the solvent. State Diagram of the ABA Block Copolymer Aqueous System Obtained by Simulations. On the basis of the criteria described in Figures S13 and S14, the state diagram of the ABA-type copolymer aqueous system was obtained and presented in Figure 11A. Six states described in detail in the sections above were exhibited in the diagram. At the lowtemperature range, states of less-bridged micelle, bridged micelle, and gel(bridge) appeared in sequence with an increase in φ. The shape of the state window of the gel(bridge) indicated that the gel(bridge) was a normal gel, corresponding to the normal gel observed in experiments. Once φ was higher than about 0.075, a thermogel containing two states, gel-1 and gel-2, appeared with an increase in temperature. The shape of the thermogel window is consistent with that in the state diagram obtained from the test-tube inversion method (Figure 3B). On the basis of the analysis of the internal structures (Figure 10) and the shape of the thermogel window (Figure 12), an excellent correlation was found between the two states of the thermogel obtained via simulations and the two states of the thermogel found via FRET experiments and rheological measurements. Some typical snapshots of the ABA copolymer aqueous system are shown in Figure 12B. In order to have a comprehensive understanding of the system, snapshots of one path with an increase of volume fraction φ and three paths with an increase of temperature are presented. From Figure 12B, the internal structures of different states and their evolutions can be directly observed, which affords a general physical picture about the responding of the ABA copolymer aqueous system to concentration or temperature. For convenience of comparison among ABA-, BAB-, and AB-type copolymers, the state diagrams of different copolymer systems are drawn in combination ways, as depicted in Figures S15−S17. In contrast to the AB copolymer aqueous system,

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DISCUSSION As an injectable hydrogel, the PEG/PLGA thermogel shows great potential in clinic.21,66,67 In such a material family, the thermogel of the ABA triblock copolymer exhibits stable performance, high modulus, and adjustable degradation, which makes this material very promising. However, even this N

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Figure 13. (A) Schematic of the state diagrams of the ABA and AB or BAB copolymer aqueous systems. (B) Differences and similarities of the thermogellings of the ABA and AB or BAB copolymers. (C) Schematic of the key structural evolution triggering thermogelling (top) and the prerequisites of the molecular design for a thermogellable copolymer (bottom).

Semibald micelles are based on the crew-cut micelles. The self-assembled structure of the thermogellable ABA copolymer in water at low temperatures is a crew-cut micelle. Hydrophobic PLGA block and hydrophilic PEG block constitute mainly the micellar core and corona, respectively. Among different neighboring micelles, the bridge structure forms spontaneously, leading to many multimicelle aggregates at the sol state. With an increase in temperature, the reversed thermosensitive PEG block43 results in the collapse of the micellar corona. The collapse of a thin corona might lead to the partial exposure of the micellar core, corresponding to the formation of the semibald micelle. Because of the hydrophobicity of the exposed areas of the core, the semibald micelle is unstable. Therefore, semibald micelles aggregate in order to reduce the total area of the exposure, and the hydrophobic channel forms at the position of the core contact. This evolution is similar to that of the diblock copolymer aqueous system demonstrated in our previous work.40 For the ABA triblock copolymer aqueous system, there are two thermogel states appearing in sequence with an increase in temperature. Because of the obstacle of the bridge, the semibald micelle first forms in some areas without bridge, and then the hydrophobic channel forms in these areas spontaneously. With the help of the bridge structures already existing in abundance, a small number of hydrophobic channels might lead to the formation of a whole network. This state is called gel-1, and its main cross-linking point is the hydrophilic bridge. With further increase in temperature, the more collapse of the corona leads to the formation of the semibald micelle in many areas rich in bridges. Therefore, the number of hydrophobic channels increases dramatically, whereas the number of bridges decreases. The hydrophobic

material has still not been clinically applied, rooting in unresolved technical details. In order to overcome these difficulties, a clear illustration of the internal structure and mechanism of this material is very necessary. Although it is common for amphiphilic copolymers to self-assemble into micelles in water, the thermogelation upon heating is not usual. The structural studies of this infrequent thermogelation might stimulate the soft matter science. In this work, we synthesized a series of PLGA-PEG-PLGA block copolymers as models to investigate the thermogelling behavior (Figure 2). These copolymer aqueous systems exhibited different sol−gel transition behaviors upon heating (Figure 3). The self-assembly morphologies at different temperatures were characterized by a series of experiments (Figures 4 and 5). FRET experiments and rheological measurements under heating−cooling cycles were carried out to investigate the internal structures of the aggregates (Figures 6 and 7). In order to directly observe the structure and reveal the inner mechanism, the computer simulations were operated (Figures 8−12). The structure and the mechanism of the thermogelling system are discussed as follows, as well as the comparison between the thermogels of the ABA copolymer and AB or BAB copolymer. Finally, the prerequisites of the molecule design for a thermogellable copolymer will be proposed. Semibald Micelle, Hydrophobic Channel, and Two Thermogel States in the Thermogelling of the ABA Copolymer Aqueous System. On the basis of the relative thicknesses between core and corona, Eisenberg classified micelles into starlike micelles and crew-cut micelles.41,42 In order to interpret the thermogelling, we put forward the concept and the term of semi-bald micelles. O

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Figure 13C, the semibald micelle is the precursor of the thermogelling. Once the semibald micelle is formed, the hydrophobic channel and the further thermogelling occur spontaneously if the polymer concentration is above CGC. Therefore, any strategy favoring the formation of the semibald micelle promotes the thermogelling thermodynamically. On this basis, the prerequisites of molecular design for a thermogellable copolymer are proposed in Figure 13C and described as follows. First, the amphiphilic property is necessary for the hierarchical self-assembly of the copolymer in water. Besides, for the formation of the semibald micelle, a thin corona is essential, and thus before thermogelling, the self-assembly structure should be the crew-cut micelle. The hydrophilic block constituting the corona should thus be short, which becomes another prerequisite in the molecular design. From our demonstrations on the effect of the molecular weight on thermogelling (Figure S12), this point has been well illustrated. Furthermore, the reversed thermosensitivity of the corona is needed and provides an evolution force responding to the temperature stimulus of the system. At low temperatures, the strong hydrophilicity of the hydrophilic block ensures the dispersion of the isolated micelles and makes the sol injectable; at high temperatures, the weak hydrophilicity of the hydrophilic block promotes the collapse of the corona, resulting in the formation of the semibald micelle and the further thermogelling. For clinical applications, a reasonable thermosensitivity is necessary to make the sol−gel transition occur around body temperature. Finally, the interaction between the hydrophobic and hydrophilic segments should be attractive. A good compatibility between PEG and PLGA has been confirmed. Thus, in our simulations, the default reduced energy ε*AB between the hydrophobic A bead and hydrophilic B bead was set negative. We also investigated the effect of the ε*AB on thermogelling, as shown in Figure S20. With other parameters unaltered, when * = −0.2, that is, the interaction between A and B beads was εAB attractive, a network with the hydrophobic channel was well * = 0.0 or εAB * = 0.2, reproduced; however, when εAB corresponding to no interaction or even repulsion between A and B beads, the self-assembly structure was still a crew-cut micelle or bridged micelle. From the perspective of the formation of the semibald micelle, these results are comprehensible. The repulsion between the core and corona is adverse to the corona collapse, which hinders the formation of the semibald micelles and the further aggregation. Furthermore, the results in our simulations explain why the thermogel structure of Pluronic is only from micelle jamming without any real cross-linking point, for the Huggins parameter between PEO and PPO is high in water, corresponding to a strong repulsion.68,69 As investigated in our previous work, if the hydrophobicity of the micellar core is much stronger, the network will still form at higher temperatures, although the interaction between the hydrophobic segment and hydrophilic segment is repulsive. However, the network is a wormlike micelle network, instead of a micelle network with a narrow hydrophobic channel. This wormlike micelle structure has been found in a Pluronic P85 aqueous system at 70 °C,69,70 a methoxy poly(ethylene glycol)b-poly(trimethylene carbonate) aqueous system at 40 °C,71 and some other soft matter systems.72−74

channel becomes the dominant cross-linking point in this new state, called gel-2. The hindering of the bridge on the formation of the semibald micelle endows gel-1 and gel-2 with a relatively obvious boundary. Different from some thermogels formed by micelle jamming,35,37 the thermogel of the PEG/PLGA block copolymer has the real physical cross-linking point of a hydrophilic bridge or a hydrophobic channel. The existence of the real cross-linking point explains why the thermogel could maintain its integrity for a long time after the formed physical hydrogel is put into a large amount of water. Differences and Similarities of Thermogellings of the ABA Copolymer and AB or BAB Copolymer. Under a reasonable control of the molecular structures, the thermogellings of the ABA copolymer and AB or BAB copolymer can be compared. Combined with the schematic state diagrams based on our experiments and simulations (Figure 13A), the main differences and similarities of the thermogellings of these copolymers are listed in Figure 13B. In contrast to the thermogels of the AB or BAB copolymer with only one thermogel state, the thermogel of the ABA copolymer has two states, termed as gel-1 and gel-2. The dominant cross-linking point in gel-1 is the hydrophilic bridge, and the dominant cross-linking point in gel-2 is the hydrophobic channel. For the only one thermogel state of the AB or BAB copolymer, the cross-linking point is merely the hydrophobic channel. In terms of the thermogel properties, the CGC of an ABA copolymer aqueous system is much lower than that of an AB or BAB copolymer system. Similar cases happen for Tgel and Tprecipitate. In addition, from the rheological measurements (Figure 3), the maximum moduli of the ABA copolymer thermogel are much higher than those of the AB copolymer thermogel. There is a distinct decay of the moduli around the transition position between gel-1 and gel-2, as illustrated with the FRET experiments. These differences in moduli can be understood by the different cross-linking points of the thermogels. The bridge structure linked by covalent bonds may have a higher strength than the hydrophobic channel constituted mainly by hydrophobic aggregation. Compared with the differences, the similarities of different systems are more important, which can lead to a universal understanding of the thermogelling. On the basis of our findings, for both ABA copolymer aqueous system and AB or BAB copolymer aqueous system, the key evolution triggering the thermogelling was the formation of the hydrophobic channel evolved from the semibald micelles. For the ABA copolymer aqueous system, the small number of hydrophobic channels plays a crucial role in gel-1 formation: the newly formed channels link the numerous bridged micelles together and lead to the formation of a whole network. Although the hydrophilic bridge is the dominant cross-linking point, the hydrophobic channel plays a role of the final driving force of the transition. In the gel-2 state, the effect of the hydrophobic channel is more obvious and acts as the dominant cross-linking point in the network. For the thermogel of the AB or BAB copolymer, the importance of the hydrophobic channel is more significant because it is the only physical cross-linking point in the system. The hydrophobic channel is spontaneously evolved from the semibald micelles. Thus, the formation of the semibald micelle is the key to thermogelling of all these copolymer aqueous systems. Universal Prerequisites of Molecular Design for a Thermogellable Copolymer. As schematically shown in P

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CONCLUSIONS

ACKNOWLEDGMENTS This work was financially supported by NSF of China (grant nos. 21774024 and 51533002) and National Key R&D Program of China (grant no. 2016YFC1100300).

The internal structure and mechanism of thermogelling of the ABA block copolymer were investigated by the combination of experiments and simulations. Two states of thermogel of the ABA copolymer, termed as gel-1 and gel-2, were found in the present study. The hydrophilic bridge and the hydrophobic channel acted as the main cross-linking points for gel-1 and gel-2, respectively. The mechanism of thermogelling was analyzed at the molecular level, and the formation of the hydrophobic channel evolved from the semibald micelles was illustrated as the key evolution triggering the thermogelling. On the basis of our structural studies, the prerequisites of the molecular design for a thermogellable copolymer were proposed. Findings in this study can not only illustrate the thermogelling behavior of the amphiphilic block copolymer but also afford the guidance for the corresponding molecular design, which is of significance to further research and the clinical development of this kind of new biomaterials. As a fundamental research in soft matter science, the structure and mechanism model in this study might be stimulating for understanding other hydrogels and other stimuli-responsive systems.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.9b00534. Dynamic strain sweep rheological results of some PEG/ PLGA hydrogels and a Pluronic F127 hydrogel, dynamic frequency sweep rheological result of the Tri2 thermogel, dynamic frequency sweep rheological results and corresponding scaling exponents, cross-correlation functions obtained from 3D DLS, FRET signal of a Tri2 copolymer aqueous system as a function of temperature, FRET signals of Tri2 copolymer aqueous systems with different concentrations, rheological results of some PEG/PLGA copolymer aqueous systems and a Pluronic F127 aqueous system under a heating−cooling cycle, schematic illustrations of the aggregate-total and aggregate-core in simulations, molecular weight effect on the sol properties and the thermogelling behaviors, criteria of different states in simulations, comparison of state diagrams of different copolymer aqueous systems, bridge effect on the sol properties and the thermogelling behaviors, and importance of the attraction between the hydrophilic and hydrophobic blocks on thermogelling (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Lin Yu: 0000-0001-7660-3367 Jiandong Ding: 0000-0001-7527-5760 Notes

The authors declare no competing financial interest. Q

DOI: 10.1021/acs.macromol.9b00534 Macromolecules XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.macromol.9b00534 Macromolecules XXXX, XXX, XXX−XXX