Semi-bald Micelles and Corresponding Percolated Micelle Networks

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Semi-bald Micelles and Corresponding Percolated Micelle Networks of Thermogels 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: As an injectable and biodegradable hydrogel, thermogels of amphiphilic block copolymers of polyester and polyether in water show great potential in biomedical fields. It is challenging to reveal the mechanism behind the reversed thermogelling with sol−gel transition upon heating. Herein, a computer simulation and corresponding experiments are combined to examine aqueous systems of amphiphilic diblock copolymer of methoxypoly(ethylene glycol) and poly(D,L-lactideco-glycolide). We synthesized the copolymer via ring-opening polymerization and characterized the thermogelling behavior of its aqueous solution by 3D dynamic light scattering and diffusing wave spectroscopy. Fluorescence resonance energy transfer and 13C NMR spectroscopy etc. were also adopted to explore the structure change during thermogelation. A dynamic Monte Carlo simulation was performed for a corresponding multichain system. A new type of micelle, the semi-bald micelle, was first proposed as the precursor for thermogelling and was confirmed from both simulations and experiments. We demonstrate that the thermogel structure is a percolated micelle network with hydrophobic channels that evolved from the semi-bald micelles. The thermogelling mechanism is discussed at the chain level. On the basis of the mechanism study, we put forward the molecular design principle of the thermogels.

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thermogels in biomedical fields such as sustained delivery of various drugs,11−14 tissue engineering,15,16 prevention of postoperative adhesion,17,18 and some other applications.19 Despite much success in animal experiments, such a promising kind of biomaterial has never been used in clinics as an approved medical product. A bottleneck comes from less controllability of the amazing physical gelation. While most amphiphilic copolymers can self-assemble into micelles in water, the reverse thermogelling is rare and puzzling. It has been known that thermogelling is influenced by molecular factors such as block length20,21 and others.22−26 But the unified mechanism of sol−gel transition upon heating is unknown, and thus the principle of corresponding molecule design is an open question. The internal structures of various gels constitute important scientific topics.27−31 However, the structure and mechanism of the thermogelling system at the nanoscale level are still elusive, although the system has attracted attention for about 20 years32−36 since the pioneering work by Kim and Jeong et al. in 1997.37 The block copolymers of polyester and polyether have no significant ionic interaction or hydrogen bond interaction, and thus cross-linking points of the physical hydrogel are unclear. In light of thermodynamics, we know

INTRODUCTION Hydrogels constitute a class of soft matter with infinite network and high water content.1−4 While this class of matter is very important, many critical aspects about its structure or mechanism are still shrouded in mystery, especially for some physical hydrogels without obvious cross-linking points, which significantly hinders the effective regulation on demand. Herein we utilize a thermogel as our model hydrogel, owing to not only its still puzzling internal structure but also its great potential in biomedical applications. Concentrated aqueous solutions of some amphiphilic block copolymers composed of poly(ethylene glycol) (PEG) and biodegradable polyester such as poly(D,L-lactide-co-glycolide) (PLGA) undergo a reversible sol−gel transition upon heating.5−8 If the transition temperature lies between room temperature and body temperature, the sol can be conveniently mixed with drugs or cells, the mixture can be injected into the body, and a hydrogel is spontaneously formed after injection into mammals. The sol transforms into a physical hydrogel free of any chemical reaction in vivo. Compared with the type of commercialized polymeric surfactant Pluronic composed of nonbiodegradable poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO−PPO−PEO) with the duration usually overnight in a large amount of water after gel formation, the thermogel composed of PEG and polyester is biodegradable, and the duration of the gel can be tailored from 1 week to 3 months.9,10 These properties potentiate © XXXX American Chemical Society

Received: May 13, 2018 Revised: July 27, 2018

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sol−gel transition happens with the occurrence of a percolated micelle network, which belongs, in light of statistical physics, to a percolation phase transition. In this study, both experiments and simulations were performed to explore the thermogelling mechanism of block copolymers with appropriate amphiphilicity in water. The approach details are given in the Supporting Information (subsections S1.1−S1.15 and Figures S1−S5).

that the hydrophobic interaction might be the main driving force of the reverse physical gelation, yet it is critical how physical cross-linking points are formed along with the hydrophobic association. For the linear ABA-type triblock copolymer, where A and B represent hydrophobic and hydrophilic blocks, respectively, Lee’s group speculated that the formation of a “bridge” of a copolymer chain with two hydrophobic end blocks inserted into two micelles was the key to the thermogelation.38 While the bridge does enhance the gelation sometimes, this speculation is inapplicable to other thermogelling copolymers such as BAB-type triblock copolymer or diblock copolymer. The present study is focused on the internal structure of the thermogel. Herein, a diblock copolymer, methoxypoly(ethylene glycol)-b-poly(D,L-lactide-co-glycolide) (mPEG− PLGA), was employed as the model amphiphilic copolymer. Those bridges could not be formed in diblock copolymers. Thus, our understanding of the sol−gel transition upon heating, if available, could reflect a universal mechanism behind the thermogelling process, which might be of general instructive significance for the corresponding molecular design. Our basic idea is that the thermogel might be composed of a percolated micelle network. A critical question arises as to how two micelles could be connected with each other with hydrophilic coronae. Eisenberg’s group suggested star-like and crew-cut micelles.39−41 We here put forward a new type of micelle termed the semi-bald micelle or semi-bare micelle. In a semi-bald micelle, the micellar core is partially exposed to water (Figure 1A). The evolution process of thermogelation is

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RESULTS Synthesis of MPEG−PLGA Copolymers and Thermogelling of the Aqueous Solutions. The diblock copolymer mPEG−PLGA was synthesized via ring-opening polymerization of the monomers D,L-lactide (LA) and glycolide (GA) using mPEG as an initiator (Figure S1). The number-average molecular weight (Mn) and molar ratio of LA and GA in the PLGA block were calculated from the 1H NMR spectrum (Figure 2A), based on the peaks with chemical shift at 3.50 ppm (CH3O of mPEG), 4.91 ppm (CH2 of GA), and 5.28 ppm (CH of LA) and the known Mn of mPEG (∼750). The molar mass dispersity ĐM of the diblock copolymer was measured by GPC (Figure S6). The parameters of mPEG− PLGA obtained are listed as follows: Mn 750−1870, LA/GA 6:1, ĐM 1.35. The copolymer aqueous systems exhibited three macroscopic states in the phase diagram determined with the test tube inversion method (Figure 2B). Once the copolymer concentration was higher than critical gelation concentration (CGC), the sol−gel transition as well as gel−precipitate transition occurred upon heating, exhibiting the lower transition temperature (gelation temperature, Tgel) and the upper transition temperature (precipitate temperature, Tprecipitate). Dynamic rheological experiments were also performed to investigate the sol−gel transition (Figure S2). The gelation point is defined as the intersection where the storage modulus (G′) exceeds the loss modulus (G″). The resultant Tgel at 25 wt % copolymer concentration in Figure S2B was consistent with that from the phase diagram in Figure 2B. For the PEG/PLGA thermogel, it has been known that an increase of the ratio of PEG/PLGA increases Tgel and even makes the sol−gel transition disappear.20,21 In this work, we also synthesized the other two mPEG−PLGA copolymers under a given PEG length (∼750) but shorter PLGA blocks to investigate the effect of block length on thermogelling. The corresponding molecular parameters are presented in Figure S7. Both direct observations with the test tube inversion method and quantitative characterizations with dynamic rheological experiments (Figure S8) indicated that the system after shortening the hydrophobic blocks maintained a sol state among a wide range of temperatures but disfavored the formation of the physical hydrogel; the viscosity of the sol increased only at very high temperature, but the system was still far from a gel. The relative lengths of the two blocks influence the relative sizes of micellar core and corona. Our experiments indicate that the thick corona in a star-like micelle is less beneficial for the thermoinduced aggregation and gelation. In experiments, it is difficult to precisely control many factors affecting thermogelling. Thus, copolymers with different block lengths were further investigated via Monte Carlo simulations with results presented in a following subsection.

Figure 1. Basic idea of the present structure study. (A) Schematic illustration of three types of micelles with the semi-bald micelle being put forward for the first time. The orange and blue colors indicate hydrophobic and hydrophilic blocks, respectively. (B) Schematic of the thermogelling process.

schematically presented in Figure 1B. The micelle at a low temperature might be a crew-cut one. With an increase of temperature, the corona collapses due to the reverse thermosensitivity of the hydrophilic block PEG. If the collapsed corona in an original crew-cut micelle cannot totally wrap the core, a semi-bald micelle is formed. Owing to the hydrophobic property of the micellar core, the exposed areas of different micellar cores might contact to reduce the total exposed area, which promotes the aggregation. Then, a hydrophobic channel is formed and acts as a physical crosslinking point between semi-bald micelles. A thermodynamic B

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Figure 2. Synthesized block copolymer and the macroscopic phase diagram of its aqueous system as functions of concentration and temperature. (A) Chemical structure of synthesized mPEG−PLGA and the corresponding 1H NMR spectrum. The peaks at 5.28, 4.91, 3.80, 3.50, and 1.80 ppm were assigned to the CH of LA, CH2 of GA, CH2 of mPEG, CH3O of mPEG, and CH3 of LA, respectively. (B) Phase diagram of the mPEG− PLGA aqueous system measured with the test tube inversion method. The upper row shows typical images of an aqueous copolymer system (25 wt %) at the indicated temperatures.

Observation of Clusters and Monitoring of the Evolution of Cluster Size upon Heating with Transmission Electron Microscopy (TEM) and Three-Dimensional Dynamic Light Scattering (3D DLS). The amphiphilic copolymers first self-assembled into micelles when the copolymer concentrations were over the critical micelle concentration (CMC). The CMC of the diblock copolymer in water was about 0.025 wt %, as determined by UV spectra of a hydrophobic dye 1,6-diphenyl-1,3,5-hexatriene (DPH) and presented in Figure S9. To further observe the morphology of micellar cluster associated with the sol−gel transition, we performed TEM experiments of a 1 wt % copolymer aqueous solution at four temperatures. Here we employed a sample preparation method involving quick freezing and lyophilization to keep sample morphology during solvent evaporation. From Figure 4A, the diblock copolymer chains formed well-dispersed spherical micelles at 5 and 25 °C. Larger clusters were observed at 37 °C and exhibited a network structure. The aggregated clusters were thickened at 60 °C. The hydrodynamic radii (Rh) of clusters were measured from 3D DLS, with results shown in Figure 4B. The cluster size increased obviously from 37 °C, which was well consistent with the TEM observations. Traditional light scattering mandates the measurement and analysis of single scattered light and is unavailable for concentrated solutions or turbid systems with multiple scattering. 3D DLS can directly detect both of these two cases.43 The theory of this instrument is described in the Supporting Information and summarized schematically in Figure S4. In our experiments, a technique of modulated 3D cross-correlation light scattering was introduced to enhance the signal-to-noise ratio, as explained in Supporting Information subsection S1.10. Normalized cross-correlation functions obtained from 3D scattering at low and high concentrations are presented in Figure S10. For a low concentration solution (1 wt %), the increased scattering at high temperatures reflected the formation of larger clusters. For a concentrated system (25 wt %, higher than CGC), the signal became stronger and more

Studies of Thermoreversibility of Sol−Gel Transition by Diffusing Wave Spectroscopy (DWS). As a physical gel, PEG/PLGA thermogel exhibits a thermoreversible behavior as presented schematically in Figure 3A. Nonetheless, the degelling process is rarely investigated quantitatively, and it is not easy to efficiently carry out a continuous dynamic detection of a hydrogel, a classical nonergodic system. While some traditional light scattering instruments can detect a gel system without disturbing the sample, the measurement has to be sufficiently long for the ensemble average and is thus faced with the difficulty to monitor a dynamic process. DWS can solve the problem well.42 The theory and the structure of the instrument are presented schematically in Figure S3 and described in detail in the Supporting Information. A significant advantage of the DWS instrument used in this work is the echo mode for the measurement of a nonergodic system. With this mode, the echo DWS can collect thousands of independent speckles for ensemble average in a short echo period (∼0.025 s). The corresponding results at two typical copolymer concentrations are shown in Figures 3B and 3C. For the system with a concentration lower than CGC, the correlation functions at large relaxing times increased significantly with temperature from 25 to 45 °C (Figure 3B), indicating the formation of large clusters. The signal from 0 min after heating to 45 °C almost overlapped with the signal after 60 min. In contrast, the de-aggregating process was much slower. Only after 12 h, the signal was recovered and then kept almost unchanged. For the system with a concentration higher than CGC, the signal at large relaxation time was much stronger (Figure 3C). At 45 °C (higher than Tgel), the correlation function maintained almost a horizontal line, corresponding to the formation of a gel network. The gelling and the de-gelling were again a rapid process and a slow process, respectively (Figure 3C). While the PEG/PLGA thermogel was confirmed to be thermoreversible, the gelling and de-gelling dynamic processes were found to be asymmetric. The de-gelling process was much slower, implying the existence of the strong physical interactions in the thermogel. C

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Figure 3. Quantitative characterization of thermoreversibility of the PEG/PLGA thermogel and the corresponding relaxation processes. (A) Schematic of thermoreversible sol−gel transition in the aqueous system of the copolymers of PEG and PLGA. (B) Correlation functions detected via DWS at indicated time scales during the heating process (left) and the recooling process (right) for the copolymeric aqueous system with concentration of 10 wt % (CGC). Other experimental parameters at 25 wt % were the same as those at 10 wt %. As the correlation function did not decay to nearly zero among the set time range of the multi-τ analysis, the echo mode for a nonergodic system started. Thus, the profiles were divided into two parts separated by the vertical dashed lines as indicated in the figures.

irregular at 37 °C (Tgel) than those at low temperatures, indicating the existence of larger and highly irregular clusters. The PEG block might play a more important role in thermogelling than the PLGA block. The collapse of the PEG coronae upon heating may afford a strong evidence for the mechanism of the temperature-induced sol−gel transition. In theory, the evolution of micelles can be detected via combination of static light scattering (SLS) and DLS;44 bonding a fluorescence molecule sensitive to the environmental polarity to the corona block might be an alternative

method.45,46 However, considering the size of the micelle (a dozen nanometers according to Figure 4B) and the very thin corona in our system, the slight change of the corona might be covered by the measurement error, and a chemical modification of the PEG block by a solvatochromic dye may interfere with the physical gelling significantly. Here, we analyzed the evolution of PEG indirectly by a cloud point measurement. The results in Figure S11 confirm that the PEG aqueous solution is a system of a lower critical solution temperature (LCST). Therefore, PEG exhibits some hydroD

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Figure 4. Mesoscopic structures of the synthesized copolymer in water as a function of temperature. (A) TEM images of mPEG−PLGA aqueous system (1 wt %) at indicated temperatures. In the image of 5 °C, typical isolated micelles are amplified at the top right. (B) Hydrodynamic radii (Rh) of clusters measured with 3D DLS.

Energy Transfer (FRET). To detect the connected micellar structure in the thermogel, we employed the FRET technique. FRET is a facile and straightforward strategy to detect the nanoscale distance and its change between clusters.48−52 The detected distance of the FRET pair in this study is within 5 nm.53 As schematically presented in Figure 5A, the FRET effect is a nonradiative process, in which an excited fluorescent donor transfers energy to a fluorescent acceptor in the ground state via a long-range dipole−dipole interaction. The FRET efficiency (η) is related to r, the distance between donor and acceptor, as shown in eq 1:54

phobicity as well as hydrophilicity, and thus the hydrophobicity must be reflected stronger with increase of temperature, which implies that the PEG coronae of the PEG−PLGA micelles in water might contract upon heating. This phenomenon is consistent with previous studies.25,47 Besides, in our experiments of 3D DLS, the signal of the correlation function at 25 °C was slightly weaker than that at 5 °C (Figure S10), which provides another evidence for the collapse of the hydrophilic corona of the corresponding micelles at the higher temperature. This conclusion is further supported by the following 13C NMR experiments. Remaining of Micelles during Sol−Gel Transition as Confirmed by NMR Experiments. We also performed 13C NMR experiments to investigate the core−corona structure during the thermogelling process by monitoring the chain mobility of a highly concentrated system. The sharp 13C peaks indicated a better mobility of the polymer. The peaks collapsed if the chain was restricted. From Figure S12, the collapsed peak of CH3 of LA (16 ppm) at different temperatures (5, 25, 37, and 45 °C) suggested that the PLGA block was restricted and located in the core structure before and after thermogelling. Only at a further high temperature (60 °C) did the peak become sharp, which indicated destruction of the core−corona structure, corresponding to the precipitate at the macroscopic level. A similar evolution also occurred for the peak of the carbonyl carbon of PLGA (170 ppm). The signal peak of CH2 of repeating units of mPEG (69 ppm) remained sharp although it slightly decreased at high temperatures, which indicated that the mPEG block had good mobility in coronae and contracted slightly. In contrast, all typical signals of the copolymer in the good solvent (CDCl3) exhibited sharp peaks and were almost unchanged with temperature. The NMR experiments illustrated that the basic core−corona structure of micelles remained during the sol−gel transition upon heating, supporting the model of the micelle network for the thermogel. Interconnectivity between Micellar Cores upon Thermogelling Revealed by Fluorescence Resonance

η = R 0 6/(R 0 6 + r 6)

(1)

Here R0 is the distance at which the FRET efficiency is 50%. In this study, phenanthrene (Phe, donor) and anthracene (An, acceptor) were taken as the FRET pair. Prior to the fluorescence emission experiments, UV absorption experiments proved that the donor and acceptor were encapsulated into the micellar core due to the hydrophobic association (Figure S13). The FRET efficiency was calibrated by I402/I366, where I402 and I366 denote the fluorescence peak intensities for acceptor and donor, respectively, and the subscripts are the wavelengths of the fluorescence peaks in units of nanometers. The FRET experiments designed in this work are schematically presented in Figure 5B. Two pretreated solutions (containing only donor or acceptor) were mixed at low temperatures. At this time, the donor and acceptor were isolated by micelles with a long separation distance; thus, only a weak FRET signal was observable. Because the aggregation happened at high temperatures, the donor−acceptor separation distance decreased, leading to an increased FRET signal. Notably, while the fluorescent dye was physically encapsulated into the micellar core and may be exchanged among different clusters over time, the signal was unchanged at least 3 days as seen from the results of the control groups in the dynamic FRET experiment at 25 °C, far longer than the measurement time scale in this work. Thus, the exchange of fluorescent E

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Figure 5. FRET experiments of the thermogel of the biodegradable block copolymer composed of PEG and PLGA. (A) Schematic of the FRET effect between fluorescent donor and acceptor. The right shows the FRET pair used in this study. (B) Schematic of FRET experiments designed in this work to monitor the gelation process upon heating. (C) Fluorescence spectra for indicated systems. The excitation wavelengths in all of the four cases were 294 nm. The FRET effect appears in the mixed system and corresponds to the specific peak in donor or acceptor characteristic emission as indicated by the arrows. (D) FRET efficiency (characterized by I402/I366) of the polymeric aqueous solution (25 wt %) against temperature. The vertical dashed line indicates the sol−gel transition temperature determined by inverted-vial method as presented in the phase diagram Figure 2B.

Figure 6. FRET experiments of another temperature-sensitive block copolymer Pluronic. (A) Schematic for a fluorescence experiment of Pluronic in water upon heating. The upper row is the chemical structure of Pluronic P85. (B) FRET efficiency as a function of temperature. The concentration of P85 was 30 wt %, and those of donor and acceptor were both 0.025 mg/mL. The gelation temperature of the system was 28 °C, as indicated by the vertical dashed line.

at Tgel (Figure 5D). To exclude the influence of temperature on either kind of fluorescent molecule, we detected the system only containing donor or acceptor at 366 or 402 nm, respectively. The value of I402/I366 was then much smaller

molecules among different clusters can be ignored in this work and does not affect the conclusion. The FRET effect was observed in the mixed system (Figure 5C). In the heating experiment, I402/I366 increased significantly F

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Figure 7. Dynamic FRET experiments of the thermogel. (A) Schematic of two possible dynamic processes in the designed experiments. (B) FRET efficiency for mPEG−PLGA aqueous systems (10 wt % < CGC) as a function of time. (C) FRET efficiency for mPEG−PLGA aqueous systems (25 wt % > CGC) as a function of time.

structure while the sol−gel transition in an aqueous system of the thermogellable PEG−PLGA block copolymers might be a real thermodynamic process. Or, the Pluronic hydrogel might arise just from micelle jamming, leading to a high viscosity instead of a qualitative structure transition; in contrast, the resultant thermogel of the block copolymer of PEG and PLGA has a unique internal structure with an infinite network connected by definite physical cross-linking points. To strengthen our viewpoint, we further designed a dynamic FRET experiment for the thermogel. There are two possibilities once micelles aggregate at a high temperature, as schematically presented in Figure 7. In the first case, a hydrophobic channel as the physical cross-linking point forms at Tgel, and fluorescent molecules can exchange between different micellar cores via the channel, so the FRET effect maintains after recooling the system to room temperature. In the second case, where no hydrophobic channel forms, the fluorescent molecules are always isolated in different micellar cores and no molecular exchange happens, and thus the FRET effect disappears once the system is recooled. To identify the thermogel structure of mPEG−PLGA, dynamic FRET experiments were performed with a heating and recooling cycle. From the results in Figures 7B and 7C, after a sufficiently long time for the complete recovery of the system (Figure 3), significant FRET effects still maintained, which was in accordance with the first case indicated in Figure 7A, i.e., different micellar cores contacted during the thermogelling process owing to the formation of the hydrophobic channel between semi-bald micelles. Our welldesigned dynamic FRET experiment strongly strengthened that the micelles in the thermogels have hydrophobic channels acting as physical cross-linking points. Simulations of Semi-bald Micelles and the Effect of the Length of Hydrophilic Block. We employed dynamic Monte Carlo simulations to explore the thermogelling behavior

and decreased monotonically with temperature (Figure S14B), different from the results obtained from the FRET experiments of the mixture. Taken all these results into consideration, the aggregation of clusters around Tgel was confirmed. The FRET experiment was also adopted in aqueous systems of other two mPEG−PLGA copolymers with shorter PLGA blocks. The fluorescence signal of mPEG−PLGA (Mn 750− 1510) increased at about 40 °C, higher than that of the thermogellable system with mPEG−PLGA (Mn 750−1870), and the value of I402/I366 was much lower, indicating only a relatively weak aggregation. For mPEG−PLGA with much shorter PLGA block (Mn 750−980), the FRET signal maintained almost a constant without any increase upon heating, indicating no further aggregation at all. Further combining with the rheological measurements presented in Figure S8, it is conclusive that the relatively thick corona in a star-like micelle is less beneficial for the thermoinduced aggregating and gelling. FRET measurements were also performed in a traditional amphipathic copolymer system, Pluronic P85. The gelation temperature for P85 was 28 °C at a concentration of 30 wt %. The FRET efficiency exhibited almost a constant within the detected temperature range (Figure 6), which suggested no significant change of distance between micelles containing fluorescent molecules before and after gelation. It has been known that the gelation of P85 followed the mechanism of close packing of micelles.55 In such a case, the formation of new micelles during heating increased the total number of micelles, resulting in the close packing and jamming-induced apparent gelation. That is why the gelling process of Pluronic had little influence on the preformed micelles encapsulating fluorescent molecules. Our series of FRET experiments imply that the apparent gelling of Pluronic might not accompany a thermodynamic phase transition with an essential change of the internal G

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Figure 8. Input energy parameters of copolymer chains used in dynamic Monte Carlo simulations, and output phases and condensed-state * and εAB * at indicated structures. (A) Schematic for criteria of different phases and self-assembly structures. (B) Phase or state diagrams of εAV temperatures. The open, green, blue, red, brown, and gray tilted squares refer to the states or phases of random coils, irregular micelles, spherical micelles, percolated network of semi-bald micelles, worm-like micelle network, and precipitate, respectively. The red circles indicate the default group of the energy parameters in our simulations. (C) Typical snapshots of the different phases in the phase diagrams. H

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Figure 9. Default energy parameters of the thermogels and the output in examination of chain length effects. (A) Coarse-grained model and default simulation parameters in the dynamic Monte Carlo simulations. “A” (orange bead), “B” (blue bead), and “V” represent hydrophobic segment, * and hydrophilic segment, and solvent, respectively. The dotted curve on the right corresponds to the relationship between reduced energy εBV temperature T. The circles represent temperatures simulated in the present work. (B) The evolution process of crew-cut micelles with a chain model of A24B8 at 37 °C. (C) Demonstration of the importance of a short PEG chain for the formation of semi-bald micelle. Two other cases are given by elongating hydrophilic block only and elongating both hydrophilic and hydrophobic blocks proportionally.

In dynamic Monte Carlo simulations, the time t was measured in units of Monte Carlo step (MCS). One MCS means the trial that every bead has been randomly selected once on average. A cluster in the present simulation is defined as follows: if any contact occurred between any two hydrophobic beads (nearest-neighbor or next-nearest-neighbor) from two model chains, these two chains belong to one cluster. The aggregation meant the fusion between different clusters, i.e., the contact of different micellar cores constituted by the hydrophobic blocks, which was based on the results of our FRET experiments. The keys to such computer experiments are an appropriate window of simulation conditions, an appropriate mapping of molecular parameters, and practicable criteria of different phase states on computers. We first made great efforts to calculate phase diagrams under series of reduced energies among block A, block B, and the solvent. The criteria of different phases in our computer simulations of multichain systems are schematically presented in Figure 8A. The weightaverage molecular weight of cluster (Mw(cluster))61 and region of all clusters (S) were employed to distinguish different phases. The critical points between sol and gel and between gel and precipitate defined in our simulations were the divergence point in the curve of Mw(cluster)/Nbead versus temperature (here Nbead denotes the number of total beads in the system) and the inflection point in the curve of S versus temperature, respectively. The two parameters are described in detail in the Supporting Information. According to these two parameters, the evolution process of the system with temperature could be divided into three main phases: sol, gel, and precipitate. In each main phase, there

further. Self-avoiding chains were modeled in a two-dimensional (2D) cubic lattice system. The Larson fluctuation model56,57 and partial-reptation algorithm58,59 were utilized as the microrelaxation modes to enhance the simulation efficiency of the multichain system. To mimic the simulation system to an infinite space, the periodic boundary condition was applied in both directions (x, y). Metropolis sampling60 was adopted as the sampling method. The corresponding details of simulations are described in the Supporting Information. Based on coarse graining of diblock copolymer mPEG− PLGA, the model chain was represented by AxBy, where A and B are hydrophobic segment (PLGA) and hydrophilic segment (mPEG), respectively, and the subscripts x and y denote the numbers of segments (beads) of the corresponding blocks. A24B8 was selected as the default chain model. Besides, solvent in the simulation system was represented by a vacancy (V). Pairwise nearest-neighbor and next-nearest-neighbor interactions were considered. In simulations, the energy parameter we inputted directly was reduced energy εij*, where the subscripts i and j represent species in the system. The relationship between εij* and energy εij is εij* = εij /(kBT )

(2)

Here kB is the Boltzmann constant and T is the Kelvin temperature. Resorting to the analysis of energy parameters in the Supporting Information, we obtained the relationship * and temperature T, and thus we between reduced energy εBV * in the simulation to model the could modulate the value of εBV system at different temperatures. I

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Figure 10. Criteria of sol−gel transition and gel−precipitate transition in the computer experiments and the macroscopic phase diagram of the aqueous system of the multiple coarse-grained amphiphilic chains as functions of concentration and temperature. (A) Mw(cluster)/Nbead as a function of temperature for systems with indicated φ, where Mw(cluster) is the weight-average molecular weight (MW) of cluster and Nbead is the number of total beads in the system. (B) Region of all clusters (S) as a function of temperature for systems with indicated φ. (C) Phase diagram obtained from simulations.

bald micelles aggregated, and at last the percolated network of semi-bald micelles spanned the whole system. We provide another two cases as examples to illustrate the necessity for short hydrophilic block for the formation of semibald micelles (Figure 9C). For A24B40 (elongating hydrophilic blocks only) and A72B24 (elongating both blocks proportionally), the corona also collapsed at high temperatures, but the semi-bald micelle did not form because the thick coronae prevented micelles from further aggregating. For these three types of model chains (A24B8, A24B40, and A72B24), fractions of B beads exposed to vacancy (f BV) and the thicknesses of the coronae (dcorona) were calculated. The results are shown in Figure S18C,D. These two parameters for the default block copolymer A24B8 were smaller than those for the other two chain model systems, which confirmed a more significant tendency for collapse of the hydrophilic block and more favorable for the formation of semi-bald micelles. Previous experiments from our group and other groups have indicated that thermogelling might disappear in the case of long copolymers.20,21 The present studies as reflected in Figures S7 and S8 further confirmed the importance of the block length. Our simulations agree with these reports and interpret the underlying reasons very well. Phase Diagram of Thermogels Obtained from Our Simulations. The thermodynamic equilibrium state of the system was reached after the multiple chains were relaxed for a sufficiently long time at a given temperature. Weight-average molecular weight of cluster (Mw(cluster)) and region of all

might also be some subphases: micelles and random coils in the sol state, and a percolated network of semi-bald micelles and worm-like micelle network in the gel state. The micellar subphase further contained spherical micelles and irregular micelles. Phase diagrams of ε*AV and ε*AB were obtained under various reduced energy parameters at different temperatures. The simulation results are presented in Figure 8B. Six phases appeared in the phase diagrams, and the typical snapshots are demonstrated in Figure 8C. The criteria for different phases are described in Figures S16 and S17. Corresponding to the physical gel state at the macroscale level, the structure of the system was a network (Figure 8A). From the phase diagram, a percolated micelle network of semi-bald micelles would exist if a network formed in the system with ε*AB < 0; i.e., hydrophobic and hydrophilic blocks had a good compatibility. It has been known that the compatibility of PEG and PLGA is good,62,63 which is consistent with our structure model of the thermogel. We adopted the default simulation parameters in Figure 9A to investigate the thermogelling process further. A single micelle in Figure 9 was extracted from the corresponding snapshot in Figure S18. At low temperatures, the crew-cut micelle formed by self-assembly of A24B8 chains. Figure 9B shows the evolution process of the crew-cut micelle at 37 °C. The corona of the micelle first collapsed, and then the micellar core was partially exposed, resulting in the formation of the semi-bald micelle. After being relaxed for a longer time, semiJ

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Figure 11. Mesoscopic structures of the simulated amphiphilic copolymer chains in water as a function of temperature. (A) Typical snapshots of simulation systems (chain model A24B8, φ = 0.35) at indicated temperatures. (B) Corresponding cluster size distribution.

of all beads. As the gravity was not introduced in our simulation, the form of precipitate might be a large aggregate probably suspending in the middle instead of always sinking to the bottom. The distribution of cluster size (the number of model chains in a cluster) was calculated and is shown in Figure 11B. The cluster size increased with temperature. The gradually broad distribution suggested a progressive aggregation process at 5, 25, and 37 °C. The sharp peak at 60 °C indicated a global aggregation of the system. We further calculated the structure factor to quantify the aggregated structure in the simulation system. The computational process of this parameter is presented in Figure S19. A larger value of q (modulus of the scattering vector q) corresponds to a more microscopic structure. The static scaling exponent υ at the chain level was calculated from the slope in the large q region (υ = −1/slope). In theory, the exponent for self-avoiding multiple chains at the athermal state is between 0.5 and 0.75, depending on concentration.64 Our simulation outputs in Figure S19B were well consistent with the theory, which justified our calculation. The structure factors of A beads (all of hydrophobic blocks) and B beads (all of hydrophilic blocks) were calculated when the system reached equilibrium at different temperatures. Besides the static scaling exponent at the chain level, we also calculated the static scaling exponent at the domain level from the moderate q region. A larger υ means more extension of chains or less aggregation extent of domains. From the results of A beads in Figure S20A, the value of υ was almost unchanged with temperature at the domain level except for 60 °C, indicating that the micellar cores maintained during the thermogelling process. The evolution process for B beads at the domain level also illustrated that the corona structure maintained until precipitated at a high temperature (60 °C) (Figure S20B). The gradually decreased υ value for B beads at

clusters (S) for systems of different concentrations (volume fractions, φ) were suggested to characterize the thermodynamic equilibrium states. The calculation processes for these two parameters are described in detail in Figure S16 and the corresponding subsection in the Supporting Information. Some results are shown in Figure 10. From Figure 10A, the value of Mw(cluster)/Nbead (Nbead: the number of total beads in the system) increased and approached 1 with an increase of temperature for a given φ, illustrating enhanced aggregation. Furthermore, the curves of Mw(cluster)/Nbead versus temperature at different concentrations indicated that the aggregation temperature decreased with an increase of φ. The region of all clusters (S) as a function of temperature is presented in Figure 10B. The rapidly decreased value of this parameter at high temperatures reflected the global contract for region of all clusters, indicating a precipitate. The gelation point and the precipitate point defined in the simulations were determined from the divergence point of Mw(cluster)/Nbead−temperature curve and the inflection point of S−temperature curve, respectively. Based on the gelation point and the precipitate point for systems with different φ’s, the phase diagram was obtained (Figure 10C). As φ increased, the lower transition temperature (Tgel) decreased and the upper transition temperature (Tprecipitate) increased, consistent with the change trends in the phase diagram obtained from experiments (Figure 2B). Simulations of the Percolated Micelle Network Underlying Thermogelling. Typical snapshots at thermodynamic equilibrium states are presented in Figure 11A. Similar to the images from TEM, many micelles dispersed in the system at low temperatures (5 and 25 °C). At 37 °C, higher than Tgel from the phase diagram in Figure 10C, a percolated network of semi-bald micelles spanned the whole system, corresponding to the gel state at the macroscale level. The precipitate formed at 60 °C due to the hydrophobic interaction K

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Figure 12. Microscopic view of chain mixing during the sol−gel transition of the simulated amphiphilic copolymers in water. (A) Number of contacts as a function of temperature. “A”: hydrophobic bead or segment; “B”: hydrophilic bead or segment. (B) Schematic for details of the structures in the representative states to illustrate the evolution process for AA, BB, and AB pairwise contacts. The upper row in (B) shows the typical states in the process, and the broad horizontal arrow indicates a heating process. The boxes below present the magnified schematics of the small boxes in the upper states.

schematic of the evolution process is presented in Figure 12B. Here, AA, BB, and AB represent the numbers of contacts for the corresponding bead pairs. It is interesting that the evolutions of the parameters for different pairwise contacts were interrelated. With temperature increased, the less hydrophilic property of block B led to the collapse of the block and promoted the contact of B beads. Meanwhile, the less hydrophilic property of block B was in favor of the mixing between A and B (ε*AB < 0); thus, the number of AB pairs increased. The competitive relation between AA and AB was significant at low temperatures when there was almost no aggregation for clusters. The increase of AB led to the decrease of AA. Nevertheless, AA increased at high temperatures as more aggregation occurred with the formation of the hydrophobic channel and A beads from different micelle contacted together. When the precipitate formed, AA further increased due to the exclusion of solvent. The detailed chain configurations and contact pairs are hard to be determined from experiments, which also illustrates the

the chain level indicated that the hydrophilic block contracted. This value was larger than 0.5 at 60 °C, which can be understood by that B blocks were still in a good “solvent” constituted by A beads due to mixing trends between A and B. Adjustment of Chain Configuration during Thermogelling. Our computer simulations also shed some light on the thermogelling at the chain level. First, the mean-squared endto-end distance was calculated to characterize the size of the coil. The results are presented in Figure S21A. The evolution process of this parameter upon heating indicated that block A slightly extended and block B contracted at high temperatures, which was consistent with the results obtained from structure factors (Figure S20). Meanwhile, the size of the whole model chain also decreased with an increase of temperature (Figure S21A). To analyze the system in more detail, we calculated the pairwise contacts to reveal the behavior of the copolymer segments (Figure 12A). A pairwise contact was defined as two beads in nearest-neighbor or next-nearest-neighbor. The L

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The percolated network of semi-bald micelles with hydrophobic channels has a clear physical cross-linking point, and thus the thermogel is a thermodynamic hydrogel instead of an aqueous system with apparently high viscosity formed simply by micelle jamming. The physically cross-linking structure explains why the thermogel, once formed and then put into a large amount of water, could still maintain its integrity for a long time, which is very important for the potential applications in biomedicine and other fields. Prerequisites for the Formation of the Percolated Micelle Network. Thermogelling is influenced by numerous factors, yet a general physical picture is lacking. On the basis of this study of the thermogelling mechanism, we figure out the prerequisites for the formation of the percolated network to guide the corresponding molecular design. Figure 13 summarizes three prerequisites for chain structure and interaction energy related to the species of monomer directly, mainly based on the default simulation parameters and the effect of block length (Figure 9), the energy phase diagram (Figure 8), and the significantly increased AB contacts upon heating (Figure 12). First, the amphipathic property of the copolymer is necessary for copolymer chains to self-assemble into micelles. But, merely a micelle formation is far away from sufficient for thermogelling. Crew-cut micelles should be ready for the transformation into semi-bald micelles at the sol−gel transition. Therefore, the short length of hydrophilic block is essential for the formation of semi-bald micelle (Figure 9) and for further thermogelling. Second, the hydrophilic block is of reverse thermosensitivity, which provides a driving force for transformation of crew-cut micelles to semi-bald micelles upon heating. The parameter should be moderate to further enable the occurrence of thermogelation, indicated in Figures 8, 9, and 13B. As clinic applications are concerned, it is also expected to have an appropriate sol−gel transition temperature, which is usually expected between room temperature and body temperature. For the formation of the percolated network of semi-bald micelles, an extra prerequisite is the mixing tendency between hydrophobic and hydrophilic blocks. In simulations, a definite mixing tendency means ε*AB< 0, and otherwise the percolated network of semi-bald micelle cannot form, as indicated in Figure 8 and also in Figure 13B. The networks in simulations could be classified into two types: the percolated network of semi-bald micelle and the worm-like micelle network. Compared with the worm-like micelle network, the percolated network of semi-bald micelle has a larger A−B interface as schematically presented in Figure 13A. When the repulsion occurs between hydrophobic and hydrophilic blocks, the interfacial tension between core and corona becomes stronger, which is unfavorable for a large interface and thus not beneficial for the formation of the percolated network of semibald micelles. The molecular design of a thermogellable polymer with a percolated network of semi-bald micelles should meet the above three prerequisites. Revealing them in this study might be very helpful for comprehensive fundamental research and potential industrialization of thermogels.

advantage of the present work with combination of both experiments and simulations in studies of the thermogelling mechanism.

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DISCUSSION Although thermogels of amphiphilic copolymers have been investigated for many years and showed vast potential for biomedical applications,65,66 this material still remains at the stage of laboratory research. A crucial bottleneck of its clinical application is the lack of a general guideline for molecular design, which needs a clear demonstration of the internal structure and the mechanism of the thermogelling system. In this study, mPEG−PLGA with the thermogelling property was synthesized as a model amphiphile (Figure 2). The correlation functions during the gelling and de-gelling processes of the thermogel were investigated by DWS with the echo technique (Figure 3). We employed TEM to observe the cluster morphology related to the sol−gel transition at different temperatures (Figure 4). FRET experiments (Figures 5−7) and computer simulations of multichains (Figures 8−12) were also carried out. The structure and the mechanism of the thermogelling system are discussed as follows, and the prerequisites for molecule design will be proposed finally. Semi-bald Micelles and a Percolated Network with Hydrophobic Channels. We hypothesize that in the thermogelling system the type of micelle at low temperatures is a crew-cut micelle on the basis of the copolymer structure and the demonstration of simulations (Figure 9). The thin corona of the crew-cut micelle is constituted by the PEG block. PEG is of reverse thermosensitivity to temperature according to our present experiments (Figure S11) and some previous reports.25,47 Therefore, the PEG corona could collapse upon heating. As the thin corona collapses, the micellar core might be partially exposed to water, leading to a semi-bald micelle. It is comprehensible that a long hydrophilic block is unfavorable for the formation of semi-bald micelle, as the micellar core might be wrapped by the thick collapsed corona. The hydrophobic property of the exposed area of the core makes the semi-bald micelle unstable in water, which promotes the aggregation of micelles. Hence, the formation of semi-bald micelle is, in our opinion, the premise for the thermogelling in such a kind of intelligent system. The exposed hydrophobic areas of the cores from different semi-bald micelles must contact to reduce the total exposed area, and thus the hydrophobic channel might form at the core-to-core location, as demonstrated in Figure 5 about FRET experiments. When a percolated network spans the whole system, the hydrophobic channel acts as a physical crosslinking point. Moreover, based on 13C NMR spectra (Figure S12), the simulation snapshots (Figure 11), and the calculated structure factors from our simulations (Figure S20), the integral micelle structure is maintained during the sol−gel transition. In this case, the hydrophobic channel was narrow and different from a worm-like micelle network. Currently, patchy copolymer micelles have attracted much attention due to the novel inhomogeneous surface and the abundant further aggregated morphology.67−69 These micelles are usually composed of triblock terpolymer with one block self-assembled as the core and the other two blocks constituting the microphase-separated corona. While our semi-bald micelles are different from the patchy micelles, they share similar trends of further aggregation among micelles.

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CONCLUSIONS We shed light on the internal structure of mPEG−PLGA thermogel and the underlying physics with sol−gel transition upon heating. A new type of micelle termed the semi-bald M

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The findings in this work can not only explain the interesting thermogelling phenomenon but also possess guidance of molecular design of this type of new material. The mechanism proposed might also be stimulating to understand other responsive systems and corresponding self-assembly dynamics. The research strategy of combining experiments and simulations of multichain systems is instructive for structural studies of other hydrogels or soft matter.

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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.8b01014. Supplementary Methods and Results (both experiments and simulations) including Figures S1−S21; schematic for synthesis of mPEG−PLGA copolymer, dynamic rheological experiment of the default block copolymer mPEG−PLGA (Mn 750−1870), theory of DWS, theory of 3D DLS, model and methods in simulations, the GPC profile of mPEG−PLGA (Mn 750−1870), characterizations of mPEG−PLGA with shorter PLGA blocks, dynamic rheological experiments of mPEG−PLGA with shorter PLGA blocks, CMC of the default diblock copolymer mPEG−PLGA (Mn 750−1870), the normalized cross-correlation function obtained from 3D DLS, the results of cloud points of two PEG homopolymers in water, the 13C NMR spectra at varied temperatures, UV absorption of fluorescent molecules, fluorescence emission of only donor or acceptor in FRET, FRET results of mPEG−PLGA copolymers with shorter PLGA blocks, criteria of sol, gel and precipitate states in computer simulations, dynamic Monte Carlo simulations of the evolution processes for different types of micelles, theoretical structure factors and some demonstrated simulated results, change of mean-squared endto-end distances of the two blocks and the whole copolymer chains upon heating during the simulated sol−gel−precipitate transitions (PDF)

Figure 13. Summary of the physical picture of the thermogel and the prerequisites to enable thermogelling of amphiphiles. (A) Schematic of the semi-bald micelle and the corresponding percolated micelle network with hydrophobic channels. (B) Prerequisites for the formation of a percolated network of semi-bald micelles. The demonstrated chain model was A24B8. Micelle snapshots were extracted from the system with size of 128 × 128 and concentration * and absolute temperature was of 0.35. The relationship between εBV * = 2.20 − 704/T. (1) Besides amphiphilicity of copolymer set as εBV for self-assembly in a selective solvent, crew-cut micelles should be formed in the sol state. (2) The hydrophilic block should have reasonable temperature response at body temperature (37 °C, i.e., ε*BV = −0.07) to guarantee the formation of the physical gel in vivo. In * indicates more hydrophilicily for B simulations, a smaller value of εBV beads. As a demonstration, only irregular micelle or precipitate instead * = −0.3) or less (εBV * = 0.1) of gel was formed with more (εBV hydrophilic response for corona (other reduced energy parameters * = −0.2, εAV * = 0.2). (3) Mixing tendency between were set as εAB hydrophobic and hydrophilic blocks is important for the formation of a percolated network of semi-bald micelles. In our simulations, a * < 0. definite mixing tendency was reflected by the energy εAB * > 0, the Otherwise, if repulsion occurs instead of mixing, i.e., εAB percolated network of semi-bald micelles could not form. As a * = 0.4, εAV * = 0.8, and varying εBV * by demonstration (keeping εAB temperature), spherical micelle or worm-like micelle network formed at low temperature (5 °C) or high temperature (60 °C), respectively.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (J.D.). ORCID

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by NSF of China (Grants 21774024 and 51533002) and National Key R&D Program of China (Grant 2016YFC1100300). We thank Professor Ming Jiang for his guidance of FRET techniques.

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micelle is proposed in this study. The percolated network of semi-bald micelles is demonstrated as the structure of the thermogel, and the hydrophobic channels serve as the physical cross-linking points. The evolution process during the thermogelling process of the amphiphilic block copolymers in water is revealed, and the prerequisites for the formation of the percolated network of semi-bald micelles are put forward.

REFERENCES

(1) Seliktar, D. Designing cell-compatible hydrogels for biomedical applications. Science 2012, 336, 1124−1128. (2) Hoffman, A. S. Hydrogels for biomedical applications. Adv. Drug Delivery Rev. 2012, 64, 18−23. (3) Buwalda, S. J.; Boere, K. W. M.; Dijkstra, P. J.; Feijen, J.; Vermonden, T.; Hennink, W. E. Hydrogels in a historical perspective: N

DOI: 10.1021/acs.macromol.8b01014 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules From simple networks to smart materials. J. Controlled Release 2014, 190, 254−273. (4) Vanparijs, N.; Nuhn, L.; De Geest, B. G. Transiently thermoresponsive polymers and their applications in biomedicine. Chem. Soc. Rev. 2017, 46, 1193−1239. (5) Jeong, B.; Kim, S. W.; Bae, Y. H. Thermosensitive sol-gel reversible hydrogels. Adv. Drug Delivery Rev. 2002, 54, 37−51. (6) He, C.; Kim, S. W.; Lee, D. S. In situ gelling stimuli-sensitive block copolymer hydrogels for drug delivery. J. Controlled Release 2008, 127, 189−207. (7) Yu, L.; Ding, J. D. Injectable hydrogels as unique biomedical materials. Chem. Soc. Rev. 2008, 37, 1473−1481. (8) Moon, H. J.; Ko, D. Y.; Park, M. H.; Joo, M. K.; Jeong, B. Temperature-responsive compounds as in situ gelling biomedical materials. Chem. Soc. Rev. 2012, 41, 4860−4883. (9) Jeong, B.; Lee, K. M.; Gutowska, A.; An, Y. H. H. Thermogelling biodegradable copolymer aqueous solutions for injectable protein delivery and tissue engineering. Biomacromolecules 2002, 3, 865−868. (10) Yu, L.; Zhang, Z.; Zhang, H.; Ding, J. D. Biodegradability and biocompatibility of thermoreversible hydrogels formed from mixing a sol and a precipitate of block copolymers in water. Biomacromolecules 2010, 11, 2169−2178. (11) Zentner, G. M.; Rathi, R.; Shih, C.; McRea, J. C.; Seo, M. H.; Oh, H.; Rhee, B. G.; Mestecky, J.; Moldoveanu, Z.; Morgan, M.; Weitman, S. Biodegradable block copolymers for delivery of proteins and water-insoluble drugs. J. Controlled Release 2001, 72, 203−215. (12) Chang, G. T.; Ci, T. Y.; Yu, L.; Ding, J. D. Enhancement of the fraction of the active form of an antitumor drug topotecan via an injectable hydrogel. J. Controlled Release 2011, 156, 21−27. (13) Chen, Y. P.; Luan, J. B.; Shen, W. J.; Lei, K. W.; Yu, L.; Ding, J. D. Injectable and thermosensitive hydrogel containing liraglutide as a long-acting antidiabetic system. ACS Appl. Mater. Interfaces 2016, 8, 30703−30713. (14) Ci, T. Y.; Shen, Y. N.; Cui, S. Q.; Liu, R. L.; Yu, L.; Ding, J. D. Achieving high drug loading and sustained release of hydrophobic drugs in hydrogels through in situ crystallization. Macromol. Biosci. 2017, 17, 1600299. (15) Lee, K. Y.; Mooney, D. J. Hydrogels for tissue engineering. Chem. Rev. 2001, 101, 1869−1879. (16) Zhang, Z.; Lai, Y. X.; Yu, L.; Ding, J. D. Effects of immobilizing sites of RGD peptides in amphiphilic block copolymers on efficacy of cell adhesion. Biomaterials 2010, 31, 7873−7882. (17) Zhang, Z.; Ni, J.; Chen, L.; Yu, L.; Xu, J. W.; Ding, J. D. Biodegradable and thermoreversible PCLA-PEG-PCLA hydrogel as a barrier for prevention of post-operative adhesion. Biomaterials 2011, 32, 4725−4736. (18) Yuan, B. M.; He, C. L.; Dong, X. M.; Wang, J. C.; Gao, Z. L.; Wang, Q.; Tian, H. Y.; Chen, X. S. 5-Fluorouracil loaded thermosensitive PLGA-PEG-PLGA hydrogels for the prevention of postoperative tendon adhesion. RSC Adv. 2015, 5, 25295−25303. (19) Yu, L.; Xu, W.; Shen, W. J.; Cao, L. P.; Liu, Y.; Li, Z. S.; Ding, J. D. Poly(lactic acid-co-glycolic acid)-poly(ethylene glycol)-poly(lactic acid-co-glycolic acid) thermogel as a novel submucosal cushion for endoscopic submucosal dissection. Acta Biomater. 2014, 10, 1251− 1258. (20) Shim, M. S.; Lee, H. T.; Shim, W. S.; Park, I.; Lee, H.; Chang, T.; Kim, S. W.; Lee, D. S. Poly(D,L-lactic acid-co-glycolic acid)-bpoly(ethylene glycol)-b-poly (D,L-lactic acid-co-glycolic acid) triblock copolymer and thermoreversible phase transition in water. J. Biomed. Mater. Res. 2002, 61, 188−196. (21) Yu, L.; Chang, G. T.; Zhang, H.; Ding, J. D. Temperatureinduced spontaneous sol-gel transitions of poly(D,L-lactic acid-coglycolic acid)-b-poly(ethylene glycol)-b-poly(D,L-lactic acid-co-glycolic acid) triblock copolymers and their end-capped derivatives in water. J. Polym. Sci., Part A: Polym. Chem. 2007, 45, 1122−1133. (22) Yu, L.; Zhang, H.; Ding, J. D. A subtle end-group effect on macroscopic physical gelation of triblock copolymer aqueous solutions. Angew. Chem., Int. Ed. 2006, 45, 2232−2235.

(23) Zhang, H.; Yu, L.; Ding, J. D. Roles of hydrophilic homopolymers on the hydrophobic-association-induced physical gelling of amphiphilic block copolymers in water. Macromolecules 2008, 41, 6493−6499. (24) Chen, L.; Ci, T. Y.; Li, T.; Yu, L.; Ding, J. D. Effects of molecular weight distribution of amphiphilic block copolymers on their solubility, micellization, and temperature-induced sol-gel transition in water. Macromolecules 2014, 47, 5895−5903. (25) Chen, L.; Ci, T. Y.; Yu, L.; Ding, J. D. Effects of molecular weight and its distribution of PEG block on micellization and thermogellability of PLGA-PEG-PLGA copolymer aqueous solutions. Macromolecules 2015, 48, 3662−3671. (26) Vidyasagar, A.; Ku, S. H.; Kim, M.; Kim, M.; Lee, H. S.; Pearce, T. R.; McCormick, A. V.; Bates, F. S.; Kokkoli, E. Design and characterization of a PVLA-PEG-PVLA thermosensitive and biodegradable hydrogel. ACS Macro Lett. 2017, 6, 1134−1139. (27) Fu, C. L.; Sun, Z. Y.; An, L. J. Relationship between structural gel and mechanical gel for ABA triblock copolymer in solutions: A molecular dynamics simulation. J. Phys. Chem. B 2011, 115, 11345− 11351. (28) Zhou, H. X.; Schon, E. M.; Wang, M. Z.; Glassman, M. J.; Liu, J.; Zhong, M. J.; Diaz, D. D.; Olsen, B. D.; Johnson, J. A. Crossover experiments applied to network formation reactions: Improved strategies for counting elastically inactive molecular defects in PEG gels and hyperbranched polymers. J. Am. Chem. Soc. 2014, 136, 9464−9470. (29) Zhou, C.; Toombes, G. E. S.; Wasbrough, M. J.; Hillmyer, M. A.; Lodge, T. P. Structure of two-compartment hydrogels from thermoresponsive ABC triblock terpolymers. Macromolecules 2015, 48, 5934−5943. (30) Zhong, M. J.; Wang, R.; Kawamoto, K.; Olsen, B. D.; Johnson, J. A. Quantifying the impact of molecular defects on polymer network elasticity. Science 2016, 353, 1264−1268. (31) Abbadessa, A.; Landin, M.; Blenke, E. O.; Hennink, W. E.; Vermonden, T. Two-component thermosensitive hydrogels: Phase separation affecting rheological behavior. Eur. Polym. J. 2017, 92, 13− 26. (32) Jeong, B.; Bae, Y. H.; Kim, S. W. Thermoreversible gelation of PEG-PLGA-PEG triblock copolymer aqueous solutions. Macromolecules 1999, 32, 7064−7069. (33) Lee, S. J.; Bae, Y.; Kataoka, K.; Kim, D.; Lee, D. S.; Kim, S. C. In vitro release and in vivo anti-tumor efficacy of doxorubicin from biodegradable temperature-sensitive star-shaped PLGA-PEG block copolymer hydrogel. Polym. J. 2008, 40, 171−176. (34) Tarasevich, B. J.; Gutowska, A.; Li, X. S.; Jeong, B. M. The effect of polymer composition on the gelation behavior of PLGA-gPEG biodegradable thermoreversible gels. J. Biomed. Mater. Res., Part A 2009, 89A, 248−254. (35) Singh, N. K.; Lee, D. S. In situ gelling pH- and temperaturesensitive biodegradable block copolymer hydrogels for drug delivery. J. Controlled Release 2014, 193, 214−227. (36) Khorshid, N. K.; Zhu, K. Z.; Knudsen, K. D.; Bekhradnia, S.; Sande, S. A.; Nystrom, B. Novel structural changes during temperature-induced self-assembling and gelation of PLGA-PEGPLGA triblock copolymer in aqueous solutions. Macromol. Biosci. 2016, 16, 1838−1852. (37) Jeong, B.; Bae, Y. H.; Lee, D. S.; Kim, S. W. Biodegradable block copolymers as injectable drug-delivery systems. Nature 1997, 388, 860−862. (38) Lee, D. S.; Shim, M. S.; Kim, S. W.; Lee, H.; Park, I.; Chang, T. Y. Novel thermoreversible gelation of biodegradable PLGA-blockPEO-block-PLGA triblock copolymers in aqueous solution. Macromol. Rapid Commun. 2001, 22, 587−592. (39) Gao, Z. S.; Varshney, S. K.; Wong, S.; Eisenberg, A. Block copolymer “crew-cut” micelles in water. Macromolecules 1994, 27, 7923−7927. (40) Zhang, L. F.; Eisenberg, A. Multiple morphologies and characteristics of ’’crew-cut’’ micelle-like aggregates of polystyrene-bO

DOI: 10.1021/acs.macromol.8b01014 Macromolecules XXXX, XXX, XXX−XXX

Article

Macromolecules poly(acrylic acid) diblock copolymers in aqueous solutions. J. Am. Chem. Soc. 1996, 118, 3168−3181. (41) Yu, Y. S.; Eisenberg, A. Control of morphology through polymer-solvent interactions in crew-cut aggregates of amphiphilic block copolymers. J. Am. Chem. Soc. 1997, 119, 8383−8384. (42) Zakharov, P.; Cardinaux, F.; Scheffold, F. Multispeckle diffusing-wave spectroscopy with a single-mode detection scheme. Phys. Rev. E 2006, 73, 011413. (43) Block, I. D.; Scheffold, F. Modulated 3D cross-correlation light scattering: Improving turbid sample characterization. Rev. Sci. Instrum. 2010, 81, 123107. (44) Wu, C.; Zhou, S. Q. Thermodynamically stable globule state of a single poly(N-isopropylacrylamide) chain in water. Macromolecules 1995, 28, 5388−5390. (45) Hua, Z.; Keogh, R.; Li, Z.; Wilks, T. R.; Chen, G. S.; O’Reilly, R. K. Reversibly manipulating the surface chemistry of polymeric nanostructures via a “grafting to” approach mediated by nucleobase Interactions. Macromolecules 2017, 50, 3662−3670. (46) Madsen, J.; Madden, G.; Themistou, E.; Warren, N. J.; Armes, S. P. pH-Responsive diblock copolymers with two different fluorescent labels for simultaneous monitoring of micellar selfassembly and degree of protonation. Polym. Chem. 2018, 9, 2964− 2976. (47) Jeong, B.; Windisch, C. F.; Park, M. J.; Sohn, Y. S.; Gutowska, A.; Char, K. Phase transition of the PLGA-g-PEG copolymer aqueous solutions. J. Phys. Chem. B 2003, 107, 10032−10039. (48) Xiang, M. L.; Jiang, M.; Zhang, Y. B.; Wu, C.; Feng, L. X. Intermacromolecular complexation due to specific interactions 0.4. The hydrogen-bonding complex of vinylphenol-containing copolymer and vinylpyridine-containing copolymer. Macromolecules 1997, 30, 2313−2319. (49) Jiwpanich, S.; Ryu, J. H.; Bickerton, S.; Thayumanavan, S. Noncovalent encapsulation stabilities in supramolecular nanoassemblies. J. Am. Chem. Soc. 2010, 132, 10683−10685. (50) Wang, R. B.; Peng, J.; Qiu, F.; Yang, Y. L. Enhanced white-light emission from multiple fluorophores encapsulated in a single layer of diblock copolymer micelles. Chem. Commun. 2011, 47, 2787−2789. (51) Robin, M. P.; Osborne, S. A. M.; Pikramenou, Z.; Raymond, J. E.; O’Reilly, R. K. Fluorescent block copolymer micelles that can selfreport on their assembly and small molecule encapsulation. Macromolecules 2016, 49, 653−662. (52) Huang, P. S.; Song, H. J.; Zhang, Y. M.; Liu, J. J.; Cheng, Z.; Liang, X. J.; Wang, W. W.; Kong, D. L.; Liu, J. F. FRET-enabled monitoring of the thermosensitive nanoscale assembly of polymeric micelles into macroscale hydrogel and sequential cognate micelles release. Biomaterials 2017, 145, 81−91. (53) Gan, D. J.; Lyon, L. A. Interfacial nonradiative energy transfer in responsive core-shell hydrogel nanoparticles. J. Am. Chem. Soc. 2001, 123, 8203−8209. (54) Yuan, L.; Lin, W. Y.; Zheng, K. B.; Zhu, S. S. FRET-based small-molecule fluorescent probes: Rational design and bioimaging applications. Acc. Chem. Res. 2013, 46, 1462−1473. (55) Ruel-Gariepy, E.; Leroux, J. C. In situ-forming hydrogels review of temperature-sensitive systems. Eur. J. Pharm. Biopharm. 2004, 58, 409−426. (56) Larson, R. G.; Scriven, L. E.; Davis, H. T. Monte Carlo simulation of model amphiphilic oil-water systems. J. Chem. Phys. 1985, 83, 2411−2420. (57) Larson, R. G. Monte Carlo lattice simulation of amphiphilic systems in two and three dimensions. J. Chem. Phys. 1988, 89, 1642− 1650. (58) Hu, W. B. Structural transformation in the collapse transition of the single flexible homopolymer model. J. Chem. Phys. 1998, 109, 3686−3690. (59) Ding, J. D.; Carver, T. J.; Windle, A. H. Self-assembled structures of block copolymers in selective solvents reproduced by lattice Monte Carlo simulation. Comput. Theor. Polym. Sci. 2001, 11, 483−490.

(60) Metropolis, N.; Rosenbluth, A. W.; Rosenbluth, M. N.; Teller, A. H.; Teller, E. Equation of state calculations by fast computing machines. J. Chem. Phys. 1953, 21, 1087−1092. (61) Nguyenmisra, M.; Mattice, W. L. Micellization and gellation of symmetrical triblock copolymers with insoluble end blocks. Macromolecules 1995, 28, 1444−1457. (62) Li, T. Q.; Zhang, J. Y.; Schneiderman, D. K.; Francis, L. F.; Bates, F. S. Toughening glassy poly(lactide) with block copolymer micelles. ACS Macro Lett. 2016, 5, 359−364. (63) Takhulee, A.; Takahashi, Y.; Vao-soongnern, V. Molecular simulation and experimental studies of the miscibility of polylactic acid/polyethylene glycol blends. J. Polym. Res. 2017, 24, 8. (64) deGennes, P.-G. Scaling Concepts in Polymer Physics; Cornell University Press: Ithaca, NY, 1979. (65) Ko, D. Y.; Shinde, U. P.; Yeon, B.; Jeong, B. Recent progress of in situ formed gels for biomedical applications. Prog. Polym. Sci. 2013, 38, 672−701. (66) Cui, S. Q.; Yu, L.; Ding, J. D. Injectable thermogels based on block copolymers of appropriate amphiphilicity. Acta Polym. Sin. 2018, 863−881. (67) Rosenfeldt, S.; Luedel, F.; Schulreich, C.; Hellweg, T.; Radulescu, A.; Schmelz, J.; Schmalz, H.; Harnau, L. Patchy wormlike micelles: solution structure studied by small-angle neutron scattering. Phys. Chem. Chem. Phys. 2012, 14, 12750−12756. (68) Groeschel, A. H.; Schacher, F. H.; Schmalz, H.; Borisov, O. V.; Zhulina, E. B.; Walther, A.; Mueller, A. H. E. Precise hierarchical selfassembly of multicompartment micelles. Nat. Commun. 2012, 3, 710. (69) Parmar, I. A.; Shedge, A. S.; Badiger, M. V.; Wadgaonkar, P. P.; Lele, A. K. Thermoreversible sol-gel transition of aqueous solutions of patchy polymers. RSC Adv. 2017, 7, 5101−5110.

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DOI: 10.1021/acs.macromol.8b01014 Macromolecules XXXX, XXX, XXX−XXX