Galactose-Functionalized Double-Hydrophilic Block Glycopolymers

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

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Galactose-Functionalized Double-Hydrophilic Block Glycopolymers and Their Thermoresponsive Self-Assembly Dynamics Jing Quan,*,†,§ Fa-Wei Shen,†,§ Hao Cai,† Yi-Na Zhang,† and Hua Wu*,‡ †

Key Laboratory of Science and Technology of Eco-Textiles, Ministry of Education, and College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, P. R. China ‡ Institute for Chemical and Bioengineering, Department of Chemistry and Applied Biosciences, ETH Zurich, 8093 Zurich, Switzerland

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S Supporting Information *

ABSTRACT: Glycopolymers with large galactose units are attractive in biological processes because of their ability to selectively recognize lectin proteins. Recently, thermoresponsive double-hydrophilic block glycopolymers (TDHBGs) have been designed, which allow sugar residues to expose or hide via the lower critical solution temperature (LCST)-type phase transition. In this work, we first synthesize a new type of TDHBGs, composed of a thermoresponsive poly(di(ethylene glycol)methyl ether methacrylate) block and a galactose-functionalized, poly(6-O-vinyladipoyl-D-galactose) (POVNG) block. The LCST can be tuned by varying the size of the POVNG block. Then, we have systematically investigated their thermoresponsive selfassembly behavior, using static and dynamic light scattering techniques, combined with transmission electron microscopy (TEM) imaging. It is found that the TDHBGs possess both micellization and LCST-type transition, and there exist strong interactions between them, depending on the concentration and structure of the TDHBGs. It is particularly interesting that for the same type of TDHBGs under different conditions, such interactions result in rich morphologies of the formed micelles (or nanoparticles) such as spheres, hollow spheres, prolate ellipsoids, crystal-like, and so on, thus potentially enriching their biological applications by noting that they are hepatomatargeting glycopolymers.

1. INTRODUCTION Saccharide−protein interactions are one class of the most important molecular recognition in living organisms.1,2 Such biological interactions are rather complex and result from the accumulated effect of various weak molecular interactions such as electrostatic interactions, hydrophobic interactions, hydrogen bonding, and so on.3,4 Though these individual molecular interactions are weak, the accumulated interactions are typically strong and reversible due to the essential role played by the multivalent effect.5 In recent years, such saccharide− protein interactions have been widely explored toward creating different bioactive structures for drug delivery, gene therapy, pathogen detection, inhibitors of toxins, lectin-based biosensors, and so on.4,6−12 Along this line, glycopolymers with pendent sugar moieties have received great attention because of their large amount of valences, leading to enhanced multivalent effects in molecular recognition.2,10−18 Although the multivalent interactions are rather similar for different proteins, the distance between the saccharide recognition sites is often very specific for each protein.19−21 Thus, in the practical applications, it is particularly desired to have generally valid methodologies that can synthesize glycopolymers with tunable distance between the saccharide recognition sites. © XXXX American Chemical Society

In the typical applications of drug delivery, it is required that the glycopolymers self-assemble in aqueous media to form micelles or nanoparticles. It is found that the micelles or nanoparticles formed by double-hydrophilic block glycopolymers offer a number of attractive properties, including high stability, prolonged circulation in the blood, and enhanced accumulation in tumor tissue.22−26 Further, thermoresponsive double-hydrophilic block glycopolymers (TDHBGs) have been designed to contain pendant sugar residues that can be exposed or hidden via an externally controlled coil-to-globule phase transition around the lower critical solution temperature (LCST).11−16,27−32 Once the temperature is above the LCST, the glycopolymers self-assemble into micelles, and conversely, the micelles dissociate into individual molecules below the LCST. The reversible display of specific sugar moieties in the TDHBG can easily be regulated by increasing or decreasing the system temperature, and thus such systems are deemed to be promising for protein binding and specific recognition. The LCST value of a thermoresponsive copolymer can be properly Received: May 8, 2018 Revised: August 15, 2018 Published: August 16, 2018 A

DOI: 10.1021/acs.langmuir.8b01516 Langmuir XXXX, XXX, XXX−XXX

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Scheme 1. (A) 6-O-Vinyladipoyl-D-galactose by Controllable Chemoenzymatic Reaction; (B) Synthesis of TDHBG by RAFT Polymerization

acrylate) (PDEGMA) block and a galactose-functionalized poly(6-O-vinyladipoyl-D-galactose) (POVNG) block. The LCST was tuned by varying the molar fraction of 6-Ovinyladipoyl-D-galactose (OVNG), that is, the size of the POVNG block, inside the polymer. Then, we systematically investigate their thermoresponsive self-assembly behavior, using both static light scattering (SLS) and dynamic light scattering (DLS) techniques, combined with transmission electron microscopy (TEM) imaging, to demonstrate the existence of strong interactions between the micellization and LCST-type transition, depending on the concentration and structure of the macromolecules.

modulated by adjusting the relative amounts of monomers used in synthesis.29−33 However, although various studies have shown that the double-hydrophilic block glycopolymers can assemble into different structures such as nanospheres, wormlike micelles, vesicles, and so on,22,23,34−36 in the case of TDHBGs, their self-assembling behavior has never been systematically investigated. In this case, due to the presence of two possible transitions, micellization and LCST-type phase transition, one would expect that their interactions could generate much richer self-assembling phenomena. For example, Pasparakis and Alexander have shown16 that a TDHBG [poly(2-glucosyloxyethyl methacrylate)-b-poly(diethylene glycol methacrylate)] self-assembled into vesicles of 500 nm before LCST, while the size of the formed vesicles reduced to 300 nm when the temperature increases above LCST. Therefore, the aim of this work is twofold. First, we synthesize a new type of TDHBGs, composed of a thermoresponsive poly(di(ethylene glycol)methyl ether meth-

2. EXPERIMENTAL SECTION 2.1. Materials. The monomer, di(ethylene glycol)methyl ether methacrylate (DEGMA), was purchased from Sigma-Aldrich. Alkaline protease from Bacillus subtilis (EC 3.4.21.14, powder, 100 U mg−1) was obtained from Wuxi Xuemei Technological Co. (Wuxi, China). Adipic acid, azobisisobutyronitrile (AIBN, 97%), and galactose were obtained from Sinopharm Chemical Reagent Co. (Shanghai, China). B


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Figure 1. 1H NMR spectra of (a) DEGMA, (b) PDEGMA, (c) OVNG, and (d) TDHBG-2 in D2O. AIBN was further purified by recrystallization from a 95% water solution. 2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) was procured from Nanjing Robiot Co. (Nanjing, China). The reversible addition−fragmentation chain-transfer (RAFT) agent, cyanomethyl methyl(4-pyridyl)carbamodithioate (CMPCD), Ricinus communis agglutinin II (RCA120), trifluoromethanesulfonic acid (TfOH), concanavalin A (Con A), and phosphate-buffered saline (0.01 M, pH 7.4) were obtained from Sigma-Aldrich (Shanghai, China). All the used solvents were of analytical grade and were dried by storing them on activated 4 Å molecular sieves for 24 h prior to use. All of the other reagents were used as received. Water was distilled before use. 2.2. Synthesis of Galactose-Functionalized Monomers. The galactose-functionalized monomer, 6-O-vinyladipoyl-D-galactose (OVNG), was synthesized using adipic acid as the linker, through controllable regioselective enzymatic transesterification,37 as illustrated in Scheme 1A. It consisted of two steps. Step 1: Formation of adipic acid vinyl ester. The adipic acid vinyl ester was synthesized using a protocol similar to that reported in our previous work.29 Typically, 0.36 mol (52.6 g) of adipic acid and 0.25 g of mercury(II) acetate were dissolved in 80 mL of vinyl acetate. After stirring the mixture for 30 min at room temperature, 0.2 mL of

concentrated sulfuric acid was added, and the solution was refluxed for 8 h at 70 °C. Then, the mixture was cooled to room temperature, and sodium acetate (1.5 g) was added to quench the catalyst. The solution was filtered and concentrated. The crude products were purified by silica gel column chromatography (mobile phase: petroleum ether/ethyl acetate, 9:1, v/v) to get pure adipic acid vinyl ester. Step 2: Galactose functionalization. 0.066 mol (4 g) galactose and 0.022 mol (12 g) adipic acid vinyl ester were dissolved in 100 mL of anhydrous pyridine, and in the presence of the catalyst (1.5 g of alkaline protease), the mixture was stirred for 4 days at 50 °C. After being cooled down to room temperature, the mixture was filtered and concentrated. The crude products were purified via silica gel column chromatography with ethyl acetate as the eluent. 2.3. Synthesis of the New TDHBGs. The synthesis of the new TDHBGs is also composed of two steps, as illustrated in Scheme 1B. Step 1: RAFT polymerization for the PDEGMA block. In a typical experiment, 1.2 × 10−2 mol (2.26 g) of DEGMA were first added into a 25 mL one-neck round-bottom flask, and 2.4 × 10−5 mol (4.2 mg) of AIBN were introduced. Then, 4.00 mL of N,N-dimethylformamide (DMF) was added to dissolve the monomers, and finally 1.44 × 10−4 mol (2.48 × 10−2 g) of TfOH and 1.2 × 10−4 mol (2.68 × 10−2 g) of C


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Langmuir the RAFT agent, CMPCD, were added. This corresponds to the molar ratio of DEGMA/CMPCD at 100:1. The mixture was then degassed by three freeze−thaw cycles and sealed under vacuum. The polymerization proceeded under stirring at 70 °C for 1 h. The obtained polymer, PDEGMA, was precipitated in hexane for three times, collected by filtration, and then dried under reduced pressure in a drying oven at room temperature. Slightly yellowish PDEGMA solids were obtained with a conversion of DEGMA to PDEGMA equal to 59%. The synthesized PDEGMA block was characterized by gel permeation chromatography (GPC) and 1H NMR. The GPC analysis curve in tetrahydrofuran (THF) is shown in the Supporting Information (SI), from which the molar masses, Mn and Mw, and the Mw/Mn ratio of the PDEGMA block are 4.43 × 104, 5.37 × 104, and 1.21, respectively, indicating low dispersity (Đ). The 1H NMR spectra of DEGMA and PDEGMA are shown in Figure 1a,b. The signals of DEGMA at 6.00 (dd, 2H) and 5.57 (dd, 1H) have disappeared, and the vinyl groups are absent. 1 H NMR of DEGMA (D2O, 400 MHz) δ (ppm): 6.00 (s, 1H, CH2C), 5.57 (s, 1H, CH2C), 4.18 (t, 2H, −CH2−), 3.67 (t, 2H, −CH2−), 3.57 (t, 2H, −CH2−), 3.47 (t, 2H, −CH2−), 3.22 (s, 3H, −OCH3), 1.78 (s, 3H, CH2 C−CH3). 1 H NMR of PDEGMA (D2O, 400 MHz) δ (ppm): 4.18 (t, 2H, −CH2−), 3.67 (t, 2H, −CH2−), 3.57 (t, 2H, −CH2−), 3.47 (t, 2H, −CH2−), 3.22 (s, 3H, −OCH3), 1.78 (s, 3H, −CH3). Thus, the first PDEGMA block was synthesized successfully. Step 2: Grafting the galactose-functionalized block to the PDEGMA block. It was done through RAFT polymerization of the galactose-functionalized monomer directly on the PDEGMA block again. It was found that the reactivity of the galactose-functionalized monomer is much lower than that of DEGMA. Thus, the polymerization time was substantially prolonged. As an example, 6.8 × 10−6 mol (0.30 g) of purified PDEGMA and 2.1 × 10−6 mol (0.35 mg) of AIBN were added into a 25 mL one-neck round-bottom flask, followed by adding 5.71 × 10−4 mol (0.21 g) of OVNG. The mixture was dissolved in 2 mL of DMF, degassed by three freeze−thaw cycles, and sealed under vacuum. The polymerization was carried out under stirring at 70 °C for 48 h. The TDHBG, PDEGMA-b-POVNG, was precipitated three times in anhydrous diethyl ether, and the final dried product was in the form of a slightly yellowish powder. Following the above procedure, we have synthesized three TDHBGs with the initially set molar fractions of the OVNG monomer in total monomers (OVNG + DEGMA), f OVNG = 22.2, 26.3, and 31.3%, and the obtained TDHBGs are referred to as TDHBG-1, TDHBG-2 and TDHBG-3, respectively. The 1H NMR spectra of OVNG and TDHBG-2 are shown in Figure 1c,d. 1 H NMR of OVNG (D2O, 400 MHz) δ (ppm): 7.12 (dd, 1H, −CH), 5.18 (d, 0.8H, 1-Hα), 4.91 (dd, 1H, CH2), 4.66 (dd, 1H, CH2), 4.57−3.35 (m, 5H, Hα and Hβ of galactose circle), 2.45 (m, 4H, −CH2−CO), 1.58 (m, 4H, −CH2). 1 H NMR of TDHBG-2 (D2O, 400 MHz) δ (ppm): 4.57−3.35 (m, 5H, Hα and Hβ of galactose circle), 3.90 (m, H of −CH(CH3)2), 2.35 (m, H of −CO−CH2CH2−), 1.55 (m, H of −CO−CH2CH2−), 1.00 (d, H of −CH(CH3)2). In the 1H NMR spectrum of TDHBG-2, the signals of OVNG at 7.12 (dd, 1H, −CH), 4.91 (dd, 1H, CH2), and 4.66 (dd, 1H, CH2) have practically disappeared, and the vinyl groups are absent. Thus, the grafting of the POVNG block is successful. It should be noted that since the structure of the OVNG monomer is rather peculiar, we are unable to find a suitable internal standard to quantify the molar mass of the three TDHBGs using GPC. Thus, we have tried to estimate the molar fractions of OVNG in the three TDHBGs based on the integral values of the signals at δ = 2.40−2.35 (2H of OVNG) and δ = 3.37−3.23 (3H of DEGMA), and the obtained f OVNG values, as well as the molar masses, Mn, are listed in Table 1. As can be seen, the f OVNG value is around 50% of the values based on the feed. This is consistent with the conversions determined by gravimetric analysis in Table 1 (last column). Thus, the estimated f OVNG and Mn values are reliable. 2.4. Preparation of the TDHBG Micelles. The synthesized TDHBGs have good solubility in water below their LCST. Thus, the

Table 1. Properties of the Three Synthesized TDHBGs TDHBG

f OVNG (%) based on feed

f OVNG (%) based on NMR

Mna (×104)

conversionb (%)

TDHBG-1 TDHBG-2 TDHBG-3

22.2 26.3 31.3

11.0 12.2 14.6

5.54 5.70 5.91

52 57 51

a Mn calculated from NMR data. gravimetric analysis.

b

Conversion determined by

polymer micelles were prepared by dissolving directly the polymers in water. Double-distilled water was used and filtered using a 0.22 μm syringe filter to remove any potential dust. We first prepared a solution for the TDHBGs at a concentration of 1 mg mL−1, and then this was diluted to get the TDHBG solutions at the other concentrations. 2.5. Characterization Methods. Nuclear magnetic resonance (NMR). 1H NMR spectra were recorded on a Bruker DRX spectrometer (400 MHz, Bruker, Rheinstetten, Germany). Samples were prepared by dissolving 5 mg of the purified TDHBG in 0.5 mL of D2O. Gel permeation chromatography (GPC). Molar masses (Mw and Mn) and molar mass distributions were determined by GPC on a Waters LS measurement system using tetrahydrofuran (THF) as the eluent, at a flow rate of 1.0 mL min−1 and a column temperature of 35 °C. Laser light scattering (LLS). An LLS system (BI-200SM, Brookhaven Instruments, Holtsville, NY, with a solid state laser, λ = 532 nm) was used to perform the static and dynamic light scattering measurements to determine the gyration radius (Rg) and hydrodynamic radius (Rh) of the formed glycopolymer micelles at different temperatures. It should be mentioned that the temperature values related to the light scattering experiments are measured directly inside the light scattering chamber, instead of the set values in the thermostat. The Rg value was estimated based on the Guinier plot38,39 2 ji (qR g) zyz I(q) zz = expjjjj− j I(0) 3 zz k {

(1)

where I(q) and I(0) are the scattered intensities at q and q = 0, respectively, and q is the magnitude of the wavevector, q=

4πn0 ji θ zy sinjj zz λ k2{

(2)

with θ being the scattering angle, λ being the wavelength of the radiation in vacuum, and n0 being the refractive index of the medium. The Rh value was determined based on the translational diffusivity, D, measured by the dynamic light scattering (cumulant algorithm) at θ = 90°, using the Stokes−Einstein equation40 Rh =

kT 6πηD

(3)

where k is Boltzmann’s constant, T is the absolute temperature, and η is the viscosity of the dispersing liquid. To be sure that the micellization is at equilibrium, we held the sample at each temperature typically for 1 h prior to the measurements. Transmission electron microscopy (TEM). TEM micrographs were obtained using a JEM-2100 microscope (JEOL, Tokyo Japan). For the sample preparation, the carbon-coated copper grids were treated by plasma cleaner (HARRIK plasma, PDC-002) to form a hydrophilic surface. The grids were first preheated to the same temperature as the micelle solution, and then the micelle solution (10 μL) was dropped to the grids and dried in an oven again at the same temperature as the micelle solution. Lectin-binding assay. The lectin agglutination activity of the TDHBGs was evaluated by measuring the change in the turbidity of a buffered solution of the polymer with RCA120 or Con A. The aqueous buffer solution (pH = 7.4) for lectin-binding assay was prepared D


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Langmuir containing 10 mM HEPES, 0.15 M NaCl, 0.1 mM CaCl2, and 0.01 mM MnCl2. The final solution contained 6 mg mL−1 glycopolymer and 3 mg mL−1 RCA120/Con A. The turbidity was measured using a UV−vis spectrophotometer (LAMBDA 35, PerkinElmer, Waltham, MA) at a wavelength of 600 nm (25 °C). The interaction tests were repeated at least three times to ensure good reproducibility.

increases, and it reaches a plateau after about 8 min. Conversely, no significant increase in absorbance can be monitored in the case of Con A because of its selective binding to glucose and mannose but not to galactose.45 Therefore, the multivalent galactose block in our synthesized TDHBG is clearly active in biorecognition, allowing selective interactions between the multivalent galactose and the lectin RCA120. 3.2. Micellization and Thermoresponsive Transition of TDHBG-1. Thermoresponsive double-hydrophilic block copolymers have attracted attention due to their aggregation behavior triggered by temperature; the temperature-induced aggregation behavior was found to be greatly dependent on copolymer monomer composition, concentration, and heating progress, affecting the LCST value, aggregation behavior, size, and morphology of the aggregates.48,49 For the abovesynthesized TDHBGs, we believe that some behaviors would be similar to those reported in the literature. Let us first examine the micellization and thermoresponsive transitions of TDHBG-1 with f OVNG = 11.0% in Table 1, observed from both the static and dynamic light scattering techniques. Figure 3

3. RESULTS AND DISCUSSION 3.1. Synthesized New TDHBGs. The new TDHBGs were synthesized by combining enzymatic catalysis with RAFT polymerization, as described in Section 2.3. The RAFT agent, CMPCD, was used not only because of its ability to control the reaction but also due to its low toxicity.41 During the synthesis of the first PDEGMA block, we have controlled the conversion of DEGMA to PDEGMA at 59%. It should be mentioned that the conversion of DEGMA to PDEGMA can reach a value larger than 90%. In fact, we have measured the Mn of PDEGMA as a function of the conversion. Considering that for our next application stage (hepatoma-targeting therapy and drug delivery), we would like to have the Mn of the PDEGMA block in the range of 4−5 × 104 g mol−1, which from our measured Mn-conversion curve corresponds to the conversion around 60%. This is the reason why we have decided on such polymerization conditions. Of course, in real applications, we will tune the RAFT agent/monomer ratio to get the desired Mn and in the mean time to have a high conversion. As mentioned in the introduction, glycopolymers with a large quantity of galactose units are very attractive in biological processes because of their ability to selectively recognize lectin proteins,42−44 and RCA120 is a known specific lectin for the selective binding of glycopolymers containing galactose moieties.45 To prove that the biofunctionality of the galactose moieties is still active after the RAFT polymerization, we have performed the lectin-binding ability of our synthesized TDHBGs, using TDHBG-2 as an example. The RCA120 and Con A lectins have been used to assess the binding with TDHBG-2 at T = 25 °C. The lectin (RCA120 or Con A) concentration was set at 3 mg mL−1, and the TDHBG-2 concentration was 6 mg mL−1. Turbidity assay was used,46,47 and the absorbance changes at a wavelength of 600 nm were recorded on a UV−vis spectrophotometer. As shown in Figure 2, the turbidity of RCA120 increases as the conjugation time

Figure 3. Scattering intensity curves at different temperatures for the aqueous dispersion of TDHBG-1 at C = 0.20 mg mL−1.

shows the typical scattering intensity curves measured at different temperatures at C = 0.20 mg mL−1. It is seen that in the low temperature range (T < 33.2 °C) each curve has two distinct regions, which are divided more or less around q = 6 × 10−3. For the region of q < 6 × 10−3, the data indicate something with Rg > 200 nm, and the shape of the intensity curve does not change significantly with temperature initially but disappears at higher temperatures. We believe that this is related to some unknown long-range correlations among the polymer molecules50 because this did not disappear even after the dispersion was filtered by a 0.1 μm filter. We call the phenomenon the long-range correlations because the scattering pattern is located in the small q range. It could be also due to the adsorption of a very small amount of the polymer molecules on the cell surface of the SALS or few air bubbles. Therefore, for T < 33.2 °C, the scattering intensity data in the region of q < 6 × 10−3 have been ignored in the following analysis. For the region of q > 6 × 10−3 and T < 33.2 °C in Figure 3, the intensity curve moves upward as the temperature increases, but the corresponding gyration radius, Rg (≈30 nm), does not change significantly with T, as shown in Figure 4a. When one

Figure 2. Turbidity assays to prove the binding of RCA120 (red circle) and Con A (black squares) to the synthesized TDHBG, TDHBG-2. E


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probably related to increase in the hydrophobicity of the hydrophobic groups as the temperature increases, leading to slight shrinkage of the micelles. In addition, in the temperature region T < 11 °C, the measured Rg value in Figure 4a is only around 16 nm and independent of T, which is significantly smaller than that of micelle-1. This should correspond to individual molecules of TDHBG-1. Therefore, T ≈ 10 °C is basically the critical micellization temperature (CMT) of TDHBG-1 at C = 0.20 mg mL−1. In the region of T ≥ 33.2 °C in Figure 3, as the temperature further increases, the upward moving of the entire intensity curve accelerates, such that the intensity values in the low q range increase drastically and the effect of the “unknown longrange correlations,” which was severe in the low temperature range, now vanishes. The bending of the curve moves toward small q range, and it follows that the Rg value starts to increase sharply with temperature in Figure 4a, reaching a plateau (Rg ≈ 120 nm) for T ≥ 40 °C. Therefore, for T > 32 °C, the LCSTtype thermoresponsive transition starts, and a new type of micelles, referred to as micelle-2, is formed from micelle-1, with a size much larger than that of micelle-1. We define the LCST value in the middle of the transition region between micelle-1 and micelle-2, and we have LCST = 35.5 °C at C = 0.20 mg mL−1. To understand the morphology of micelle-1 and micelle-2 at C = 0.20 mg mL−1, let us analyze the hydrodynamic radius (Rh) in Figure 4a and the TEM images in Figure 5a−c. Compared to the corresponding Rg values in the same figure, in the region where micelle-1 is formed, the Rh value is initially much larger than the Rg value and then decreases as temperature increases. It is again related to the presence of the unknown long-range correlations, whose effect is very difficult to avoid for dynamic light scattering measurements. With increase in temperature, since more and more micelles are formed, the unknown long-range correlations effect progressively vanishes, and finally, we have the result that the Rh value is basically equal to the Rg value. Figure 5a shows the TEM image of micelle-1, formed at C = 0.20 mg mL−1 and T = 25 °C, and reveals a rather spherical shape with an average radius of about 30 nm, consistent with the Rg and Rh values. Since for a sphere, Rg = (3/5)0.5R and Rh = R > Rg, where R is the radius of the sphere, the equal size of Rg and Rh may indicate that the sphere of micelle-1 is somewhat hollow.38 Unlike micelle-1, in the region of micelle-2 in Figure 4a, the Rh value is only half of the Rg value, and micelle-2 is definitely not spherical. In fact, the TEM images of micelle-2 in Figure 5b,c, taken at T = 45 °C and C = 0.20 mg mL−1, show a rather long shape, which could be considered to be prolate ellipsoids, although some of them are slightly curved. Thus, it is not surprising to have Rg ≈ 2Rh, because for prolate ellipsoids, the Rg/Rh ratio is typically in the range of 1.36−2.24.51 It is particularly interesting to explore how the transition of micelle1 to micelle-2 occurs, leading to such different sizes and shapes. Let us observe the amplified image in Figure 5c, where two peculiarities are essential: one very thin, rodlike micelle in the middle of the image and one of micelle-2 located at the bottom-right angle, with a similar thin, rodlike micelle that partially joins to it, like its branch. The thin, rodlike micelle is most probably restructured from the hollow spherical shape of micelle-1 during the LCST-type transition, and along its axis, there must be aligned hydrophobic and hydrophilic patches. The hydrophobic patches are unstable in water, and the formed rodlike micelles tend to aggregate to reduce the surface

Figure 4. (a) Gyration radius, Rg, and hydrodynamic radius, Rh, of the micelles as a function of temperature in the aqueous dispersion of TDHBG-1 at C = 0.20 mg mL−1. (b) Effect of the TDHBG-1 concentration on the micellization behavior.

considers that although TDHBG-1 is double-hydrophilic, their backbone is still hydrophobic. TDHBG-1 is very similar to an amphiphilic surfactant. Thus, when its concentration reaches the critical micellization concentration or the temperature reaches critical micellization temperature (CMT), it forms micelles. In addition, it should be also noted that differences in hydrophilicity between the two blocks,22−24 as well as possible hydrogen bonding,25 can also contribute to the formation of the micelles. The amount of micelles increases as the temperature increases, while the size of the micelles keeps constant in this case. The scattering intensity at zero angle, I(0) reported in Figure S1 in SI, supports the argument, and it increases with T in the corresponding T region, due to an increased number of micelles. The micelles formed in this region are referred to as micelle-1. Thus, although TDHBGs were usually considered to exist as individual molecules at temperatures below LCST, TDHBG-1 does form micelles below LCST. This is rather similar to the thermoresponsive double-hydrophilic block copolymer, poly(ethylene oxide)-bpoly(N-isopropylacrylamide), which also forms micelles below LCST.48 Note that for T > 28 °C, with increase in temperature, a slight decrease in Rg was observed in Figure 4a. This is most F


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Figure 5. TEM images of the self-assembled micelles from TDHBG-1: (a) micelle-1 at C = 0.20 mg mL−1 and 25 °C; (b, c) micelle-2 at C = 0.20 mg mL−1 and 45 °C; (d) micelle-3 at C = 0.02 mg mL−1 and 45 °C. The insets sketch the possible structures of the micelles.

that the micellization and thermoresponsive transition for this TDHBG at C ≥ 0.20 mg mL−1 behave basically the same. This conclusion is important in practical applications because the TDHBG concentration should typically be much larger than 0.20 mg mL−1. In fact, we have looked at the thermoresponsive transition at the TDHBG concentrations until 16 mg mL−1, but, as expected, the system becomes very turbid and cannot be characterized by DLS and SLS when the temperature is larger than LCST. From visual observation, even at such high concentrations, the dispersion is still a milky uniform liquid. On the other hand, toward lower concentrations, at C = 0.02 mg mL−1, the situation is rather different. First, in the micelle-1 region in Figure 4b, the Rg values at C = 0.02 mg mL−1 are constant, around 16 nm, which are basically equal to those in the individual molecule region at C = 0.20 mg mL−1. This means that at C = 0.02 mg mL−1, TDHBG-1 is in the form of individual molecules, without forming micelle-1. Second, similar to the cases of C = 0.20 and 0.50 mg mL−1, at C = 0.02 mg mL−1, when T ≥ 33.2 °C, the Rg value also starts to increase sharply as the temperature increases. Thus, this corresponds to the LCST-type thermoresponsive transition, but now the transition is from individual molecules, instead of micelle-1. It is interesting that the plateau value of Rg reached at high temperature is only ∼80 nm, much smaller than that (∼120 nm) reached at C = 0.20 and 0.50 mg mL−1. It means that the formed micelles are neither micelle-1 nor micelle-2,

energy, leading to micelle-2, which is a bundle of a limited number of the rodlike micelles, as evidenced in Figure 5c. More evidence about the bundle structure of micelle-2 can be found in additional TEM images of higher resolution in Figure S2 in the SI. The size of micelle-2 reaches a plateau in Figure 4a, confirming that the average number of rodlike micelles in each bundle (micelle-2) is limited. It indicates that the external surface of micelle-2 is hydrophilic, completely covered by the galactose groups, and thus further aggregation becomes less likely. Figure 4b compares the Rg values at three concentrations of TDHBG-1, C = 0.50, 0.20, and 0.02 mg mL−1. It is seen that the Rg data at C = 0.20 and 0.50 mg mL−1 are basically overlapped in the regions of micelle-1 and micelle-2, and this is true also for the Rh data as reported in Figure S3 in SI. It should be noted that in the transition region from micelle-1 to micelle-2, the Rg (Rh as well) values are rather different between C = 0.20 and 0.50 mg mL−1. It arises because when the temperature is relatively low, the transition needs substantial time to reach the equilibrium and depends also on the TDHBG concentration. It is evidenced in Figure S4 in SI, where are shown the Rh values and counts of the scattering intensity as a function of the equilibrium time at C = 0.20 mg mL−1 and T = 35.8 °C. It is seen that the Rh value and the counts continue increasing with time without reaching equilibrium even after 6 h. The results in Figure 4b indicate G


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Langmuir Table 2. Micelle Size and LCST Data of the Three Synthesized TDHBGs TDHBG

CMT (°C)

Rg (nm), ind. mol.

Rg (nm), micelle-1

Rh (nm), micelle-1

Rg (nm), micelle-2

Rh (nm), micelle-2

LCST (°C)

TDHBG-1 TDHBG-2 TDHBG-3

11a 17a >23a

16 19 21

34 28 27b

34 29 18b

120 140 64b

62 138 80b

35.5 31.4 30.3

At C = 0.20 mg mL−1. bAt C = 1.00 mg mL−1.

a

which are referred to as micelle-3. Figure 5d shows the TEM image of micelle-3 taken at T = 45 °C, and these micelles are obviously rather different from those in Figure 5b at C = 0.20 mg mL−1. They have rather sharp edges, typical of crystals, and their shape varies from potatoes to triangles to cylinders. The Rg/Rh ratio is ∼1.5 for micelle-3 (Rh reported in Figure S2 in the SI), smaller than that (∼2) for micelle-2. From the above results, we can conclude that the size (and shape) of the micelles formed after the LCST-type transition depends on whether the transition starts from individual molecules or micelle-1. This feature offers the opportunity in practical applications to tune the morphology of the micelles of the same TDHBG through varying its concentration. Although the micellization process and the micelle structure may depend on the concentration of TDHBG-1, the LCST values estimated from Figure 4b at different concentrations are basically identical. This feature is expected because the LCSTtype phase transition is a property of a given TDHBG, independent of its concentration. 3.3. Effect of OVNG Molar Fraction on Micellization and LCST-type Transition. For the remaining two TDHBGs in Table 2, TDHBG-2 and TDHBG-3, with f OVNG = 12.2 and 14.6%, respectively, similar light scattering studies have been performed. Figure 6a shows the Rg values as a function of temperature for TDHBG-2 at three concentrations, C = 0.20, 0.10, and 0.02 mg mL−1. The results are rather similar to those in Figure 4b for TDHBG-1. The Rg curves in Figure 6a in the regions of micelle-1 and micelle-2 overlap at C = 0.20 and 0.10 mg mL−1, indicating that the micellization and the LCST-type transition are the same under the two concentrations, which should be true also for even higher concentrations. On the other hand, when the Rg values are compared to the Rh values, as reported in Figure S5 in the SI, the Rg/Rh ratio is close to unity not only for micelle-1 but also for micelle-2. This is different from that of TDHBG-1 in Figure 4a, which is close to 2 for micelle-2. It clearly indicates that the shape of micelle-2 is different for TDHBG-2 than for TDHBG-1. Figure 7a shows the TEM picture of micelle-2 from TDHBG-2 at C = 0.20 mg mL−1 and T = 45 °C. It is seen that micelle-2 is rather spherical, and from the contrast, it is most probably hollow, consistent with the Rg/ Rh ratio that is close to unity. At C = 0.02 mg mL−1, similar to that of TDHBG-1, no micelle-1 is formed, and the LCST-type transition occurs directly from the individual molecules to micelle-3, which has a substantially smaller Rg value with respect to micelle-2. The Rg/Rh ratio is also close to unity (data not shown), and in fact, the TEM image of micelle-3 shown in Figure 7b confirms its hollow spherical shape. Figure 6b shows the Rg curves for TDHBG-3 at the concentrations C = 1.00, 0.50, and 0.20 mg mL−1. For this TDHBG, we have increased the concentration level with respect to the previous two cases because, as can be seen from Figure 6b, until C = 0.50 mg mL−1, we are unable to observe the formation of micelle-1. Thus, we have to further increase the concentration to C = 1.00 mg mL−1. At this concentration,

Figure 6. Effect of the OVNG molar fraction (f OVNG) in the TDHBG on the micellization and thermoresponsive transitions: (a) for TDHBG-2 (f OVNG = 12.2%) and (b) for TDHBG-3 (f OVNG = 14.6%).

we can indeed observe the formation of micelle-1, but in the low temperature range, we are unable to observe the CMT. It arises because the concentration is too high such that the CMT is smaller than the minimum temperature that our setup can reach. Compared to Figures 4b and 6a in the cases of TDHBG1 and -2, Figure 6b in the case of TDHBG-3 exhibits significant differences. In the previous two cases, the size of micelle-3 is much smaller than that of micelle-2 and depends on the polymer concentration. In Figure 6b, the size of micelle-2 formed at C = 1.00 mg mL−1 is rather comparable to that of micelle-3 formed at C = 0.50 mg mL−1, and at further higher temperatures, it somewhat decreases to a size similar to that of micelle-3 formed at C = 0.20 mg mL−1. On the other hand, as reported in Figure S6, the Rg/Rh ratio decreases with C, from H


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Langmuir

may indicate that micelle-1 cannot transfer directly to micelle2, and it first self-disassembles to individual molecules and then forms micelle-2 after the LCST. In this case, from our above definition, micelle-2 should be considered to be micelle-3. Such a phenomenon is mostly related to the fact that as the size of the POVNG block increases, the fraction of the hydrophilic galactose groups in the molecule increases, unfavorable in forming micelles, and the formed micelles are more fragile. This result indicates that in practical applications, the fraction of the hydrophilic galactose groups in the TDHBGs should be designed to balance between the mechanical strength and protein-binding capacity of the micelles. Table 2 compares the different properties of the three TDHBGs obtained from the above light scattering investigations. It is seen that the Rg values of the individual molecules are 16, 19, and 21 nm, respectively, for TDHBG-1, -2, and -3. It is consistent with the increase in the size of the POVNG block, that is, increasing with f OVNG. The CMT value increases and the Rg and Rh values of micelle-1 decrease as f OVNG increases, again indicating that the POVNG block is unfavorable in forming micelles. It is due to the increase in hydrogen bonds between hydroxyl groups of galactose and water, which retards the process of phase transition with increase in f OVNG. The LCST value decreases as f OVNG increases in the given f OVNG range. At f OVNG = 11.0%, the LCST value is 35.5 °C, and we can expect that by slightly reducing f OVNG, one can have a TDHBG with the LCST around 37 °C, useful for biomedical applications.52−54 It should be mentioned that self-assembly of block copolymers depends on the respective volume fractions of the blocks. In the present cases, since galactose is rather large in volume, it follows that increase in f OVNG leads to substantial increase in the volume fraction of POVNG in the block glycopolymer. Therefore, f OVNG is a good factor for tuning the self-assembly behavior.

Figure 7. TEM images of (a) micelle-2 at C = 0.20 mg mL−1 and 45 °C and (b) micelle-3 at C = 0.02 mg mL−1 and 45 °C, in the case of TDHBG-2.

4. CONCLUDING REMARKS In this work, we have synthesized a new type of thermoresponsive double-hydrophilic block glycopolymers (TDHBGs) via controlled RAFT polymerization, which are composed of a thermoresponsive, poly(di(ethylene glycol)methyl ether methacrylate) (PDEGMA), block and a galactose-functionalized, poly(6-O-vinyladipoyl-D-galactose) (POVNG), block. After having verified the strong activity of the POVNG block in lectin binding, we have systematically investigated the thermoresponsive self-assembly behavior of the TDHBGs, using static and dynamic light scattering techniques, as well as TEM imaging. It is found that the new TDHBGs possess both micellization and the lower critical solution temperature (LCST)-type phase transition. In particular, at relatively high concentrations, starting from individual molecules, as temperature increases, the TDHBGs first form micelles after reaching the critical micellization temperature (CMT), with an Rg value around 30 nm, which are very similar to those of amphiphilic surfactants, referred to as micelle-1. Then, as temperature increases to reach the LCST, a new type of micelles is formed, with substantially larger sizes, which are referred to as micelle-2. The shape of micelle-2 depends on the size of the POVNG block and can be either prolate ellipsoids or hollow spheres or nonhollow spheres, but does not depend on the TDHBG concentration.

Rg/Rh ≈ 1 at C = 0.20 mg mL−1 to Rg/Rh ≈ 0.92 at C = 0.50 mg mL−1 and then to Rg/Rh ≈ 0.80 at C = 1.00 mg mL−1. This means that with C increasing from 0.20 to 1.00 mg mL−1, the micelle structure changes from a hollow sphere to a nonhollow sphere. Thus, even though the Rg values are rather comparable at the three concentrations, the real mass of the micelle at C = 1.00 mg mL−1 is larger. The above behaviors for TDHBG-3 are rather similar to the self-assembling behaviors of poly(ethylene oxide)-b-poly(N-isopropylacrylamide) observed by Zhao et al.49 In their cases, the size and morphology of the micelles formed after the LCST also depend on the polymer concentration. Another interesting observation in Figure 6b is located at the transition from micelle-1 to micelle-2 at C = 1.00 mg mL−1. Before the LCST, the Rg value first decreases with T to reach the same Rg value of the individual molecules. Although we also observed a slight decrease in Rg for TDHBG-1 and -2 in the same region, as can be seen in Figures 4 and 6a, the Rg values are always larger than those of the individual molecules. We believe that this is related to the PDEGMA block, which would become less and less hydrated as the temperature progressively increases even before the LCST. It follows that the PDEGMA chains start to shrink, leading to the decrease in the Rg value. In the case of TDHBG-3, since the Rg value has reduced to the same Rg value as the individual molecules, this I


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Multivalency as a chemical organization and action principle. Angew. Chem., Int. Ed. 2012, 51, 10472−10498. (5) Taylor, M. E.; Drickamer, K. Introduction to Glycobiology; 3rd ed.;Oxford University Press: Oxford, 2006. (6) Marradi, M.; Chiodo, F.; Garcia, I.; Penades, S. Glyconanoparticles as multifunctional and multimodal carbohydrate systems. Chem. Soc. Rev. 2013, 42, 4728−4745. (7) Ribeiro-Viana, R.; Sánchez-Navarro, M.; Luczkowiak, J.; Koeppe, J. R.; Delgado, R.; Rojo, J.; Davis, B. G. Virus-like glycodendrinanoparticles displaying quasi-equivalent nested polyvalency upon glycoprotein platforms potently block viral infection. Nat. Commun. 2012, 3, No. 1303. (8) Richards, S.-J.; Jones, M. W.; Hunaban, M.; Haddleton, D. M.; Gibson, M. I. Probing bacterial-toxin inhibition with synthetic glycopolymers prepared by tandem post-polymerization modification: role of linker length and carbohydrate density. Angew. Chem., Int. Ed. 2012, 51, 7812−7816. (9) Luczkowiak, J.; Muñoz, A.; Sánchez-Navarro, M.; Ribeiro-Viana, R.; Ginieis, A.; Illescas, B. M.; Martín, N.; Delgado, R.; Rojo, J. Glycofullerenes inhibit viral infection. Biomacromolecules 2013, 14, 431−437. (10) Xiao, Y.; Sun, H.; Du, J. Sugar-breathing glycopolymersomes for regulating glucose level. J. Am. Chem. Soc. 2017, 139, 7640−7647. (11) Yilmaz, G.; Becer, C. R. Glyconanoparticles and their interactions with lectins. Polym. Chem. 2015, 6, 5503−5514. (12) Lou, S.; Gao, S.; Wang, W.; Zhang, M.; Zhang, J.; Wang, C.; Li, C.; Kong, D.; Zhao, Q. Galactose-functionalized multi-responsive nanogels for hepatoma-targeted drug delivery. Nanoscale 2015, 7, 3137−3146. (13) Li, X.; Chen, G. Glycopolymer-based nanoparticles: synthesis and application. Polym. Chem. 2015, 6, 1417−1430. (14) Kiessling, L. L.; Grim, J. C. Glycopolymer probes of signal transduction. Chem. Soc. Rev. 2013, 42, 4476−4491. (15) Chen, G.; Amajjahe, S.; Stenzel, M. H. Synthesis of thiol-linked neoglycopolymers and thermo-responsive glycomicelles as potential drug carrier. Chem. Commun. 2009, 1198−1200. (16) Pasparakis, G.; Alexander, C. Sweet talking double hydrophilic block copolymer vesicles. Angew. Chem., Int. Ed. 2008, 47, 4847− 4850. (17) Kumar, J.; McDowall, L.; Chen, G.; Stenzel, M. H. Synthesis of thermo-responsive glycopolymers via copper catalysed azide−alkyne ‘click’ chemistry for inhibition of ricin: the effect of spacer between polymer backbone and galactose. Polym. Chem. 2011, 2, 1879−1886. (18) Yilmaz, G.; Messager, L.; Gleinich, A. S.; Mitchell, D. A.; Battaglia, G.; Becer, C. R. Glyconanoparticles with controlled morphologies and their interactions with a dendritic cell lectin. Polym. Chem. 2016, 7, 6293−6296. (19) Rini, J. M.; Hardman, K. D.; Einspahr, H.; Suddath, F. L.; Carver, J. P. X-ray crystal structure of a pea lectin-trimannoside complex at 2.6 A resolution. J. Biol. Chem. 1993, 268, 10126−10132. (20) Ling, H.; Boodhoo, A.; Hazes, B.; Cummings, M. D.; Armstrong, G. D.; Brunton, J. L.; Read, R. J. Structure of the shigalike toxin i b-pentamer complexed with an analogue of its receptor Gb3. Biochemistry 1998, 37, 1777−1788. (21) Zhang, R.-G.; Scott, D. L.; Westbrook, M. L.; Nance, S.; Spangler, B. D.; Shipley, G. G.; Westbrook, E. M. The threedimensional crystal structure of cholera toxin. J. Mol. Biol. 1995, 251, 563−573. (22) Casse, O.; Shkilnyy, A.; Linders, J.; Mayer, C.; Häussinger, D.; Völkel, A.; Thünemann, A. F.; Dimova, R.; Cölfen, H.; Meier, W.; Schlaad, H.; Taubert, A. Solution behavior of double-hydrophilic block copolymers in dilute aqueous solution. Macromolecules 2012, 45, 4772−4777. (23) Schmidt, B. V. K. J. Double hydrophilic block copolymer selfassembly in aqueous solution. Macromol. Chem. Phys. 2018, 219, No. 1700494. (24) Park, H.; Walta, S.; Rosencrantz, R. R.; Körner, A.; Schulte, C.; Elling, L.; Richtering, W.; Böker, A. Micelles from self-assembled double-hydrophilic PHEMA-glycopolymer-diblock copolymers as

At relatively low TDHBG concentrations, the LCST is smaller than the CMT, and the LCST-type phase transition proceeds from individual molecules, instead of micelle-1. In this case, the LCST-induced micelles are different from those formed from micelle-1, which are referred to as micelle-3. The shape of micelle-3 depends not only on the size of the POVNG block but also on the TDHBG concentration, which can be either crystal-like or spherical or hollow spherical. In the range of the OVNG molar fraction in the TDHBG, from 11.0 to 14.6%, the LCST value decreases as the OVNG molar fraction increases, from 36 to 30 °C, and this can obviously be further tuned by changing the OVNG molar fraction. With the tunable LCST value, together with the rich morphologies of the formed micelles, we believe that the synthesized TDHBGs would have potential applications in biological processes. In fact, since these DHBGs are hepatomatargeting glycopolymers, the applications along this line have already started in our lab.

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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.langmuir.8b01516.

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GPC analysis curves; further experimental data and TEM images related to micellization and thermoresponsive transition of the synthesized TDHBGs (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (J.Q.). *E-mail: [email protected] (H.W.). ORCID

Fa-Wei Shen: 0000-0001-7588-5219 Hua Wu: 0000-0002-2805-4612 Author Contributions §

J.Q. and F.-W.S. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the Natural Science Foundation of China (Grant No. 21303014), the Swiss National Science Foundation (Grant No. 200020_165917), the State Key Laboratory of Molecular Engineering of Polymers (Fudan University), the Key Laboratory of Science & Technology of Eco-Textiles of the Ministry of Education, and the Fundamental Research Funds for the Central Universities.

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REFERENCES

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