Glycopolymers are synthetic polymers featuring pendant carbohydrate moieties on a non-carbohydrate main chain ^[1]. Their good water solubility,low toxicity and biocompatibility render them a wide range of applications,such as drug delivery ^[2], vaccines ^[3],fluorescent probes ^[4],etc. To further extend these bio-applications to a much boarder area,one feasible method is to hybridize them with inorganic nanomaterials ^{[5, 6]}. The corresponding "sweet" hybrid nanomaterials have shown great promise in the areas of sensor ^[7],magnetic resonance imaging ^[8] and vaccines ^[3],which made them receive quite a few attentions.

However,in most cases,thiol-functionalized glycopolymers were directly bound to gold nanoparticles (AuNPs) via Au-S bond. When surface of AuNP is covered with thiol-containing polymers, the nanoparticle only provides a scaffold and the property of AuNP itself could not be fully incorporated ^[9]. One convenient way to solve this problem is to stabilize AuNPs without using thiol group. In literature,monosaccharide on the surface of dendrimers was proved to stabilize AuNPs in water ^[10]. Recently,we also have demonstrated that AuNPs can be generated in situ and stabilized by glyco-inside vesicles ^[11]. This study provided a new route to generate rather "free" AuNPs within self-assembled micelles or vesicles,which might be beneficial for their further bio-applications. But in these studies,full manipulation on morphology of the hybrid nanomaterial was not achieved,which might hamper our further exploration. Thus,this paper aims at generating nanomaterials stabilized by glycopolymers with precise control on the morphology. By controlling the thickness of glyco-shell,Janus "sweet" nanoparticle was realized,which has not been reported in literature. In the end,catalysis property of these nanomaterials was also investigated. 2. Results and discussion 2.1. Synthesis and characterization of triblock glycopolymers

To control the morphology of hybrid nanomaterials ^[12] and even make Janus ones,polymer scientists have developed some intelligent strategies ^{[13, 14, 15]}. Among them,properties of the polymeric micelles,including the lengths of different stabilizing blocks and softness of stabilizing micelle cores were also employed ^[16]. However,glyco-NPs have not been utilized for preparation of Janus hybrid particles. Considering the bulky sugar pendant groups on the main chain of glycopolymers,we suppose that the effect from glyco-block might be much more significant than the previous non-sugar units. Thus two tri-block copolymers are designed,i.e. their middle blocks are glyco-blocks with hydrophobic and hydrophilic blocks on their both sides. By controlling the ratio between the sugar block and the hydrophobic block,different morphologies of hybrid nanomaterials (glyco-AuNPs) were expected after in situ reduction of precursor HAuCl₄.

Glycopolymers were synthesized via the combination of reversible addition-fragmentation chain transfer (RAFT) polymerization and post-polymerization modification. As shown in Scheme 1,the triblock glycopolymer contains three components, from left to right,a hydrophilic poly(oligo(ethyleneglycol) methacrylate) (POMA) block,a hydrophilic glyco-block (PMal) and a hydrophobic polystyrene block (PS). The length of POMA in the two series of triblock glycopolymers was similar. However,the ratio of PMal:PS was designed to vary,which might help us to demonstrate the contribution of the middle glyco-block.

Scheme 1

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Scheme 1.Synthetic route of amphiphilic triblock glycopolymer POMA-b-PMal-b-PS.

Briefly,oligo(ethylene glycol) methacrylate (OMA,M_n = 300 g mol^-1) was polymerized by RAFT polymerization using cumyl dithiobenzoate (CDB) as chain transfer agent (CTA),yielding P1-1 and P2-1. As shown in Table 1,the degree of polymerization (DP) of POMA was obtained by comparison of the integral areas of ethylene glycol peak at 4.02 ppm and that of the phenyl group of CTA at 7.0-8.0 ppm in ¹H NMR. Then,the obtained POMA was used as macro chain transfer agent (macro-CTA) to prepare the second block poly(glycidyl methacrylate) (PG),yielding two diblock copolymers P1-2 and P2-2. Similarly,the DP of PG block was calculated by comparing the ¹H NMR peak of epoxy unit of PG at 2.64 ppm and that of ethylene glycol at 4.02 ppm. The triblock copolymer POMA-b-PG-b-PS was further prepared using the previous diblock copolymer (P1-2 and P2-2) as macro-CTA and styrene as monomer,yielding P1-3 and P2-3,with their DP obtained from the integral area of PS at 6.5-7.5 ppm. All of these copolymers showed narrow polydispersity indexes (PDI),measured by gel permeation chromatography (GPC). And the characterization results are shown in Table 1 and Figs. S1 and S2 in Supporting information. It is worth to mention that ¹H NMR results were used to determine DP,since PEG was used as calibration standard for GPC analysis,which was not effective to characterize glycopolymers due to great structural inconsistency.

Table 1

Table 1 Mn and PDI of precursors of the final triblock copolymers.

Table 1 Mn and PDI of precursors of the final triblock copolymers.

Combination of ring-opening reaction and click reaction was used in post-polymerization modification. P1-3 and P2-3 were treated with NaN₃ and acetic acid at 80℃ to obtain azidecontaining copolymer P1-4 or P2-4,by the ring opening of epoxide from the glycidyl methacrylate. Disappearance of the epoxy units in ¹H NMR spectra and the appearance of azide at 2105 cm^-1 in Fourier transform infrared spectra (FT-IR) demonstrated the complete transformation from epoxide to azide (Fig. S3 in Supporting information). After click reaction with propargyloxyb- β-maltopyranoside,the strong peak of azide at 2105 cm^-1 disappeared (Fig. S3),while the peak at 3340 cm^-1 increased due to the hydroxyl groups from maltose. This result indicated the complete conversion to POMA-b-PMal-b-PS. ¹H NMR was also used to characterize the triblock glycopolymers (Fig. S3). Finally,a pair of triblock glycopolymers was determined as POMA₂₂-b-PMal₅₄-b- PS₁₇ (P1) with PMal to PS ratio as 3:1,and POMA₂₀-b-PMal₂₆-b- PS₁₀₈ (P2) with PMal to PS ratio as 1:4. As control,POMA₂₂-b- PPA₅₄-b-PS₁₇ (P3) was obtained by the same strategy but propargyl alcohol was used to click with azide-containing polymer P1-4 (FTIR and ¹H NMR in Fig. S4 in Supporting information) instead. What is more,diblock copolymer PMal₄₇-b-PS₁₅₈ (P4) was also synthesized (GPC,FT-IR and 1H NMR characterization data are shown in Fig. S5 and S6) via the similar strategy and used as another control. 2.2. Preparation of glyco-NPs

Self-assembly of glycopolymers were performed by selective solvent method. Typically P1 or P2 was firstly dissolved in 1 mL DMAc (dimethylacetamide,10 mg mL^-1),then 8 mL deionized water was added into the solution quickly. After dialysis,the final concentration was fixed at 0.83 mg mL^-1 in which self-assembled structures were obtained. For clarity,M1 and M2 were used to represent the nanostructures formed by the corresponding glycopolymer precursors P1 and P2,respectively. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to characterize these structures (Fig. 1 and Fig. S7 in Supporting information). DLS measurements showed that h> (hydrodynamic radius) of M1 and M2 was 18 nm and 20 nm, respectively. TEM pictures confirmed their micellar morphologies (Fig. S7). According to the chemical structure of P1 and P2,M1 and M2 were proposed as micelles with PS as the dense core,soluble glyco-block PMal as the shell,and short POMA as the corona outside the shell. M1 and M2 at the same concentration were characterized by ¹H NMR with equal amount of sodium 2,2- dimethyl-isotope 2-silapentane-5-sulfonate (DSS) as internal standard. As shown in Fig. S8 in Supporting information,the integral ratio of triazole group (M1 to M2),as a marker of sugar moieties was about 2.5:1. This result indicated that the amount of solvated sugar units from M1 were much larger than that of M2 in water,demonstrating the glyco-shell of M1 was thicker than that of M2 as we designed,although their overall h> seemed similar. Similarly,M3 and M4 formed micelles with h> around 30 nm and 26 nm,respectively (Fig. S9 in Supporting information).

Fig. 1

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Fig. 1.(a) DLS and (b) cartoon scheme of M1 before and after reduction of HAuCl4; (c) DLS and (b) cartoon scheme of M2 before and after reduction of HAuCl4.

M1 and M2 were used for in situ AuNP formation. Chloroauric acid (HAuCl₄) was firstly mixed with the glyco-NPs by stirring at room temperature overnight. UV-vis spectrum of the mixture proved that no self-reduction happened during this process (Fig. S10 in Supporting information). Then NaBH4 (3.2 equiv. to HAuCl₄) was then added and the mixture turned brown in several seconds. Appearance of a distinct characteristic absorption peak at 515 nm in the UV-vis spectrum (Fig. S11 in Supporting information) confirmed the formation of AuNPs. TEM images also demonstrated the presence of AuNPs,i.e. the black dots with diameters smaller than 10 nm (Fig. 2). The magnified images of the AuNPs showed an obvious crystal lattice and the inter-planar spacing of the lattice was near 0.224 nm (Fig. 2b and d),which agreed well with the (1 1 1) lattice spacing of AuNPs. The EDX (energy dispersive X-ray) spectrum showed that there were strong peaks for elemental gold indicating that these nanoparticles were composed of Au (Fig. S12 in Supporting information). It was quite interesting to find that M1 with a thick sugar shell had many AuNPs with diameter around 3 nm(Fig. 2a and b with inset cartoon). While most of M2 with thin sugar shells had only one AuNP with diameter around 7 nm(Fig. 2c and d with inset cartoon),demonstrating the feature of Janus particles. This "one-micelle-one-AuNP" hybrid structure could be easily found under TEM (Fig. 2c and d) and more TEM images were presented in Fig. S13 in Supporting information. Interestingly,after reduction of HAuCl₄,the h> of M1 increased to 43 nm (Fig. 1) compared to the original 18 nm. However,the h> of hybridized M2 kept constant,which was around 27 nm. As a control,by using triblock copolymer P3 with the same backbone but no sugar inside, the micelle M3 could not stabilize AuNPs,which were much larger than those inside the glyco-micelles and easily aggregated (Fig. S14 in Supporting information). Moreover,the same experiment was carried out for micelles formed by diblock copolymer without POMA (P4),resulting in precipitates after reduction (Fig. S15 in Supporting information). Therefore,this result indicated that the three-layer design of micelles with the glyco-layer in the middle was crucial to stabilize AuNPs.

Fig. 2

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Fig. 2.TEM images of different glyco-AuNPs. (a), (b) M1 with many AuNPs inside (inset: cartoon scheme); (c), (d) M2 with only one AuNP inside (inset: cartoon scheme).

To investigate the role of glyco-shell thickness in nanoparticle hybridization,different amounts of HAuCl₄ were employed to form glyco-AuNPs,while the amount of the glyco-NPs and reducing reagent NaBH₄ were constant. To the aqueous solution of glyco- NPs (1 mL,0.83 mg mL^-1),different amount of HAuCl₄ (50 mg mL^-1) was added (1 μL,5 μL and 10 μL),followed by incubation and reduction. As shown in Fig. S16 in Supporting information,the size of AuNPs encapsulated by M1 was all around 3 nm (Fig. S16(a)-(c)),and more AuNPs were found inside the micelles with the increasing concentration of HAuCl₄. On the contrary,the encapsulated amount of AuNPs by M2 only slightly increased with increasing concentration of HAuCl₄ (Fig. S16(d)- (f)). The result indicated that the Janus particle morphology was greatly related to the thickness of glyco-shell,not directly determined by the concentration of HAuCl₄ and NaBH₄. At an appropriate concentration of HAuCl₄ and NaBH₄,Janus particles would be observed in the presence of M2 (Fig. 2c and d). For the mechanism,we supposed that HAuCl₄ might locate in the sugar layer and protected by the POMA corona. After reduction,if the sugar layer was very thin,only a few Au seeds could be loaded in the shell and only one seed can grow up forming nanoparticle. However,when the sugar shell became thicker,more Au seeds would be inside the shell resulting in formation of much more AuNPs. 2.4. Catalytic reduction of 4-nitrophenol (4-NP)

To evaluate the catalytic activity of the glyco-AuNPs,model reduction reaction of 4-nitrophenol (4-NP) to 4-aminophenol (4- AP) in the presence of an excess amount of NaBH₄ was studied. AuNPs were widely used as catalyst for this reaction (Fig. S17 in Supporting information) by relaying electrons from donor BH₄ -to acceptor 4-NP ^[17],when both of them were adsorbed on surface. The reaction process was monitored by UV-vis spectrometry. As shown in Fig. S18c in Supporting information,the reaction could not happen in the absence of glyco-AuNPs. After addition of the catalyst,the characteristic peak of 4-NP at 400 nm started to decrease,while a new peak of 4-AP at 295 nm appeared (Fig. S18). After a while,the peak of 4-NP was no longer observed and the solution color changed from yellow to colorless,indicating that the catalytic reduction of 4-NP proceeded completely. The rate constant k was calculated using the rate law for the first-order kinetics: ln(A_t/A₀) = -kt,where A_t represented absorbance of 4-NP at any time,A₀ represented the starting absorbance of 4-NP and k was rate constant. Fig. 3 showed a good linear correlation of ln(A_t/A₀) versus time,therefore the kinetic reaction rate constant was estimated as 4.0 × 10^-3 s^-1 for M1 and 2.2 × 10^-3 s^-1 for M2. Both the them were larger than citrate-capped AuNPs (1.2 ± 0.1 × 10^-3 s^-1) with diameter of 3.5 nm and poly(N-isopropylacrylamide)- b-poly(4-vinyl pyridine) (PNIPAM-b-P4VP) stabilized AuNPs (1.5 × 10^-3 s^-1) with diameter of 3.3 nm,which located in the dense core of P4VP ^{[18, 19]}. 3. Conclusion

A novel amphiphilic brush triblock glycopolymer POMA-b- PMal-b-PS was prepared through the combination of RAFT polymerization and post-polymerization modification. Micelles prepared by this new glycopolymer were used to in situ formation of AuNPs. Different morphologies including Janus particles and raspberry-like particles were obtained by changing the ratio of PMal and PS.

Fig. 3

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Fig. 3.Plot of ln(At/A0) versus time for the reduction of 4-NP. Black line: M1 with AuNPs; Red line: M2 with AuNPs.

Acknowledgments

Ministry of Science and Technology of China (No. 2011CB932503332),National Natural Science Foundation of China (No. 91227203,21474020 and 51322306),and the Shanghai Rising-Star Program (No. 13QA1400600) are acknowledged for their financial support.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2015.05.022.