1. Introduction

Using anisotropic nanoparticles as building blocks for bottomup fabrication of devices and functional structures is a fascinating objective within the field of nanotechnology. Recently,anisotropic micelle of crystalline-coil copolymers have attracted much attention. The crystallization of a crystalline-coil block copolymer as the driving force is a facile method for preparing a variety of anisotropic nano-aggregates ^{[1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14]}. The crystallization processes not only have important influence on the morphologies of the micelles from a crystalline-coil copolymer,but also are crucial to other properties,such as stability. In our previous works,the amphiphilic crystalline-coil block copolymer based on PPDO can self-assemble into anisotropic micelles with star anise-like morphology in water ^{[13, 14, 15, 16, 17]}. A ‘‘crystallization induced hierarchical assembly’’ mechanism was used for explaining the formation of the anisotropic micelles ^[14]. This kind of anisotropic semicrystalline micelle with special morphology is also a very good candidate for constructing hierarchical nanostructures such as core-shell nanofibers ^[18]. In many applications,e.g. biomedicine and drug delivery systems,the stability of micelles in water is a key issue; owing to the supramolecular nature of micelles,polymeric micelles and unimers are known to coexist at equilibrium above the critical micelle concentration (CMC) ^[19]. It is very difficult to keep the polymeric micelles intact and stable when it is diluted within the biological media,where it is below the CMC or interacts with surfactant proteins. The extremely low polymer concentration or interaction with surfactant proteins could accelerate the dissociation of polymeric micelles under physiological conditions ^[20].

Recently,much effort has been devoted to improving stability of polymeric micelles. Cross-linking of polymeric micelles is a important strategy to increase particle stability. However,in many cases,crosslinking is performed utilizing covalent bonding within the micelle. The micelle is often achieved with an irreversible stabilization mechanism. Moreover,it is relatively difficult to control the cross-linking process,which does not lend itself to tunable micellar properties. In addition,in some crosslinked micelles,the crosslinks are physically located in the core of the micelle where drugs are loaded,which may interfere with pharmaceutical drug action or drug release from the micelle ^[21]. It is found that the modification of core-forming blocks of amphiphilic copolymers with hydrophobic alkyl chains or aromatic moieties can enhance stability of polymeric micelles ^{[22, 23]}. However,relatively large amounts of the hydrophobic moieties, which may inevitably change the nature of the copolymer,were needed for enhancing the micelle stability. Thus,stability of polymeric micelles used in biological media remains an issue that needs to be settled. In this work,we developed a novel strategy for enhancing the stability of semicrystalline micelles based on amphiphilic diblock copolymers of PPDO by introducing pyrene moieties at the chain end of PPDO blocks. The aggregation of pyrene moieties induced by π-π interactions obviously enhances the micelle stability and crystallization rate of the semicrystalline micelles of the Py-PPDO-b-PEG diblock copolymer because it may serve as a heterogeneous nucleation agent for crystallization of PPDO blocks. 2. Experimental

PDO (99.9%) with a melting point of 25 ℃ was provided by the Pilot Plant of the Center for Degradable and Flame-Retardant Polymeric Materials (Chengdu,China),and dried over CaH₂for 48 h and distilled under reduced pressure before use. Poly(ethylene glycol) monomethyl ether (MPEG,degree of polymerization = 12, 16,45,M_n= 0.55 kDa,0.75 kDa,2 kDa) were purchased from Sigma-Aldrich. 1-Pyrenemethanol was purchased from Alfa Aesar. Stannous octoate (SnOct₂) (95%) was purchased from Sigma- Aldrich,and was stored in glass ampoules under nitrogen after dilution with anhydrous toluene. Nile Red (analytical grade) was purchased from Sigma-Aldrich. Polyethylene glycol-block-poly (p-dioxanone) diblock copolymers functionalized with (Py-PPDOb-PEG) or without pyrene moieties (PPDO-b-PEG) were synthesized. Other reagents with AR grade were purchased from Aladdin Reagent Company (Shanghai,China) and Bodi Chemical Factory (Tianjin,China) and were used after removing water. 2.1. Preparation of Py-PPDO-b-PEG copolymer The Py-PPDO-b-PEG copolymer was synthesized by a coupling approach reported before ^{[10, 24]}. The synthesis process is described briefly as follows. First,the hydroxyl-terminated Py-PPDO precursor was prepared under nitrogen by typical bulk ring-opening polymerization of PDO using 1-pyrenemethanol as an initiator,using Sn(Oct)₂ as catalyst. The light yellow powder Py-PPDO-OH precursor was obtained. The MPEG-COOH was prepared by the acylation of the hydroxyl-terminated MPEG with succinic anhydride (SA) using DMAP and TEA as the catalysts. The white powder mPEG end-hydroxyl group (MPEG-COOH) was obtained. The Py-PPDO-b-PEG copolymer was synthesized using DCC as a coupling agent. The synthesis route of Py-PPDO-b-PEG is illustrated in Scheme 1.

For comparison,the PPDO-b-PEG copolymer without a pyrene moiety was prepared under nitrogen by typical bulk ring-opening polymerization of PDO using MPEG-OH as an initiator,using Sn(Oct₎₂ as catalyst. The synthesis route of PPDO-b-PEG is illustrated in Scheme 2.

2.2. Characterization The micellar solutions of copolymers with different compositions were prepared by the direct dissolution method. The Py-PPDO-bPEG copolymer or PPDO-b-PEG copolymer was added directly to water at a certain concentration at room temperature. Then thermal treatment was performed at approximate 95 ℃ for 5 min to erase all previous thermal history. Afterwards,the temperature of system was maintained at a certain temperature for 24 h.

The ¹H NMR spectra were measured at 25 ℃ using an Avance Bruke-II NMR spectrometer under 400 MH_z (Bruker,Germany) using CDCl₃ as solvent and tetramethylsilane (TMS) as internal standard.

Gel permeation chromatography (GPC). The molecular weight distribution was measured using a Waters GPC device equipped with a 1515 pump,a Waters model 717 auto sampler,and a 2414 refractive index detector. Chloroform was used as the eluent with flow rate 1.0 mL min^-1 at 30 ℃. A calibration curve was obtained using monodisperse polystyrene standards.

The CMCs of the block copolymers in aqueous solution were determined according to reported procedures ^{[25, 26]}, employing hydrophobic Nile Red as the probe. The CMC experiments are performed as followed: a stock solution of Nile Red (4 × 10 ^-4 mol L ^-1 ) in acetone is prepared and stored chilled and protected from light. The Nile Red-loaded micelles samples were diluted with a final Nile Red concentration of 8 × 10 ^-6 mol L ^-1 and the micelle concentrations between 2 × 10 ^-5 and 0.1 mg mL^-1 in deionized water. The micelle stability experiments is performed as followed: The same amount of Nile Red-loaded Py-PPDO-b-PEG and PPDO-b-PEG micelles samples diluted with the final micelle concentration of 0.2 mg mL^-1 in deionized water. Nile Red-loaded micelles samples incubated in a shaking water bath at 25 ℃ for several months. Fluorescence emission spectra were carried out using a HORIBA JOBINYVON Fluormax-4 spectrofluorometer at room temperature at an excitation wavelength of 310 nm and the emission from 590 to 700 nm. The emission and excitation slit widths were both set to 5 nm.

The copolymer aqueous solution was heated to 95 ℃ and kept for 5 min and then cooled to different temperatures (25 ℃,40 ℃, 50 ℃ and 60 ℃) using a PolyScience temperature controller as the control. The transmittance of copolymers with different composition micelles solutions at the concentration of 1 mg mL^-1 were measured with increasing time by using a HITACHI U-1900 spectrophotometer (600 nm) with a temperature-controlled quartz sample cell holder.

The averaged hydrodynamic diameter (D_h) of the polymeric solutions with a concentration of 1 mg mL^-1 in water were determined with increasing time by using dynamic light scattering (DLS) on a Brookhaven model BI-200SM spectrometer and a 9000AT correlator using an Innova 304 He-Ne laser (1 W,532 nm) at a fixed scattering angle (u) of 90 ℃.

Differential scanning calorimetry (DSC). Thermal effects of a complete cycle of copolymer with different composition micelles in aqueous solutions were studied with a NANO DSC of TA Instruments-Waters LLC,New Castle,DE at an external pressure of 3.0 atm using deionized water as an external reference. The cell volume was 0.33 mL. The heating rate was 1 ℃ min^-1 .

Transmission electron microscopy (TEM). The morphology of the Py-PPDO-b-PEG micelle was observed by Transmission Electron Microscope (TEM,Tecnai G2 F20 S-TWIN electron microscope (FEI Co.) operated at an acceleration voltage of 200 kV. In the TEM study,the micelles were collected onto a copper grid for about 10 min at room temperature,and then recorded after staining by 2% OsO4solution for 4 min. 3. Results and discussion 3.1. Synthesis of Py-PPDO-b-PEG and PPDO-b-PEG The synthesis of a hydroxyl-terminated Py-PPDO precursor was prepared by typical bulk ring-opening polymerization of PDO using pyrenemethanol as an initiator and Sn(Oct₎₂ as catalyst. ¹H NMR (400 MH_z,CDCl₃): δ 8.16-8.29 (m,1H,CHCHC or CHCHCH), 5.93 (s,2H,CHCH₂C),4.34 (m,2H,CH₂CH₂OC(≡0O)),4.16 (s,2H, OC(≡0O)CH₂O),4.10 (s,2H,CHCH₂OC(≡0O)CH₂O),3.78 (s,2H, CH₂CH₂OC(≡0O)),3.65-3.75 (m,2H,OCH₂CH₂OH),3.65-3.75 (m,2H,OCH₂CH₂OH) (Fig. 1).

The degree of polymerization (Dp) of Py-PPDO-OH was calculated by ¹H NMR using following equation:

where A_4.09-4.25ppm and A_5.93ppm are the integrals of chemical shifts at 4.09-4.25 ppm and 5.93 ppm,respectively.

The M_n,NMRof Py-PPDO-OH was calculated by ¹H NMR using following equation:

MPEG-COOH was prepared by the acylation of the mPEG endhydroxyl group with succinic anhydride using DMAP and TEA as catalysts according to the procedure widely reported previously. MPEG-COOH precursor: ¹H NMR (400 MH_z,CDCl₃): δ 4.25 (t,2H, CH₂C(≡0)OCH₂CH₂),3.65 (s,4H,OCH₂CH₂O),3.38 (s,3H, CH₂CH₂OCOCH₃),2.65 (m,4H,C(≡0)CH₂CH₂COOH). The carboxyl group of mPEG and hydroxyl group of PPDO with an OH/COOH mole ratio of 1:1 was connected by a moderate esterification reaction in the presence of DCC as dehydrant. Py-PPDO-b-PEG copolymer: ¹H NMR (400 MH_z,CDCl₃): δ 8.16-8.29 (m,1H,CHCHC or CHCHCH), 5.93 (s,2H,CHCH₂C),4.33 (t,2H,CH₂CH₂OC(≡0)CH₂O),4.30 (t,2H, OCH₂CH₂OC(≡0)CH₂CH₂),4.24 (t,2H,CH₂C(≡0)CH₂CH₂),4.20 (s,2H,OC(≡0)CH₂O),4.10 (s,2H,CHCH₂OC(≡0)CH₂O),3.78 (s,2H, CH₂CH₂OC(≡0)),3.64 (s,4H,OCH₂CH₂O),3.37 (s,3H,CH₂CH₂OCH₃), 2.65 (s,4H,C(≡0) CH₂CH2COOCH2). The M_n,NMR of Py-PPDO-b-PEG copolymer was calculated by ¹H NMR as follows:

The PPDO-b-PEG copolymer was prepared by typical bulk ringopening polymerization of PDO using MPEG as an initiator and Sn(Oct)₂ as catalyst. ¹H NMR (CDCl₃,400 MH_z): δ 3.40 (s,CH₃OCH₂CH₂O_-),3.66 (m,-OCH₂CH₂O-),3.76 (t,COCH₂OCH_2-CH₂OH),3.82 (t,-COCH₂OCH₂CH₂O-),4.20 (s,-COCH₂OCH₂CH₂O-), 4.32 (t,-CH₂CH₂OCO-),4.37 (t,-COCH₂OCH₂CH₂O-) (Fig. 2).

The degree of polymerization (DP) of PPDO block was then calculated as follows:

where A_{4.17-4.22 ppmand} A_{4.32 ppmare} integrals of chemical shifts at 4.17-4.22 ppm and 4.32 ppm,respectively. The weight percentage of PPDO blocks of the copolymer was calculated as follows:

The copolymers were successfully synthesized and characterized by ¹H NMR spectroscopy and GPC experiment (Fig. 3),the results were listed in Table 1. The copolymers were recorded as Py-PPDO_xb-PEG_y,where x and y represent the degree of polymerization of PPDO and PEG blocks,respectively. Meanwhile,in comparison to Py-PPDO_6.5-b-PEG₁₆,the control sample of the PPDO-b-PEG copolymer without a pyrene group has similar molecular weight to that of Py-PPDO_6.5-b-PEG₁₆,and was recorded as PPDO₇-b-PEG₁₆.

The successful coupling of the Py-PPDO precursor and MPEGCOOH was verified by the disappearance of the resonance of Py-PPDO-CH₂OH (3.65-3.75 ppm) and an increase of molecular weight in the GPC experiment. Moreover,it can be found that the number average molecular weight of the Py-PPDO precursor and the copolymer obtained from ¹H NMR both showed good accordance with the value estimated by GPC.

3.2. Crystallization induced micellization of Py-PPDO-b-PEG copolymers The micellization of the amphiphilic copolymers based on PPDO is critically dependent on the crystallization of PPDO blocks. Nano DSC is an effective and sensitive method for measuring the microcalorimetry curves of amphiphilic polymers in aqueous solution. Fig. 4 displays the cooling and subsequent heating scans after erasing the thermal history of the micelle dispersion of the copolymers with or without pyrene moieties. Fig. 4A shows that for Py-PPDO-b-PEG copolymer,an exothermic peak appeared at 31 ℃ with an exothermic crystallization enthalpy value of 44.3 kJ mol^-1 during the cooling scans,which could be assigned to the crystallization of PPDO blocks of Py-PPDO-b-PEG micelles. However,no such obvious exothermic peak was observed for PPDO-b-PEG without pyrene moieties. In the subsequent heating scans (Fig. 4B),the micelle dispersion of Py-PPDO-b-PEG also showed a much stronger endothermic peak at 60.7 ℃ with an melting enthalpy value of endothermic transition of 86.1 kJ mol^-1 ,corresponding to the melting of PPDO crystals, significantly higher than that of 38.8 kJ mol^-1 of PPDO-b-PEG. Therefore,this directly demonstrated pyrene moieties could promote the crystallization of the PPDO blocks in micelles.

The micellization kinetics of PPDO-b-PEG copolymers with or without pyrene moieties under different temperature were monitored by a UV-Vis spectrometer. After heating treatment at 95 ℃,all PPDO crystalline were melted and the copolymers were homogeneously dispersed in water. When cooled to certain temperatures, the transmittance gradually decreased. The micellization of copolymers and the crystallization of PPDO blocks in micelles,especially the latter,are responsible for the decrease in transmittance. As showed in Fig. 5,the temperature plays a key role in determining of crystallization and micellization of the copolymer because supercooling is needed for homogeneous nucleation. Compared to the PPDO₇-b-PEG₁₆copolymer without pyrene moieties,the Py-PPDO_6.5-b-PEG₁₆showed much faster decreasing rate of transmittance under same temperature. Meanwhile,the equilibrium transmittance of Py-PPDO_6.5-b-PEG₁₆after a long time of deposition at a certain temperature (e.g. 25 ℃ and 40 ℃) are also much lower than those of PPDO₇-b-PEG₁₆. These phenomena suggest that the copolymer functionized with pyrene moieties may have stronger micellization ability even at high temperatures and faster crystallization rate of PPDO blocks in micelles than those of copolymer without pyrene moieties. These results also proved that the pyrene moieties could promote the crystallization of the PPDO blocks in micelles.

Fig. 6 shows the variation of micelle diameter at different temperatures after heating treatment at 95 ℃. As showed in Fig. 6, the Py-PPDO-b-PEG copolymer can self-assemble into micelles in water in very short time. When micellization occurs at 60 ℃, micelles with relatively small size (about 100 nm) are formed. As shown in the TEM image of Py-PPDO_6.5-b-PEG₁₆micelles (Fig. 7A), the copolymer formed isotropic spherical micelles suggested that the PPDO blocks did not crystallize at this temperature. Moreover, some black dots were observed in the micelles which may be attributed to the aggregation of pyrene moieties. In comparison,no micelle was observed for PPDO₇-b-PEG₁₆copolymer during DLS and TEM tests,indicating that the interaction between pyrene moieties may also promote the micellization of Py-PPDO-b-PEG copolymer at high temperature (60 ℃). The micellization at 50 ℃ led to a slightly smaller D_h of Py-PPDO_6.5-b-PEG₁₆micelles than at 60 ℃,while some anisotropic flake-like morphology of these micelles was observed in TEM image (Fig. 7B),suggesting that the PPDO blocks had already crystallized at this temperature (50 ℃). This promotion of crystallization of PPDO blocks at relatively high temperature could be attributed to the aggregation of pyrene moieties induced by π-π interactions,which may serve as the heterogeneous nucleation agent for PPDO blocks. For micellization at low temperature (25 ℃ and 40 ℃),the Dhof Py-PPDO-b-PEG micelles gradually increased and reached equilibrium after several hours,and the obtained micelles had relatively large size and star anise-like morphology (Fig. 7C and D). This phenomenon could be explained by a crystallization induced hierarchical assembly mechanism ^[14].

3.3. Micelle stability of Py-PPDO-b-PEG amphiphilic block copolymers in water The CMCs of the copolymers in water were obtained by fluorescent measurement. To avoid the effect of pyrene moieties on the characterization,widely reported Nile Red was used as the fluorescent probe,and the maximum of the Nile Red emission peak (lmax) as a function of the copolymer concentration was recorded. Fig. 8A shows the emission spectra of Nile Red in Py-PPDO_6.5-b-PEG₁₆ aqueous solution. With increasing polymer solution concentration,the fluorescence intensity markedly increases, which resulted from the transfer of Nile Red molecules from a water environment to the hydrophobic environment,indicating the formation of micelles in water. From the plot of fluorescence intensity at 621 nm versus log concentration (Log C) of the copolymer (Fig. 8B) the CMC value was obtained. The CMC values of copolymers with different compositions in water are listed in Table 2. Compared with the PPDO₇-b-PEG₁₆ copolymer,the Py-PPDO_6.5-b-PEG₁₆ has significantly lower CMC value. Furthermore,it can be seen clearly that the CMC values of copolymers decreased when the pyrene moieties content of copolymers increased. These results indicate pyrene moieties play a crucial role in the formation of micelle of Py-PPDO-b-PEG copolymer and promote the formation of micelles. When the polymer concentration falls below the CMC, polymeric micelles became unstable and easily dissociation under physiological conditions. Therefore,the low CMC value of Py-PPDO-b-PEG suggested that the copolymers with pyrene moieties have much improved micelle stability compared to those without pyrene moities.

In addition,the stability of Py-PPDO-b-PEG micelle solution was also evaluated by monitoring the variation of fluorescent intensity of Nile Red probe in micelle solutions. Nile Red is nearly insoluble in water and becomes nonfluorescent within 20-25 min. In the presence of micelles,Nile Red can transfer into the hydrophobic phase and remain very stable ^[27]. Fig. 9 shows the fluorescence intensity at 621 nm of fluorescent emission spectra of copolymers micelle solutions as a function of time. It has been clearly seen that the fluorescent intensity increased quickly at the initial stage of the measurement,suggesting that the Nile Red probe gradually transferred from a water environment into the hydrophobic core of micelles. After achieving equilibrium,the fluorescence intensity of Nile Red in Py-PPDO-b-PEG micellar solution almost remained unchanged even after four months,indicating that the Py-PPDOb-PEG micelles can keep stable for a long time. In comparison,the fluorescence intensity of Nile Red in PPDO-b-PEG micelle decreased with time. The improved micelle stability of Py-PPDO-b-PEG copolymer could be attributed to the aggregation of pyrene moieties induced by intermolecular interactions. Pyrene is one of the few condensed aromatic hydrocarbons with strong hydrophobicity and it tends to aggregate in water medium even when the temperature was higher than the melting temperature of PPDO blocks. These aggregated pyrene moieties may also serve as heterogeneous nucleation agent and promote the crystallization of PPDO blocks with low supercooling degree. Therefore,the Py-PPDO-b-PEG micelles showed much higher stability than those of PPDO-b-PEG copolymer without pyrene moieties.

4. Conclusion Amphiphilic diblock copolymer of PPDO and PEG functionalized with pyrene moieties at the chain ends of PPDO blocks (Py-PPDOb-PEG) have been successfully synthesized. The copolymer can self-assemble into anisotropic micelles in water medium induced by the crystallization of PPDO blocks. TEM,transmittance and fluorescence measurements indicated that the pyrene moieties play a crucial role in the formation of micelle of Py-PPDO-b-PEG copolymer. Comparing to PPDO-b-PEG copolymer without pyrene moieties,the Py-PPDO-b-PEG can form micelle with low supercooling degree and has relatively quick crystallization rate of PPDO blocks into micelles. The aggregation of pyrene moieties may promote both micellization of the copolymer and crystallization of the PPDO blocks by serving as heterogeneous nucleation agent. Therefore the Py-PPDO-b-PEG micelles exhibited much higher stability than those of PPDO-b-PEG micelles without pyrene moieties.

Acknowledgments This work was financially supported by the National Natural Science Foundation of China (Nos. 21274093 and 51121001), Program for Changjiang Scholars and Innovative Research Team in University of Ministry of Education of China (No. IRT1026),and National High-Tech Research and Development Program (863 Program) of China (No. 2012AA062904). The Analytical and Testing Center of Sichuan University provided TEM analysis.