Glycopolymer

3644

J. Phys. Chem. B 2008, 112, 3644-3652

Supramolecular and Biomimetic Polypseudorotaxane/Glycopolymer Biohybrids: Synthesis, Glucose-Surfaced Nanoparticles, and Recognition with Lectin Xiao-Hui Dai,†,‡ Chang-Ming Dong,*,† and Deyue Yan† Department of Polymer Science & Engineering, School of Chemistry and Chemical Technology, Shanghai Jiao Tong UniVersity, Shanghai 200240, P. R. China, and Department of Packaging Engineering, School of Mechanical Engineering, Jiangsu UniVersity, Zhenjiang 212013, P. R. China ReceiVed: NoVember 8, 2007; In Final Form: December 28, 2007

A new class of supramolecular and biomimetic glycopolymer/poly(-caprolactone)-based polypseudorotaxane/ glycopolymer triblock copolymers (poly(D-gluconamidoethyl methacrylate)-PPR-poly(D-gluconamidoethyl methacrylate), PGAMA-PPR-PGAMA), exhibiting controlled molecular weights and low polydispersities, was synthesized by the combination of ring-opening polymerization of -caprolactone, supramolecular inclusion reaction, and direct atom transfer radical polymerization (ATRP) of unprotected D-gluconamidoethyl methacrylate (GAMA) glycomonomer. The PPR macroinitiator for ATRP was prepared by the inclusion complexation of biodegradable poly(-caprolactone) (PCL) with R-cyclodextrin (R-CD), in which the crystalline PCL segments were included into the hydrophobic R-CD cavities and their crystallization was completely suppressed. Moreover, the self-assembled aggregates from these triblock copolymers have a hydrophilic glycopolymer shell and an oligosaccharide threaded polypseudorotaxane core, which changed from spherical micelles to vesicles with the decreasing weight fraction of glycopolymer segments. Furthermore, it was demonstrated that these triblock copolymers had specific biomolecular recognition with concanavalin A (Con A) in comparison with bovine serum albumin (BSA). To the best of our knowledge, this is the first report that describes the synthesis of supramolecular and biomimetic polypseudorotaxane/glycopolymer biohybrids and the fabrication of glucose-shelled and oligosaccharide-threaded polypseudorotaxane-cored aggregates. This hopefully provides a platform for targeted drug delivery and for studying the biomolecular recognition between sugar and lectin.

1. Introduction Supramolecular polypseudorotaxanes formed between cyclodextrins and polymers have attracted much attention because of their potential applications in nanotechnology, molecular machines, and drug/gene delivery vesicles.1-5 Indeed, cyclodextrins (CDs), a series of oligosaccharides with a hydrophobic and hollow truncated cavity, have been shown to generate a variety of polypseudorotaxanes through inclusion complexation with synthetic polymers. For examples, several research groups intensively investigated the polypseudorotaxanes of CDs with biocompatible and hydrophilic polymers, such as poly(ethylene oxide) homopolymer and poly(propylene oxide)-b-poly(ethylene oxide)-b-poly(propylene oxide) triblock copolymer.6-9 Likewise, increasing effort was also made for the preparation of polypseudorotaxanes from biodegradable polymers and/or branched polymers with CDs.10-15 As an extension study, the design and fabrication of novel polypseudorotaxane-based drug/gene delivery systems have been intensively investigated by several research groups. For examples, Yui et al. reported the pHsensitive complexation of linear polyethylenimine with CDs (e.g., γ-CD) and its application as a gene carrier, which exhibited low cytotoxicity and high gene expression.16 Zhang et al. synthesized a photo-cross-linkable polypseudorotaxane hydrogel with high mechanical strength and adjustable thermosensitivity.17 * To whom correspondence should be addressed. Phone: 86-2154748916. Fax: 86-21-54741297. E-mail: [email protected]. † Shanghai Jiao Tong University. ‡ Jiangsu University.

Very interestingly, Kim et al. designed a pH-sensitive polypseudorotaxane/mesoporous hybrid material for the controlled release of guest molecules.18 However, it is difficult to fabricate polypseudorotaxane-based micro- and nanoparticles for nanobiotechnology application because these polypseudorotaxanes usually do not dissolve in water and common organic solvents. Therefore, it is an urgent task to design new polypseudorotaxane-based biomaterials for nanobiotechnology. As a U.S. Food and Drug Administration (FDA) approved biomedical polymers, poly(-caprolactone) (PCL) and related biomaterials have been widely used for surgery (e.g., sutures and screws), drug delivery matrices, and tissue engineering scaffolds.19 However, both uncontrollable degradation rate (in vitro and in vivo) and the lack of cell/material compatibility became two major drawbacks for clinical applications because of the high crystallinity and the hydrophobicity of the PCL backbone.20-22 Therefore, R-CD threading onto the PCL backbone (i.e., to form PCL-based polypseudorotaxane) will play an important role in adjusting the hydrophobicityhydrophilicity balance and the crystallinity of PCL backbone,23 which might improve the biodegradation and controlled drug release properties of PCL-based biomaterials. On the other hand, biomimetic water-soluble glycopolymers (side-chain sugarcontaining polymers) have been recently used to probe the specific sugar-protein recognition processes in living cells, which hold great relevance for both drug discovery and biomaterials applications.24-26 Thus, the block copolymerization of supramolecular polypseudorotaxanes with biomimetic gly-

10.1021/jp710698c CCC: $40.75 © 2008 American Chemical Society Published on Web 03/05/2008

Polypseudorotaxane/Glycopolymer Biohybrids

J. Phys. Chem. B, Vol. 112, No. 12, 2008 3645

SCHEME 1: Synthesis of Supramolecular and Biomimetic PGAMA-PPR-PGAMA Triblock Copolymers

copolymers will provide a facile method to prepare a new class of biohybrids, which are expected to generate sugar-surfaced polymeric aggregates in aqueous solution. Indeed, much attention has been paid to the fabrication of sugar-installed polymeric nanoparticles for targeted drug delivery by the utilization of both the specific sugar-protein biomolecular recognition in living systems and the enhanced permeability and retention effect induced by the size of the nanoparticles.27,28 Therefore, the design of such supramolecular and biomimetic biohybrids will hopefully provide a platform for fabricating targeted drug delivery systems, useful for hydrophilic peptides drugs, genes, and even cells, because oligosaccharides can greatly improve the biocompatibility between synthetic biomaterials and peptides, genes, and/or cells.26-30 Since its independent discovery by Matyjaszewski and Sawamoto, atom transfer radical polymerization (ATRP) has proved to be a simple and robust strategy for synthesizing welldefined polymers and hybrids with various macromolecular architectures and compositions.30-32 Specifically, here a supramolecular polypseudorotaxane was for the first time used as a macroinitiator for synthesizing a new class of supramolecular and biomimetic polypseudorotaxane/glycopolymer bio-

hybrids by the direct ATRP of unprotected GAMA glycomonomer, as shown in Scheme 1. The molecular structures, selfassembled nanoparticles, and recognition properties of these triblock copolymers have been thoroughly characterized by means of FT-IR, 1H NMR, GPC, WAXD, DSC, UV-vis, DLS, and TEM. To the best of our knowledge, this is the first report that describes the fabrication of a novel kind of glucose-shelled and oligosaccharide-threaded polypseudorotaxane-cored polymeric nanoparticles in aqueous solution. 2. Experimental Section 2.1. Materials. 2-Aminoethyl methacrylate hydrochloride, bipyridine, 2-bromo-2-methylpropionyl bromide, 2-bromopropionyl bromide, copper(I) bromide, R-cyclodextrin (R-CD), 1,6diphenyl-1,3,5-hexatriene (DPH), D-gluconolactone, N,N,N′,N′′,N′′pentamethyldiethylenetriamine (PMDETA), and stannous octoate were purchased from Aldrich or Acros and used as received. Concanavalin A (Con A) and bovine serum albumin (BSA) were purchased from Sigma. -Caprolactone (Aldrich) and toluene were distilled from CaH2, respectively. 1,6-Hexanediol (Aldrich) was dried in vacuo at 40 °C for 24 h. D-Gluconamidoethyl

3646 J. Phys. Chem. B, Vol. 112, No. 12, 2008 methacrylate glycomonomer (GAMA) was synthesized from D-gluconolactone and 2-aminoethyl methacrylate hydrochloride according to a literature procedure (52.0% yield).33 The GAMA glycomonomer was characterized by 1H NMR and 13C NMR spectroscopy, which was in good agreement with the results reported in literature. 1H NMR (D2O) of GAMA: δ (ppm) ) 1.75 (s, 3H, -CH3), 3.38-3.70 (m, 6H, -CH2NHand -CHOHCHOHCH2OH), 3.91-3.95 (t, J ) 4 Hz, 1H, and -CHOH-), 4.13-4.19 (m, 3H, -OCH2-COCHOH-), 5.58 (s, 1H, dCH2), 6.0 (s, 1H, dCH2). 13C NMR (D2O) of GAMA: δ (ppm) ) 17.54 (-CH3), 38.25 (-CH2NH-), 62.83 (-OCOCH2-), 63.71 (-CH2OH), 70.56 (-CHOHCH2OH), 71.31 (-CHOHCHOHCH2OH), 72.39 (-NHCOCHOHCHOH-), 73.60 (-NHCOCHOHCHOH-), 127.27 (dCH2), 136.01 (Cd), 169.92 (OCO), 174.90 (NHCO). The other reagents and solvents were local commercial products and used without further purification. 2.2. Methods. Fourier transform infrared (FT-IR) spectra were recorded on a Perkin-Elmer Paragon 1000 spectrometer at frequencies ranging from 400 to 4000 cm-1. Samples were thoroughly mixed with KBr and pressed into pellet form. The polydispersity indices (Mw/Mn) of polymers were determined on a gel permeation chromatograph (GPC, Perkin-Elmer Series 200) with a refractive index detector at 30 °C. The elution phase was DMF (elution rate: 1.0 mL/min), and polystyrene was used as the calibration standard. 1H NMR spectra were recorded at room temperature on a Varian Mercury-400 spectrometer. CDCl3, DMSO-d6, and D2O were used as the deuterated solvents for the PCL precursors, PPR, and PGAMA-PPR-PGAMA block copolymers. The differential scanning calorimetry (DSC) analysis was carried out using a Perkin-Elmer Pyris 1 instrument under nitrogen flow (10 mL/min). All samples were first heated from 0 to 90 °C at 10 °C/min and held for 2-3 min to erase the thermal history, then cooled to 0 °C at 10 °C/min, and finally heated to 90 °C at 10 °C/min. Wide-angle X-ray diffraction (WAXD) patterns of powder samples were obtained at room temperature on a Shimrdzu XRD-6000 X-ray diffractometer with a Cu KR radiation source (wavelength ) 1.54 Å). The supplied voltage and current were set to 40 kV and 30 mA, respectively. Samples were exposed at a scan rate of 2θ ) 4° min-1 between 2θ ) 5 and 40°. UV-vis spectra were recorded at room temperature using a Spectrumlab54 UV-visible spectrophotometer. The mean size of aggregates was determined by dynamic light scattering (DLS) using a Malvern Nano_S instrument (Malvern, U.K.). The solution of aggregates was performed at a scattering angle of 90° and at 25 °C. All the measurements were repeated three times, and the average values reported were the mean diameter ( standard deviation. Transmission electron microscopy (TEM) was performed using a JEM-2010/INCA Oxford TEM (JEOL/OXFORD) at a 200 kV accelerating voltage. One drop of aggregate solution was deposited onto the surface of 300 mesh Formvar-carbon filmcoated copper grids. Excess solution was quickly wicked away with a filter paper. The image contrast was enhanced by negative staining with phosphotungstic acid (0.5 wt %). 2.3. Preparation of Bifunctional 2-BromopropionylTerminated Poly(-caprolactone) (PCL-Br). According to our previous publications,20 linear PCL with two hydroxyl end groups (PCL-OH) was synthesized by the controlled ringopening polymerization of -caprolactone monomer using 1,6hexanediol as initiator and stannous octoate as catalyst at 110 °C (PCL20-OH sample, 92.2% yield). 1H NMR (CDCl3, TMS) of PCL20-OH sample: δ (ppm) ) 1.32-1.47 (m, 40H, -COCH2CH2CH2CH2CH2O-),1.54-1.73(m,80H,-COCH2CH2-

Dai et al. CH2CH2CH2O-), 2.25-2.41 (m, 40H, -COCH2CH2CH2CH2CH2O-), 3.65 (t, J ) 8 Hz, 2H, -CH2OH), 4.02-4.19 (m, 40H, -COCH2CH2CH2CH2CH2O-) (see Supporting Information Figure S1). Then, the PCL20-OH was converted into ATRP macroinitiator via esterification with 2-bromopropionyl bromide. A typical example is given below. PCL20-OH precursor (468.1 mg, 0.1 mmol) was dissolved in dry CH2Cl2 (15.0 mL). To this solution was added triethylamine (0.9 mmol, 0.125 mL) under N2, and the reaction mixture was stirred for 30 min and then cooled to 0 °C. 2-Bromopropionyl bromide (0.8 mmol, 0.086 mL) in CH2Cl2 (10 mL) was added dropwise to PCL20-OH solution via a syringe under N2 and kept 1 h at 0 °C. The reaction was stirred vigorously for 36 h at room temperature, and then the mixture was washed sequentially with saturated NaHCO3 solution and distilled water. The organic layer was dried over anhydrous Na2SO4, and most of the solvent was removed using a rotary evaporator. The residue was precipitated dropwise into methanol. The resulting white solid was dried at 50 °C for 24 h in vacuo (432.5 mg, 92.4% yield). 1H NMR (CDCl3) of PCL20-Br sample: δ (ppm) ) 1.31-1.46 (m, 40H, -COCH2CH2CH2CH2CH2O-),1.58-1.74(m,80H,-COCH2CH2CH2CH2CH2O-), 1.79-1.87 (d, J ) 8 Hz, 3H, -HCCH3Br), 2.25-2.38 (m, 40H, -COCH2CH2CH2CH2CH2O-), 4.02-4.13 (m, 40H, -COCH2CH2CH2CH2CH2O-), 4.35-4.43 (q, J ) 8 Hz, 1H, -HCCH3Br) (see Supporting Information Figure S1). 2.4. Preparation of the PCL-Based Polypseudorotaxane (PPR). As a representative protocol, the polypseudorotaxane of PCL20-Br with R-CD was prepared as follows. PCL20-Br (24.7 mg) was dissolved in 2.5 mL of acetone at 50 °C, and R-CD (97.3 mg) was dissolved in 1.0 mL of distilled water at 60 °C. Then the PCL20-Br solution was added dropwise to the R-CD solution at 60 °C with vigorous stirring. After being stirred at 60 °C for 8 h, the mixture was cooled to room temperature and stirred vigorously for 32 h. The precipitated product was collected by filtration and twice washed with acetone (10.0 mL) to remove free polymer and then twice washed with distilled water (10.0 mL) to remove uncomplexed R-CD. The white powder was then dried overnight in vacuo at 60 °C to give 73.5 mg of PPR (60.2 wt. % yield). 2.5. Synthesis of PGAMA-PPR-PGAMA Triblock Copolymers. A typical procedure for the direct ATRP of unprotected GAMA glycomonomer using PPR macroinitiator is as follows: Both PPR macroinitiator (0.004 mmol, 119.6 mg) and GAMA glycomonomer (0.24 mmol, 73.9 mg) were dissolved in DMSO (0.8 mL) at 35 °C, and the solution was degassed via nitrogen purge for 30 min. Copper(I) bromide (0.032 mmol, 4.7 mg) and PMDETA (0.032 mmol, 6.8 µL) were added in turn, and the resulting solution was degassed again for 10 min and then stirred vigorously under nitrogen at 35 °C for 48 h. The mixture was precipitated into 2-propanol (20.0 mL) and then purified by heated methanol (5.0 mL) to remove the possible unreacted GAMA glycomonomer and/or homopolymer. The resulting PGAMA-PPR-PGAMA triblock copolymer was then dried overnight in vacuo at 40 °C (PGAMA-PPRPGAMA, monomer conversion ) 69.1% yield). 2.6. Preparation of Glucose-Shelled and Polypseudorotaxane-Cored Aggregates in Water. Biomimetic PGAMAPPR-PGAMA triblock copolymer (1 mg/mL) was dissolved in DMF, and distilled water was then added gradually at a speed of 10 µL/min using a microsyringe. After the appearance of a blue tint (water content: about 50 - 52 wt %) indicating the formation of aggregates, another 20-30 wt % of water was added to stabilize the aggregates. After stirring of the solution



TABLE 1: Molecular Characteristics of PCL Precursors, PPR Macroinitiator, and the As-Synthesized PGAMA-PPR-PGAMA Triblock Copolymers via ATRP samplesa PCL20-OH PCL20-Br PPR20 PGAMA4-PPR20-PGAMA4 PGAMA6-PPR20-PGAMA6 PGAMA8-PPR20-PGAMA8 PGAMA15-PPR20-PGAMA15

[GAMA]:[PPR] (mol:mol)

yield (%)

Mw/Mnb

Mn,NMR (KDa)

fPGAMAc (wt %)

1:30 1:60 1:60 1:100

92.2 92.4 65.5 58.3 69.1 71.7

1.39 1.29 1.23 1.43 1.26 1.56 1.34

4.7 5.0 30 33 34 35 39

35.8 45.6 50.6 65.0

a The subscript numbers represent the repeating units number of polymers. b Mw/Mn denotes the polydispersities of these polymers, which were determined by GPC in DMF solution. c fPGAMA denotes the weight fraction of PGAMA segments within the triblock copolymers, which was determined by 1H NMR.

for 24 h at room temperature, DMF was removed by dialyzing against distilled water for 3 days using a dialysis membrane with a MWCO of 7000 Da. Both the mean size and morphology of aggregates were determined by DLS and TEM, respectively. 2.7. Measurement of Copolymer Critical Aggregation Concentration. The critical aggregation concentration (cac) of amphiphilic PGAMA-PPR-PGAMA triblock copolymers was determined employing the hydrophobic dye solubilization method using DPH as a probe molecule.34 DPH was dissolved in methanol to produce 0.5 mM DPH methanol solution. In a quartz cell, 0.5 mL of copolymer aggregate solution and 5 µL of DPH solution were added and capped with Teflon film and then allowed to homogenize about 2 min at room temperature. To prevent the evaporation of water during the measurement, the sample cell was sealed with Teflon film. UV-vis spectra of samples were recorded in the range of 200-500 nm at room temperature. 2.8. Lectin Recognition. The lectin recognition activity of the copolymer solution was analyzed by changes in the turbidity of solution with time at 360 nm and room temperature following the addition of various concentrations of aggregate solution into Con A solution, in which its concentration was equal to 0.5 mg/mL. Similarly, BSA was used for the control experiments. 3. Results and Discussion 3.1. Synthesis of PGAMA-PPR-PGAMA Triblock Copolymers. ATRP has proved to be a simple and robust strategy for the preparation of well-defined glycopolymers with various macromolecular architectures (e.g., linear, star-shaped, and dendritic), while it usually includes additional two procedures, i.e., protection of glycomonomer and deprotection of the resulting glycopolymers.24-26 The deprotection procedure is often performed under acid and/or base conditions, which would probably induce the degradation of biodegradable PCL-based biomaterials.35 So, the direct ATRP of unprotected glycomonomer provides a convenient method to generate glycopolymers/ PCL-based biohybrids. In this work, supramolecular and biomimetic PGAMA-PPR-PGAMA triblock copolymers were synthesized by the combination of ring-opening polymerization (ROP) of -caprolactone, supramolecular inclusion reaction, and direct ATRP of unprotected GAMA glycomonomer via the following four steps, as shown in Scheme 1. First, bifunctional PCL with two hydroxyl end groups (PCL-OH) was synthesized by the controlled ROP of -caprolactone monomer according to our previous publications.20 The actual polymer molecular weight or the degree of polymerization of PCL-OH could be easily determined by means of 1H NMR spectroscopy, and the polydispersity (Mw/Mn) of PCL-OH was narrow on the basis of the GPC analysis (Table 1). Then PCL-OH terminated with two hydroxyl end groups was further functionalized by reaction with 2-bromopropionyl bromide to introduce active initiating species

for ATRP. In comparison of the 1H NMR of the resulting PCL20Br with that of the PCL20-OH precursor, the proton signals at 3.65 ppm assignable to the primary hydroxymethylene end group (HOCH2-) of PCL20-OH precursor wholly disappeared while new signals corresponding to methyl protons of 2-bromopropionate (OCOCH(CH3)Br) appeared at 1.79-1.87 ppm for the obtained PCL20-Br (see Supporting Information Figure S1). Moreover, the integral ratio of the methyl proton signal on the 2-bromopropionate end group to the repeating methylene unit of PCL20-Br was very close to the theoretical value (Ha:Hf ) 40:3). These results show that the hydroxyl end groups of PCLOH precursor were quantitatively converted into 2-bromopropionate end groups within PCL-Br. In the third step, the PCL-based polypseudorotaxane (PPR) was prepared by adding dropwise the acetone solution of PCLBr into an aqueous R-CD solution under rigorous stirring (Scheme 1). In comparison with that of R-CD (Figure S2), the 1H NMR spectrum of the PPR clearly showed that the proton peaks at 7H and 8H split from one signal (in R-CD) into two separate signals (PPR, Figure 1 a), and the relative peak height between 1H and 9H for the PPR greatly decreased in comparison with that of R-CD. This indicates that the inclusion complexation between PCL-Br and R-CD occurred,14,23 which is also clarified by the following analyses of FT-IR, WAXD, and DSC. In a comparison of the integration of peak for R-CD (1H) with that of the methylene groups of PCL-Br, the host-guest stoichiometry (i.e., CL:R-CD, mol:mol) of PPR is 1.56, calculated by the molar ratio of the monomeric repeating unit of PCL-Br to R-CD, which is similar to that reported for polypseudorotaxane of PCL and R-CD (CL:R-CD ) 1.60:1, mol:mol).14 As a note, the 2-bromo-2-methylpropionate-terminated PCL was first designed to form the polypseudorotaxane with R-CD because 2-bromo-2-methylpropionate initiating species was more efficient for ATRP than 2-bromopropionate.32 However, the 2-bromo-2-methylpropionate-terminated PCL could not form the related polypseudorotaxane with R-CD because of the large size of 2-bromo-2-methylpropionate group compared with the cavity of R-CD.14 Finally, the as-synthesized PCL-based polypseudorotaxane terminated with 2-bromopropionate-initiating species was used as a macroinitiator for the direct ATRP of unprotected GAMA glycomonomer in the following study. The direct ATRP of unprotected GAMA glycomonomer using PPR macroinitiator was performed in NMP solution at room temperature, and the results are summarized in Table 1. In a comparison with that of the PPR macroinitiator, the typical GPC curves of as-synthesized PGAMA-PPR-PGAMA triblock copolymers revealed symmetrical elution peaks at different elution times, denoting a progression of the polymer molecular weight and reasonable polydispersity index (see Supporting Information Figure S3). As a note, the polydispersities (Mw/ Mn) of these triblock copolymers slightly increased in compari-

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Figure 1. 1H NMR spectra of PPR macroinitiator in DMSO-d6 (a) and PGAMA-PPR-PGAMA in DMSO-d6 (b) and D2O (c).

son with the PPR macroinitiator. This was possibly caused by the chain transfer to the cyclodextrin or gluconamido units, which needs further investigation. In comparison with the PPR macroinitiator, the 1H NMR spectra (DMSO-d6) of PGAMA-PPR-PGAMA triblock copolymers clearly show that, besides the typical proton signals of both PCL backbone and R-CD, new proton signals appeared at both 1.70-1.90 ppm and 0.60-1.10 ppm for the backbone of the PGAMA glycopolymer, while the proton signals at 4.004.67 ppm for glucose residues overlapped with that of R-CD (Figure 1b). Notably, the methyl groups assignable to 2-bromopropionate end groups of the PPR macroinitiator at 1.791.87 ppm completely shifted to 1.03 ppm for the triblock copolymers, which suggested that the bromide-terminated PPR really played its role as a macroinitiator in the ATRP process of GAMA glycomonomer. FT-IR results of R-CD, PCL-Br, the PPR macroinitiator, and the triblock copolymers are shown in Figure 2. The spectrum of R-CD shows a broad band at 3390 cm-1 due to the symmetric and antisymmetric O-H stretching mode and other intense bands at 1151 cm-1 (C-O-C glycosidic bridge) coupled with 1031 cm-1 (C-O). PCL-Br is characterized by a distinct

Figure 2. FT-IR spectra of R-CD, PCL-Br, PPR macroinitiator, and the PGAMA-PPR-PGAMA triblock copolymers.

carbonyl stretching band at 1728 cm-1, while the spectrum of PPR macroinitiator confirmed the presence of both host and


Figure 3. WAXD patterns of R-CD, PCL-Br, PPR, and PGAMAPPR-PGAMA.

guest components. Moreover, it can be observed that the CdO band of PCL-Br at 1728 cm-1 was shifted to higher frequency at 1736 cm-1 in the PPR macroinitiator; meanwhile the broad O-H band of R-CD at 3390 cm-1 was also shifted to 3450 cm-1. These indicate that the PPR macroinitiator was formed through R-CD threading onto the backbone of PCL-Br precursor.14,23 This is further supported by the WAXD and DSC analyses below. On the other hand, Including the distinct stretching bands for R-CD and PCL backbone, the PGAMAPPR-PGAMA triblock copolymers presented the characteristic amide I and II bonds at both 1655 cm-1 and 1542 cm-1 for the linker groups within the PGAMA blocks, while the broad O-H band at about 3410 cm-1 assignable to the glucose residues of PGAMA block overlapped with that of R-CD. Meanwhile, the relative intensities of the amide bands within PGAMA block to the carbonyl bands within both PCL and PGAMA blocks increased gradually with the increasing block length of PGAMA. In all, the PGAMA-PPR-PGAMA triblock copolymers with central PCL-based polypseudorotaxane segments and outer glycopolymer segments were for the first time synthesized successfully by the combination of ROP of -caprolactone, supramolecular inclusion reaction, and the direct ATRP of unprotected GAMA glycomonomer, as shown in Scheme 1. In the ATRP copolymerization process, do R-CD molecules unthread from the backbone of PCL-Br? This is an important question and has been clarified in the following parts. WAXD is a useful method to elucidate the structure of these polypseudorotaxanes including PPR and PGAMA-PPR-PGAMA in the solid-state. Figure 3 shows the diffraction patterns of R-CD, PCL-Br, PPR, and the PGAMA-PPR-PPGAMA biohybrid. The PCL-Br sample showed prominent peaks at 21.3 and 23.6°, which was consistent with that for linear PCL crystals located at 21.4 and 23.8°. However, PPR showed new strong diffraction peaks at 19.8 and 22.4°, while the major crystalline peaks for PCL-Br disappeared. This convincingly shows that R-CD molecules were threaded onto the PCL backbone and PPR adopted a channel-type crystalline structure.14,23 Similarly, the PGAMA-PPR-PPGAMA biohybrid showed strong diffraction peaks at 19.9 and 22.6°, while no crystalline peak for PCL-Br was found. These results suggest that the R-CD molecules did not unthread from the PCL backbone in the ATRP polymerization and the central PPR segments also adopted a channeltype crystalline structure within the PGAMA-PPR-PGAMA biohybrid.


Figure 4. DSC curves of PCL-Br, PPR, and PGAMA-PPR-PGAMA in the first heating run (a), in the cooling run (b), and in the second heating run (c).

The melting and crystallization behavior of PCL-Br, PPR, and the PGAMA-PPR-PGAMA biohybrid were investigated by DSC, as shown in Figure 4. The maximal melting peak of the free PCL-Br precursor was observed at 57.4 °C in the heating and cooling runs, while no melting peak was observed for the PPR macroinitiator. These observations indicate that the crystallization of PCL within PPR was completely suppressed in the R-CD cavities.14,23 Moreover, the PGAMA-PPR-PGAMA biohybrid showed similar DSC curves compared with PPR, which also suggested that the central PCL backbone was completely threaded by R-CD molecules. 3.2. Self-Assembly Properties of PGAMA-PPR-PGAMA Triblock Copolymers. 1H NMR proved to be a convenient method to characterize the self-assembled core-shell structures of amphiphilic copolymers, and it was preliminarily used to investigate the self-assembly behavior of PGAMA-PPRPGAMA triblock copolymers in aqueous solution. In comparison with that in DMSO-d6 solvent, the 1H NMR of PGAMA15PPR20-PGAMA15 in D2O solution showed the attenuated proton signals of hydrophobic PCL segments (Figure 1b vs Figure 1c). This phenomenon was attributed to both the decreased mobility of physically associated PPR segments and the shielding effect of hydrophilic PGAMA shell to the hydrophobic PPR core. This also implies the spontaneous self-assembly of amphiphilic PGAMA-PPR-PGAMA biohybrids in aqueous solution. Then, we examined their critical aggregation concentration (cac) by employing the dye solubilization method, which was an important parameter for the thermodynamic stability of selfassembled aggregates in aqueous solution.27 1,6-Diphenyl-1,3,5hexatriene (DPH) was used as a probe molecule, and the relationship of the absorbance intensity of DPH as a function of copolymer concentration at room temperature is shown in Figure 5. The absorbance intensity values of DPH remained nearly constant below a certain concentration. Above that concentration, the absorbance intensity increased substantially, reflecting the incorporation of DPH in the hydrophobic region of aggregates. The cac value of the PGAMA-PPR-PGAMA triblock copolymers slightly increased from 0.0461 to 0.0512 mg/mL with the increasing weight fraction of hydrophilic PGAMA. This was similar to that reported in other amphiphilic block copolymers, suggesting that the self-assembled aggregates were thermodynamically stable in aqueous solution.27,28,36 Both the morphology and the average size of the selfassembled aggregates from these PGAMA-PPR-PGAMA

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Dai et al.

Figure 5. Relationship of the absorbance intensity of DPH as a function of the PGAMA-PPR-PGAMA triblock copolymer concentration at room temperature.

triblock copolymers were investigated by the techniques of TEM and DLS, as shown in Figures 6 and 7. When the repeating unit number of hydrophobic PPR block was 20, the PGAMA15PPR20-PGAMA15 triblock copolymer with long PGAMA segment (fPGAMA ) 65.0%) showed spherical micellar aggregates with an average diameter of 121 ( 12 nm, as shown in Figure 6A. These micellar aggregates were bigger than the conventional polymeric micelles usually with a diameter of 10-50 nm,27,28 which suggests that they should not have the simple core/shell micelles structure formed from the conventional amphiphilic block copolymers. As shown in Figure 6B, the magnification photo of these bigger aggregates showed that they probably formed from the intermicellar aggregation of simple core-shell micelles (core, hydrophobic oligosaccharide (R-CD) threaded polypseudorotaxane; shell, water-soluble glycopolymer), which is induced by the strong hydrogen-bond interactions among the glycopolymer shell.37-39 Moreover, the average size of these spherical micellar aggregates remained basically unchanged at least within 50 days at 4 °C (data not shown). This suggests that they were very stable in vitro, which provides them suitable for drug delivery.27,28 As the hydrophilic PGAMA block was shortened, the polymersomes and/or vesicles with an average diameter of 81 ( 4 nm were shown for the PGAMA4-PPR20PGAMA4 sample (fPGAMA ) 35.8%, Figure 6C). This can be attributed to both the decreased repulsion among the corona chains (i.e., hydrophilic PGAMA corona) and the increased surface tension resulting from the increased hydrophobicityhydrophilicity balance.27 Thus, the above results indicate that a new kind of glucose-shelled and oligosaccharide-threaded polypseudorotaxane-cored aggregates with micellar and vesicular morphologies can be conveniently fabricated by adjusting the weight fraction of PGAMA block within these PGAMAPPR-PGAMA triblock copolymers. This will hopefully provide a platform for fabricating targeted drug delivery systems, useful for hydrophilic peptides drugs, genes, and even cells, because oligosaccharides can greatly improve the biocompatibility between synthetic biomaterials and peptides, genes, and/or cells.26-30 3.3. Recognition Properties of PGAMA-PPR-PGAMA Triblock Copolymers. In living systems, the specific sugarprotein recognition events govern many psychological and pathological processes, which hold great relevance for both drug discovery and biomaterials applications.40-42 It is reported that Con A specifically recognizes D-glucopyranoside and D-man-

Figure 6. TEM photographs of the self-assembled aggregates from PGAMA-PPR-PGAMA triblock copolymers: PGAMA15-PPR20PGAMA15 (A, B); PGAMA4-PPR20-PGAMA4 (C).

nopyranoside residues with free 3-, 4-, and 6-hydroxyl groups, and the binding of Con A with glycopolymer usually results in the Con A/glycopolymer cross-linked aggregates.35,43 Thus, the



Figure 8. Interactions of Con A (CConA ) 0.5 mg/mL) and/or BSA (CBSA ) 0.5 mg/mL) with PGAMA15-PPR20-PGAMA15 (Ccopolymer ) 0.01 mg/mL).

Figure 7. Nanoparticle size distribution of the self-assembled aggregates from PGAMA-PPR-PGAMA triblock copolymers in aqueous solution at room temperature.

interaction of Con A with the PGAMA-PPR-PGAMA triblock copolymers was investigated in aqueous solution at room temperature. The turbidity of the copolymer solution increased after Con A was added, while nearly no change was observed after BSA was added (Figure 8). This suggests that the specific binding between copolymer and Con A probably occurred and resulted in the Con A/copolymer cross-linked aggregates, which can be clarified by the following DLS analysis. Similarly, the non-rotaxanated PGAMA-PCL-PGAMA triblock copolymer also presented a specific binding with Con A, as shown in Supporting Information Figure S4. The average size of the Con A/copolymer cross-linked aggregates was 1.81((0.6) × 103 nm, which was bigger than the original copolymer aggregates (121 ( 12 nm), as shown in Figure 9. However, the average size of BSA/copolymer aggregates was about 6.5 ( 0.1 nm, which mainly complied with the size of BSA molecules in solution (CBSA ) 0.5 mg/mL . Ccopolymer ) 0.01 mg/mL). This implies that no specific binding between copolymer and BSA occurred in aqueous solution at room temperature. In conclusion, the above analyses indicate that these PGAMA-PPR-PGAMA triblock copolymers had specific binding with Con A in aqueous solution. This will hopefully provide a platform for studying the biomolecular recognition between sugar and protein because the glycopolymer-based polymers with tunable architectures might present different sugar densities and spatial distribution.26

Figure 9. Nanoparticle size distribution of PGAMA15-PPR20PGAMA15/Con A (Ccopolymer ) 0.01 mg/mL, CConA ) 0.5 mg/mL) and PGAMA15-PPR20-PGAMA15/BSA (Ccopolymer ) 0.01 mg/mL, CBSA ) 0.5 mg/mL) in aqueous solution at room temperature.

4. Conclusions A new class of supramolecular and biomimetic glycopolymer/ PCL-based polypseudorotaxane/glycopolymer triblock copolymers (PGAMA-PPR-PGAMA) was successfully synthesized

3652 J. Phys. Chem. B, Vol. 112, No. 12, 2008 by the combination of ROP, supramolecular inclusion reaction, and direct ATRP of unprotected GAMA glycomonomer. The central PPR segments adopted a channel-type crystalline structure within the biohybrid, where the crystallization of PCL segments was completely suppressed within the hydrophobic R-CD cavities. Moreover, a novel class of glucose-shelled and oligosaccharide (R-CD) threaded polypseudorotaxane-cored nanoparticles self-assembled in aqueous solution, which changed from spherical micelles to vesicles with the decreasing weight fraction of hydrophilic glycopolymer segments. Furthermore, these triblock copolymers demonstrated specific biomolecular recognition with Con A in comparison with BSA. Consequently, this provides a convenient method not only for synthesizing supramolecular and biomimetic polypseudorotaxane/glycopolymer biohybrids but also for fabricating sugar-installed nanoparticles for targeted drug delivery. Abbreviations: atom transfer radical polymerization, ATRP; bovine serum albumin, BSA; 2-bromopropionyl-terminated poly(-caprolactone), PCL-Br; concanavalin A, Con A; critical aggregation concentration, cac; R-cyclodextrin, R-CD; cyclodextrins, CDs; differential scanning calorimetry, DSC; 1,6diphenyl-1,3,5-hexatriene, DPH; dynamic light scattering, DLS; Food and Drug Administration, FDA; Fourier transform infrared, FT-IR; gel permeation chromatograph, GPC; D-gluconamidoethyl methacrylate, GAMA; nuclear magnetic resonance, NMR; N′,N′′,N′′-pentamethyldiethylenetriamine, PMDETA; poly(caprolactone), PCL; PCL-based polypseudorotaxane, PPR; PCL with two hydroxyl end groups, PCL-OH; polydispersity indices, Mw/Mn; poly(D-gluconamidoethyl methacrylate)-PPR-poly(Dgluconamidoethyl methacrylate), PGAMA-PPR-PGAMA; ring-opening polymerization, ROP; transmission electron microscopy, TEM; wide-angle X-ray diffraction, WAXD. Acknowledgment. We are greatly grateful for the financial support of the National Natural Science Foundation of China (Grants 20404007 and 20674050) and Shanghai Leading Academic Discipline Project (Grant B202). We thank the reviewers for their valuable comments and grammar revisions for this manuscript. The assistance of the Instrumental Analysis Center of SJTU is also appreciated. Supporting Information Available: 1H NMR for PCL-OH, PCL-Br, and R-CD and GPC for R-CD, PPR, and PGAMAPPR-PGAMA. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Liu, Y.; Yu, L.; Chen, Y.; Zhao, Y. L.; Yang, H. J. Am. Chem. Soc. 2007, 129, 10656-10657. (2) Harada, A.; Hashidzume, A.; Takashima, Y. AdV. Polym. Sci. 2006, 201, 1-43. (3) Huang, F.; Gibson, H. W. Prog. Polym. Sci. 2005, 30, 982-1018. (4) Harada, A. Acc. Chem. Res. 2001, 34, 456-64. (5) Nepogodiev, S. A.; Stoddart, J. F. Chem. ReV. 1998, 98, 195976. (6) Araki, J.; Zhao, C.; Ito, K. Macromolecules 2005, 38, 7524-27. (7) Li, J.; Ni, P.; Zhou, Z.; and Leong, K. W. J. Am. Chem. Soc. 2003, 125, 1788-95. (8) Li, J.; Ni, P.; Leong, K. Angew. Chem., Int. Ed. 2003, 42, 69-72. (9) Jiao, H.; Goh, S. H.; Valiyaveettil, S. Macromolecules 2002, 35, 1980-3. (10) Chen, L.; Zhu, X.; Yan, D.; Chen, Y.; Chen, Q.; Yao, Y. F. Angew. Chem., Int. Ed. 2006, 45, 87-90.

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