Shell-Cross-Linked Micelles from PNIPAM-b-(PLL ... - ACS Publications

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Biomacromolecules 2010, 11, 1882–1890

Shell-Cross-Linked Micelles from PNIPAM-b-(PLL)2 Y-Shaped Miktoarm Star Copolymer as Drug Carriers Li-Ying Li, Wei-Dong He,* Jian Li, Bo-Yu Zhang, Ting-Ting Pan, Xiao-Li Sun, and Zong-Lei Ding Department of Polymer Science and Engineering, CAS Key Laboratory of Soft Matter Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China Received April 22, 2010; Revised Manuscript Received May 26, 2010

Well-defined AB2 Y-shaped miktoarm star copolymers of PNIPAM-b-(PZLL)2 and PNIPAM-b-(PLL)2 were synthesized through the combination of atom transfer radical polymerization (ATRP), ring-opening polymerization (ROP), and click chemistry, where PNIPAM, PZLL, and PLL are poly(N-isopropylacrylamide), poly(ε-benzyloxycarbonyl-L-lysine), and poly(L-lysine), respectively. Propargyl amine was employed as ROP initiator for the preparation of alkynyl-terminated PZLL. Diazide-terminated PNIPAM was obtained with an azide-containing ATRP initiator. The subsequent click reaction led to the formation of PNIPAM-b-(PZLL)2. After the removal of the benzyloxycarbonyl group, water-soluble PNIPAM-b-(PLL)2 was obtained. The core-shell micelles of PNIPAMb-(PLL)2 were formed above lower critical solution temperature of PNIPAM block. At this temperature, the shell cross-linking was performed through the reaction between glutaraldehyde and the primary amine groups of the PLL shell, affording the micelles with the endurance to temperature and pH changes. These shell-cross-linked micelles were used as drug nanocarriers and the release profile was dually controlled by the solution temperature and the cross-linking degree.

Introduction In recent years, growing attention has been paid to the stimulus-responsive polymeric micelles because of their unique core-shell structure and intelligent properties, including their responsive abilities to pH, temperature, ionic strength, and so on.1-4 Such polymeric assemblies can be prepared from amphiphilic block copolymers, which self-assemble in aqueous solution to afford different nanoscale structures including core-shell micelles. Previous studies of the micelles mainly focused on the synthesis and supermolecular self-assembly of linear AB diblock copolymers.5-11 It is reported that the critical micellization concentration, aggregation number, shape, and size of the micelles are determined by the solution conditions, the relative block length, and the molecular weight of the polymers.5,12,13 It is theoretically predicted that the chain architecture of block copolymers can also play an important role in their micellization in both organic and aqueous solution.14-18 In the category of nonlinear block copolymers, asymmetric AB2 miktoarm (Y-shaped) star copolymers have received considerable attention because of their comprehensive phase-separation behavior in either bulk or solution, based on the fact that the building blocks are linked to a single junction point. Their self-assembly in selective solvents can create novel structures with potential applications in diverse areas such as drug delivery19 and biotechnology.20 However, those traditional micelles still have limited usage in practical applications. One of the important reasons is that these micelles should be stable enough to withstand a wide pH change, solvent etching, and temperature variation. In those cases, self-assembly micelles usually have poor stability. Therefore, several research groups have focused their interest on the novel strategies to increase the stability of such * To whom all correspondence should be addressed. Tel.: 86-5513601699. Fax: 86-551-3606743. E-mail: [email protected].

nanostructures. The approaches of core-cross-linked (CCL)21-24 and shell-cross-linked (SCL)25-28 micelles were then developed to maintain their structural integrity. The latter approach was originally reported by Wooley et al. for amphiphilic block copolymers.29,30 It should be noted that shell cross-linking can also partially modulate the release of guest molecules through the adjustment of the cross-linking degree.31 Besides the importance of the shell cross-linking in drug release, the features of core domain are also of significance.32 A stimulus responsive core of SCL micelles, such as poly(Nisopropylacrylamide) (PNIPAM), can provide more sophisticated and smarter drug nanocarriers. Above or below lower critical solution temperature (LCST) of PNIPAM chains, the transition between hydrophobicity and hydrophilicity of PNIPAMcross-linked CCL and SCL micelles composed of PNIPAM-bpoly(diethylaminoethyl methacrylate) will accelerate or retard the diffusion of guest molecules out of the nanocarriers.33 However, the hydrolysis of 3-(trimethoxysilyl)propyl methacrylate units was adopted for PNIPAM cross-linking, and waterfree preparation was strictly required to perform the micelle formation. Herein, we reported the synthesis of well-defined Y-shaped miktoarm copolymers, PNIAPM-b-(PZLL)2 [poly(ε-benzyloxycarbonyl-L-lysine)] and PNIAPM-b-(PLL)2 [poly(L-lysine)] through the combination of atom transfer radical polymerization (ATRP), ring-opening polymerization (ROP), and click chemistry. SCL micelles of PNIAPM-b-(PLL)2 with PNIPAM as the core were obtained with glutaraldehyde as the cross-linking reagent. The influences of cross-linking degree and temperature on thermal response and drug release of SCL micelles were investigated.

Experimental Section Materials. N-Isopropylacrylamide (NIPAM, 97%, Kohjin Co., Japan) was purified by recrystallization from a benzene/n-hexane

10.1021/bm1004383  2010 American Chemical Society Published on Web 06/10/2010

Shell-Cross-Linked Micelles

Biomacromolecules, Vol. 11, No. 7, 2010

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Scheme 1. Synthetic Route of Y-Shaped Miktoarm Star Copolymers of PNIPAM-b-(PZLL)2 and PNIPAM-b-(PLL)2

mixture (65/35 v/v) and ε-(benzyloxy carbonyl)-L-lysine (ZLL, SigmaAldrich) was used as received. 2-Chloropropionyl chloride (98%) and bis(2-chloroethyl)amine hydrochloride (98%) were purchased from Alfa Aesar and used as received. Triethylamine was stirred with KOH for 12 h at room temperature, refluxed with benzene-4-sulfonyl-chloride, and distilled before use. N,N,N′,N′′,N′′′,N′′′-(Hexamethyltriethylenetetramine) (Me6TREN) was synthesized according to the method previously described.34 N,N,N′,N′,N′-Pentamethyl diethylenetriamine (PMDETA, 98%, Sigma-Aldrich) was distilled over NaOH prior to use. Merrifield resin (1.55 mmol/g) was purchased from Sigma-Aldrich. Tetrahydrofuran (THF) was dried over sodium/benzophenone and distillated just before use. N,N-Dimethylformamide (DMF) and nhexane were dried and distilled over calcium hydride. Triphosgene, trifluoroacetic acid (TFA), and hydrobromic acid in glacial acid (HBr/ HAc, 45% w/v) were purchased from Sigma-Aldrich. Prednisone acetate and glutaraldehyde were obtained from Tianjin Tianyao Pharmaceuticals Co. Ltd. and used as received. All other reagents were of analytical grade and used as received. Sample Synthesis. General approach to the synthesis of PNIPAMN(CH2CH2N3)2, alkynyl-PZLL, and Y-shaped miktoarm star copolymers is shown in Scheme 1. Synthesis of Bis(2-azidoethyl)amine Hydrochloride. Bis(2-chloroethyl)amine hydrochloride (5 g, 28.17 mmol), H2O (100 mL), NaN3 (18.31 g, 281.70 mmol), and one pinch of KI were added into a 250 mL round-bottom flask. The reaction mixture was allowed to stir at 60

°C. After 36 h, the mixture was cooled to room temperature and NaCl (10 g) was then added. The mixture was extracted with benzene (3 × 100 mL). The organic layer was collected and dried over anhydrous MgSO4. After the filtration, the solvent was partially removed by rotary evaporation at 25 °C. (Caution: high temperature and heavy distillation should be avoided!) The concentration of bis(2-azidoethyl)amine hydrochloride in the residual solution was measured by 1H NMR spectroscopy (∼7.5 wt %). Synthesis of N,N-Di(2-azidoethyl)-2-chloropropionylamide) (AECPA). After the solution of bis(2-azidoethyl)amine hydrochloride (2.5 g, 13.05 mmol) and triethylamine (7.91 g, 78.30 mmol) in benzene (30 mL) were cooled in an ice bath, a solution of 2-chloropropionyl chloride (8.28 g, 65.25 mmol) in benzene (10 mL) was added dropwise. The mixture was stirred at 0 °C for 1 h and at room temperature for 24 h. The resulting salt was filtered off and the filtrate was washed with saturated NaHCO3 aqueous solution (3 × 50 mL). The organic layer was dried over anhydrous MgSO4 and partially evaporated to obtain a yellow oily crude residue. The crude product was further purified by versa flash chromatography (silica column, CH2Cl2) to obtain colorless AECPA (2.4 g, yield: 74.2%). Synthesis of PNIPAM-N(CH2CH2N3)2 Via ATRP.35 NIPAM (3.0 g, 0.026 mol), Me6TREN (57 mg, 2.04 × 10-4 mol), and isopropanol (4.0 mL) were added to a polymerization tube equipped with a stirring bar. Then, CuCl (20 mg, 2.04 × 10-4 mol) in isopropanol (2 mL) was introduced, and the mixture was stirred for 15 min to allow the

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formation of the CuCl/Me6TREN complex. After that, the initiator of AECPA (50 mg, 2.04 × 10-4 mol) was charged. The polymerization tube was degassed by three freeze-pump-thaw cycles and sealed under vacuum. The reaction was carried out under vigorous stirring at 25 °C. After 8 h, the tube was opened and the mixture was diluted with saturated CuCl2/isopropanol solution. PNIPAM capped with two azide groups at one chain-end [PNIPAM-N(CH2CH2N3)2] was obtained after the precipitation in n-hexane. Further purification was performed by dialysis against deionized water for 48 h using a dialysis membrane with a cutoff molecular weight of 3500. The white solid product was obtained by lyophilization (2.71 g, yield: 90.3%). The block length of the obtained product was determined to be 130 based on 1H NMR analysis, and molecular weight distribution was obtained with a gel permeation chromatograph (GPC). Synthesis of Azide-Functionalized Resin. Azide-functionalized resin was synthesized as follows. Commercial Merrifield resin (5 g, 7.75 mmol) was dispersed in 40 mL of DMSO and NaN3 (2.4 g, 36.75 mmol) was added into the reaction flask. The reaction mixture was stirred at 50 °C for 36 h. Then, the suspension was filtrated and thoroughly washed with water. After drying in a vacuum oven at room temperature, azide-functionalized resin was obtained with quantitative yield. Synthesis of Alkynyl-PZLL. ZLL-NCA (N-carboxy-R-amino anhydride) was synthesized by the Fuchs-Farthing method using triphosgene.36 For the preparation of alkynyl-terminated PZLL (alkynyl-PZLL), freshly prepared ZLL-NCA (4.27 g, 15.25 mmol) was dissolved in anhydrous DMF (25 mL) in a baked flask. Propargyl amine (15.30 mg, 0.28 mmol) in anhydrous DMF (5 mL) was added into the solution of ZLL-NCA. The reaction mixture was allowed to stir at 30 °C under a dry N2 atmosphere. After 2 d, the mixture was precipitated into excess water. The white product was collected by filtration and dried under vacuum at room temperature for 48 h (3.97 g, yield: 93%). The block length was determined to be 44 from 1H NMR spectrum and molecular weight distribution was obtained with GPC. Synthesis of Miktoarm Star Copolymer PNIPAM-b-(PZLL)2 by Click Reaction. The mixture of PNIPAM-N(CH2CH2N3)2 (0.2 g, 1.36 × 10-5 mol) and alkynyl-PZLL (0.34 g, 3.0 × 10-5 mol) was dissolved with nitrogen-purged DMSO (10 mL) in a polymerization tube. CuBr (6 mg, 3.0 × 10-5 mol) and PMDETA (13 mg, 3.0 × 10-5 mol) were added, and the mixture was degassed by three freeze-pump-thaw cycles. The tube was sealed under vacuum and stirred at 50 °C. After 48 h, the tube was opened and little of the mixture was taken for GPC analysis. The main portion of mixture was transferred to another tube and azide-functionalized resin (0.1 g, 0.13 mmol) was added. After degassing with three freeze-pump-thaw cycles, the tube was sealed under vacuum and kept with stirring for 8 h at 50 °C. After the removal of resin by filtration, the polymer solution was dialyzed against deionized water for 48 h using a dialysis membrane with a cutoff molecular weight of 14000 g/mol. The white solid product of PNIPAMb-(PZLL)2 was obtained by lyophilization (0.35 g, yield: 71%). Synthesis of PNIPAM-b-(PLL)2. The obtained PNIPAM-b-(PZLL)2 was then subjected to hydrolysis to remove the protecting ε-(benzyloxycarbonyl) groups of PZLL as follows. PNIPAM-b-(PZLL)2 (0.35 g, 9.33 × 10-6 mol) was dissolved in TFA (5 mL) under stirring. HBr/ HAc (0.76 mL, 6 mmol HBr) was then dropwise introduced into the reaction mixture. After stirring at room temperature for 24 h, most of the solvent was removed under reduced pressure. The solid was redispersed in water and the solution pH was adjusted to 7.0 with 0.2 M NaOH solution. The polymer was purified by dialysis against deionized water for 2 d using a dialysis membrane with a cutoff molecular weight of 3500. The product was obtained as a white powder after lyophilization (0.21 g, yield: 86.9%). Preparation of PNIPAM-b-(PLL)2 Micelles and Sequential Shell Cross-Linking. At room temperature, PNIPAM-b-(PLL)2 copolymer (30 mg, 5.52 × 10-7 mol) was dissolved in deionized water (30 mL). Then, the solution was heated gradually up to 50 °C to induce the formation of the micelles with PNIAPM as the core and PLL as the shell. The micelle dispersion was kept stirring at 50 °C for 12 h to reach the micellization equilibrium.

Li et al. Subsequent shell cross-linking of the micelles was performed by the addition of glutaraldehyde into the above micelle dispersion. The mixture was stirred at 50 °C for 24 h and the molar ratio of glutaraldehyde/amine was fixed at 1:2 or 1:4, targeting a 100 or 50% cross-linking degree. Herein, the cross-linking degree was defined as the conversion of amine groups of PLL units. With shell cross-linking, the micelle morphology would remain without disassociation after the dispersion was cooled to room temperature. Loading Prednisone Acetate into PNIPAM-b-(PLL)2 Micelles. Encapsulation of prednisone acetate into PNIPAM-b-(PLL)2 micelles was performed at 50 °C by a simple method. After PNIPAM-b-(PLL)2 (30 mg) and prednisone acetate (3.5 mg) were dissolved in THF (2 mL), the solution obtained was added dropwise into 30 mL of neutral water (preheated at 50 °C) under vigorous stirring. Then, the dispersion was kept at 50 °C under stirring for 12 h to form drug-loaded micelles. Then, glutaraldehyde (0.25 or 0.5 equivalent to amine group) was added and the cross-linking reaction lasted at 50 °C for 24 h. After that, SCL drug-loaded micelle dispersion was packed into a dialysis tube (cutoff molecular weight: 8000-14000 g/mol) and subjected to dialysis against deionized water (1 L) at 50 °C. The deionized water (50 °C) was renewed every 3 h to remove THF and the unloaded free drug, until no residue of prednisone acetate was detected by ultraviolet (UV) spectroscopy at 242 nm. Prednisone Acetate Releasing from PNIPAM-b-(PLL)2 SCL Micelles. Subsequently, the drug-loaded SCL micelle dispersion in a dialysis tube was divided into two equipartitions for the evaluation of drug release at two temperatures (25 and 38 °C) in PBS buffer solution (0.01 mol/L, pH ) 7.4). At different predetermined intervals, 3.0 mL of solution was withdrawn to measure the amount of prednisone acetate released from SCL micelles using UV absorbance at 242 nm, based on the calibration from prednisone acetate solution in PBS buffer. At the same time, an equal volume of fresh medium was added to keep the constant volume. After the drug release, the solution in the dialysis tube was lyophilized. Then, the dried micelles were dissolved in DMF and the drug concentration was measured by UV absorbance at 272 nm to determine the amount of prednisone acetate remained in the micelles, using a calibration curve experimentally obtained with prednisone/DMF solutions. The total amount of prednisone loaded in the micelles was the sum of the drug released in the aqueous medium and the drug left in the SCL micelles. The cross-linking degree was varied to study its influence on the drug release behavior. The entrapment efficiency (EE) and loading content (DL) of prednisone acetate are defined as follows:

Wload × 100% Wadd

(1)

Wload Wpolymer + Wload

(2)

EE% )

DL )

where Wload, Wadd, and Wpolymer are the weights of the loaded drug by the micelles, added drug for loading, and polymer used. The data of drug loading and releasing were obtained with three parallel measurements and the experiment deviation is within 10%. Characterization of Polymers. The molecular weight and its polydispersity index were determined on a waters 150C GPC instrument equipped with three Ultrastyragel columns (500, 103 and 104 Å) in series and a RI detector at 30 °C, using monodispersed polystyrene as calibration standard. DMF was used as the eluent at a flow rate of 1.0 mL/min. All 1H NMR spectra were recorded at 25 °C on a Bruker AV300 NMR spectrometer (resonance frequency of 300 MHz) operated in the Fourier transform mode. Fourier transform infrared (FTIR) spectra were recorded on a Bruker VECTOR-22 IR spectrometer in KBr pellets. Temperature-Dependent Turbidimetry. The optical transmittance of the aqueous polymer solution and micelle dispersion at 500 nm was acquired on a Unico UV/vis 2802PCS spectrophotometer. A thermo-



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Figure 1. 1H NMR spectra of bis(2-azidoethyl)amine hydrochloride (a) and AECPA (b).

statically controlled cuvette was employed, and the heating rate was 0.2 °C/min. The lower critical solution temperature (LCST) was defined as the temperature corresponding to initially abrupt decrease of transmittance. Laser Light Scattering (LLS). A commercial spectrometer (ALV/ DLS/SLS-5022F) equipped with multi-τ digital time correlation (ALV5000) and a cylindrical 22 mW UNIPHASE He-Ne laser (λ0 ) 632.8 nm) as the light source was used. Before LLS measurement, the polymer solution and micelle dispersion were dust-free using a Millipore filter (0.45 µm). For dynamic LLS, scattered light was collected at a fixed angle of 30° for a duration of 10 min. Averaged hydrodynamic radius and hydrodynamic radius distribution were computed using cumulants analysis and CONTIN routine, respectively. In static LLS, the angular dependence of the absolute excess time-averaged scattering intensity, known as the Rayleigh ratio Rvv(q), was recorded, and we were able to obtain the weight-averaged molecular weight of micelles (Mw). The scattering angular range used was from 20 to 150°. All data were averaged over three measurements. Transmission Electron Microscopy (TEM). TEM images were recorded using a Philips CM120 electron microscope at an accelerating voltage of 200 kV. TEM samples were prepared by placing 10 µL of aqueous dispersion of free SCL micelles (1.0 mg/mL) on copper grids coated with thin films of Formvar and carbon.

Results and Discussion Synthesis and Characterization of PNIPAM-b-(PZLL)2 Miktoarm Star Copolymers. The first example of the synthesis of amphiphilic Y-shaped miktoarm star copolymers using ATRP and click chemistry was reported by Monteiro et al. and amphiphilic copolymers of polystyrene-b-[poly(acrylic acid)]2 was obtained.37 In the current study, we synthesized totally hydrophilic Y-shaped miktoarm star copolymers by the combination of ATRP, ROP, and click chemistry, and the synthetic procedure is illustrated in Scheme 1. Synthesis of PNIPAM-N(CH2CH2N3)2 Via ATRP. Bis(2azidoethyl)amine hydrochloride was synthesized by the reaction of bis(2-chloroethyl)amine hydrochloride with NaN3.38 1H NMR spectra of bis(2-azidoethyl)amine hydrochloride is shown in Figure 1a. The signals at δ ) 3.61 ppm (a) and 2.76 ppm (b) are ascribed to methylene protons of -CH2NH and -CH2N3, respectively. After the reaction with 2-chloropropionyl chloride, new signals at δ ) 1.67 ppm (c) and 4.73 ppm (d) are ascribed

Figure 2. 1H NMR spectra of PNIPAM-N(CH2CH2N3)2 (a), alkynylPZLL (b), and PNIPAM-b-(PZLL)2 (c) in CDCl3/TFA mixture.

Figure 3. FT-IR spectra of PNIPAM-N(CH2CH2N3)2 (a) and PNIPAMb-(PZLL)2 (b).

to the methyl proton of -CHCH3 and the methine proton of -ClCHCH3 (Figure 1b), respectively. The integration ratio of (a + b)/c/d is 8:3:1, indicating the successful synthesis of the ATRP initiator. When this initiator is used, ATRP of NIPAM monomer gives a dual-azide end-functionalized polymer, PNIPAM-N(CH2CH2N3)2. 1H NMR spectra and the corresponding peak assignments are shown in Figure 2a. The signals at 6.40 (a) and 4.01 ppm (b) are characteristic of PNIPAM block. The polymerization degree of PNIPAM is calculated to be 130, by comparing the integration of the methine proton peak at δ ) 4.01 ppm (b) with the methylene proton peak at δ ) 3.53 ppm (c) of the initiator (Figure 2a). FT-IR spectrum of PNIPAMN(CH2CH2N3)2 clearly reveals an absorbance peak at ∼2110 cm-1, which corresponds to the vibration of frequency of the azide group (Figure 3a).39 Synthesis of Alkynyl-PZLL. It has been well-established that many different basic initiators can be used for NCA ring-opening polymerization. However, among those initiators, only primary amines are quantitatively incorporated at the C terminus of the

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Figure 4. GPC traces of PNIPAM-N(CH2CH2N3)2 (a), alkynyl-PZLL (b), and PNIPAM-b-(PZLL)2 before (c) and after (d) treating with azidefunctionalized resin.

Figure 5. 1H NMR spectrum of Y-shaped miktoarm star copolymer PNIPAM-b-(PLL)2 in D2O at 25 °C.

polypeptide chain.40,41 In our present study, propargyl amine is used as the initiator for ring-opening polymerization of ZLLNCA in dry DMF, leading to the formation of alkynyl-PZLL. 1 H NMR spectrum of alkynyl-PZLL in CDCl3 containing ∼10% TFA (v/v) is shown in Figure 2b. The signals at δ ) 7.50-7.70 (a), 5.12 (b), 4.40 (c), 3.10 (d), and 1.20-1.80 ppm (e) are ascribed to the proton of the amide group, methylene protons of the benzyl group, the methine proton in the backbone, the methylene protons of lysine attached to the benzyloxycarbonyl group, and the protons of the other three methylene groups of lysine, respectively. The signal at δ ) 2.50 ppm (g) is assigned to the alkynyl proton. By comparing the integration of signals of d and g, the polymerization degree of alkynyl-PZLL is calculated to be 44. GPC analysis in DMF reveals the monomodal and symmetric distribution of the molecular weight for alkynyl-PZLL (Figure 4b). Its Mn,GPC is determined to be 11000 and Mw/Mn is 1.17. Synthesis of Miktoarm Star Copolymer PNIPAM-b(PZLL)2 by Click Reaction. Using “click” chemistry, PNIPAMb-(PZLL)2 miktoarm star copolymer is obtained from PNIPAMN(CH2CH2N3)2 and alkynyl-PZLL in the presence of CuBr/ PMDETA in DMSO at 50 °C. We use an excess of alkynylPZLL to achieve the complete conversion of azide groups of PNIPAM-N(CH2CH2N3)2, which results in unreacted alkynylPZLL. The excess alkynyl-PZLL can be facilely removed by clicking with azide-functionalized resin and subsequent simple filtration.42 The 1H NMR spectrum of the Y-shaped miktoarm star copolymer PNIPAM-b-(PZLL)2 is shown in Figure 2c. The signals at δ ) 3.10 ppm (d) and 4.01 ppm (b) are characteristic signals of PZLL and PNIPAM. The molar ratio of the PNIPAM unit to PZLL unit determined by the signal integral ratio of peaks (b) to (d) is 1.48, which agrees well with the expected value based on the reactant amounts. GPC analysis of the crude product with unreacted alkynylPZLL and purified miktoarm star copolymer PNIPAM-b(PZLL)2 is demonstrated in Figure 4c and d, respectively. The molecular weight of purified PNIPAM-b-(PZLL)2 shifts to a higher molecular weight region with Mn,GPC of 65000 and Mw/ Mn of 1.21, compared with that of PNIPAM-N(CH2CH2N3)2. The crude PNIPAM-b-(PZLL)2 has a shoulder peak, which

should be ascribed to the excess alkynyl-PZLL. GPC analysis reveals that the alkynyl-PZLL is removed successfully by azidefunctionalized resin. FT-IR spectrum of the obtained miktoarm star copolymer is shown in Figure 3b. From Figure 3b, we can clearly observe the complete disappearance of the characteristic azide peak at ∼2110 cm-1, further confirming the full conversion of azide groups. Combining with the proofs of GPC, 1H NMR, and FT-IR, it can be concluded that the formation of Y-shaped miktoarm star copolymer PNIPAM-b-(PZLL)2 via ATRP, ROP, and click chemistry is successful. Synthesis of PNIPAM-b-(PLL)2. The obtained PNIPAM-b(PZLL)2 was then subjected to the hydrolysis to remove benzyloxycarbonyl protecting group. A mixture of TFA and 45% w/v HBr/HAc was employed for the hydrolysis reaction according to the procedure previously reported.43-451H NMR spectra of PNIPAM-b-(PLL)2 is shown in Figure 5. The signals characteristic of benzyloxycarbonyl group of PZLL at 7.31 and 4.92 ppm disappear, indicating the complete removal of ε-(benzyloxycarbonyl) groups from PZLL. Thermal Phase Transition Behavior of PNIPAM-b(PLL)2. PNIPAM is a well-known thermoresponsive polymer that exhibits LCST at about 32 °C in aqueous solution.46,47 It is generally accepted that this phase transition is greatly affected by the molecular weight of the PNIPAM chain, the polymer concentration, and the comonomer.48 In the current study, temperature-dependent turbidimetry is employed to determine LCST values of the PNIPAM homopolymer and the PNIPAMb-(PLL)2 copolymer. Figure 6 shows temperature-dependent transmittance at λ ) 500 nm obtained for aqueous solutions of PNIPAM homopolymer and PNIPAM-b-(PLL)2 copolymer at two polymer concentrations (Cpolymer). For PNIPAM homopolymer at Cpolymer ) 2.0 and 1.2 mg/mL, we can see that the concentration dependence is not clear. They exhibit almost the same decrease tendency of transmittance due to the relatively low polymer concentrations and low molecular weight.35 When the concentration of the PNIPAM homopolymer is decreased to 1.2 mg/mL, the concentration of PNIAPM block is the same as that of the copolymer of PNIPAM-b-(PLL)2 at Cpolymer ) 2.0 mg/mL. As for PNIPAM homopolymer at Cpolymer


Figure 6. Temperature dependence of optical transmittance at 500 nm obtained for PNIPAM-N(CH2CH2N3)2 and PNIPAM-b-(PLL)2 at two polymer concentrations at pH ) 7.

) 1.2 mg/mL, we can clearly see that transmittance starts to decrease at 32 °C. As for copolymer of PNIPAM-b-(PLL)2 at Cpolymer ) 2.0 mg/mL, the transmittance starts to decrease at 37 °C. LCST of PNIPAM-b-(PLL)2 at a lower polymer concentration of 1.0 mg/mL is 42 °C. Compared with the PNIPAM homopolymer, the LCST of PNIPAM-b-(PLL)2 is a little higher, which is due to the presence of the hydrophilic PLL block. Furthermore, it is also found that the LCST of the Y-shaped block copolymer is obviously higher at lower polymer concentration, which is consistent with the results achieved by Zhu and Napper.49,50 Preparation of Shell-Cross-Linked PNIPAM-b-(PLL)2 Micelles. In recent years, a variety of methods have been employed to covalently stabilize the micellar assemblies, involving either their cores or their coronas. For our Y-shaped miktoarm star block copolymer, the PNIPAM block becomes hydrophobic when the solution temperature increases above its LCST, resulting in the formation of the micelles with PNIPAM as the core. Poly(L-lysine) is hydrophilic in neutral aqueous solution and distributes at the periphery of the micellar assembly. Along the peptide block, the amine groups of PLL can be used to cross-link and stabilize the assembly by the reaction with complementary functional groups. When the solution temperature increases to 50 °C above LCST of PNIPAM block, PNIPAM-b-(PLL)2 copolymer forms the micelles. When glutaraldehyde is used as the cross-linking reagent, PLL blocks are cross-linked. The cross-linking degree is defined as the theoretical conversion of amine groups and targeted to be 50 and 100%. The reaction was allowed to proceed for 24 h to ensure the conversion of all the amine groups. The measurements of DLS and temperature-dependent turbidimetry were performed to offer valuable information about the success of the cross-linking reaction. Figure 7a shows the hydrodynamic radius distributions, f(Rh), for the aqueous solution of the PNIPAM-b-(PLL)2 copolymer without cross-linking under pH ) 2.0, 7.0, and 12.0 at room temperature. PLL is water-soluble at neutral or acidic medium due to the protonation of amine groups. At room temperature, as pH of the copolymer solution is increased, the hydrophilicity of PLL stabilizing block decreases, then the miktoarm star copolymer will form the micelles with PLL as the core and PNIPAM chains as the shell. For these micelles, the averaged hydrodynamic radius (Rh) is determined to be 8 nm at pH ) 2.0 and 128 nm at pH ) 12.0, respectively. The results indicate that, at room temperature, PNIPAM-b-(PLL)2 can self-assemble into PLL-core micelles at pH ) 12.0, while its chains remain


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as unimers at pH ) 2.0. At pH ) 7.0, PNIPAM-b-(PLL)2 is in dynamic equilibrium between unimer and micelle, suggesting that PNIPAM-b-(PLL)2 micelles formed at 50° and pH ) 7.0 may disassemble and phase inversion would occur. As the temperature rises to 50 °C at pH ) 7.0, PNIPAM becomes hydrophobic and PLL keeps partially hydrophilic. Thus, the micelles with PNIPAM as core and PLL as shell are formed. After the cross-linking with glutaraldehyde at the temperature of 50 °C and pH of 7.0, the integrity of the micelles remains after the micelle dispersion is cooled to room temperature. Figure 7b and c show f(Rh) for the cross-linked micelles of PNIPAM-b-(PLL)2 with targeted cross-linking degree of 50 and 100% at different pH values and room temperature. As for SCL micelles, they are formed at 50 °C. After the temperature returns to room temperature, the core-shell structure of micelles with PNIPAM as the shell remains. As shown in Figure 7b, an increase in Rh from 132 nm at pH ) 12.0 to 150 nm at pH ) 2.0 is observed at room temperature for the micelles with 50% cross-linking degree. However, the expansion of PNIPAM core is strongly limited and the micelle size remains almost constant for the micelles with 100% cross-linking degree under three pH values at room temperature. The above results confirm that shell cross-linking has successfully fixed the core-shell nanostructure, and lower cross-linking density allows the micelles to expand to a greater extent. Thus, the swelling degree can be controlled by the cross-linking density of the micelles. In contrast to core cross-linking reactions, shell cross-linking reactions can eventually be accompanied with undesirable intermicellar connection. We, thus, used DLS analysis to examine the extent of potential side reaction. For that purpose, the angular dependence of averaged characteristic line width (Γ) of the micelles as a function of the squared scattering vector [q2; q ) 4πn sin(θ/2)/λ, where n is the refractive index of the solution, θ is the scattering angle, and λ (632.8 nm) is the wavelength of the incident laser light] was measured before and after the cross-linking. The measurements were performed between 30 and 130°. The plots are shown in Figure 8. After the cross-linking of the micelles at a concentration of 1.0 mg/mL, the evolution of Γ exhibits also the linear relationship with q2, which confirms the spherical shape of the micelles formed at 50 °C.51 The two lines pass through the origin, indicating that the relaxation process measured in dynamic LLS is isotropic diffusion. The slope of the line in Figure 8 leads to the average translational diffusion coefficient D, that is, D ) Γ/q2. The averaged hydrodynamic radius (Rh) of the un-crosslinked micelles formed in neutral solution at 50 °C calculated from the Stokes-Einstein equation is 104 nm, while the Rh value of 100% cross-linked micelles in neutral solution at 25 °C is 130 nm. Considering that PNIPAM chains exist in partially extended conformation at room temperature, the results suggest that the micelles maintain their integrity and the intermicellar fusion may not occur during the cross-linking. The actual morphology of the SCL micelles with a targeted cross-linking degree of 100% has been obtained by TEM observation (Figure 9). The TEM image of the SCL micelles reveals the presence of presumably spherical micelles with a diameter of 145-190 nm, which is smaller than the hydrodynamic diameter (2Rh) of ∼260 nm obtained from DLS. It is well-known that the diameter measured by TEM is typically much smaller than that obtained from DLS because the former reflects the size in the dry state. Figure 10 shows the temperature-dependent transmittance obtained for the neutral aqueous solution of the PNIPAM-b-

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Figure 7. Hydrodynamic radius distributions, f(Rh), obtained for aqueous solution of PNIPAM-b-(PLL)2 (a), 50% cross-linked micelle dispersion (b), and 100% cross-linked micelle dispersion (c): temp, 25 °C; pH 2.0, 7.0 and 12.0; Cpolymer, 1.0 mg/mL.

Figure 8. Squared scattering vector (q2) dependence of characteristic line width (Γ) for PNIPAM-b-(PLL)2 micelles in aqueous solution (1.0 mg/mL) between 30 and 130° at room temperature.

(PLL)2 copolymer and the neutral aqueous dispersion of PNIPAM-b-(PLL)2 micelles with different cross-linking degrees at a polymer concentration of 1.0 mg/mL. As seen in Figure 10, for the micelle dispersion with crosslinking degree of 50 and 100%, the transmittance starts to decrease dramatically at 38 and 37 °C and finally falls down to ∼55 and ∼80%, respectively. For the un-cross-linked copolymer, the transmittance starts to decrease at 42 °C and gets down to ∼30%. The low LCST values for SCL micelles might be caused by the relatively high local concentration of PNIPAM chains. It is reported that PNIPAM phase transition is greatly affected by the polymer concentrations.35 At higher concentrations, PNIPAM has lower LCST values. On the other hand, the transmittance of SCL micelles remains high above LCST and that of PNIPAM-b-(PLL)2 remains very low. Because shell cross-linking results in a covalently connected network attached to PNIPAM cores, the spatial hindrance prevents SCL micelles from further aggregation with each other. Drug Release from Shell-Cross-Linked Micelles. Although various types of SCL micelles have been prepared, few studies

Figure 9. TEM images of SCL micelles with targeted cross-linking degree of 100%.

about their drug release behavior have been reported.52 We can expect that SCL PNIPAM-b-(PLL)2 micelles should be excellent drug nanocarriers due to the following advantages. First, the structure of the nanocarrier is permanently stable due to the introduction of shell cross-linking. Second, as the SCL micelles possess temperature-responsive cores, the encapsulation and subsequent triggered release of drugs is possible. Finally, we can also control the permeability of the shell by the adjustment of the cross-linking degree. The drug release profiles of PNIPAM-b-(PLL)2 micelles at different temperatures were monitored using a conventional dialysis method. Figure 11 shows the time dependence of cumulative prednisone release from drug-loaded SCL micelles into buffer solution at different temperatures. The EE and DL value of the SCL micelles with 100% cross-linking degree is 21.4 and 2.5%, respectively. For that with 50% cross-linking degree, the EE and DL value is 18.6 and 2.1%, respectively. The drug loaded


Figure 10. Temperature dependence of optical transmittance at 500 nm obtained for PNIPAM-b-(PLL)2 solution and SCL micelle dispersions at pH ) 7.0.


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°C. It has been reported that when the core or the shell turns hydrophobic, the drug is squeezed out more quickly from the micelles as a result of the shrinkage of the structure.33,54-57 According to those reports, the faster cumulative release behavior under 38 °C is reasonable. Under the same circumstance, the drug release from the micelles with 50% targeted cross-linking degree exhibits faster than that with 100% targeted cross-linking degree. The decrease of the cross-linking density allows the enhanced permeability of the shell to guest molecules, leading to faster drug release. On the basis of the above discussion, we successfully establish that the SCL micelles fabricated from PNIPAM-b-(PLL)2 can act as excellent drug nanocarriers, whose structure integrity is endowed with the shell cross-linking. Moreover, as the SCL micelles have the temperature-responsive PNIPAM cores, the release profile of encapsulated guest molecules can also be dually controlled by the solution temperature as well as the cross-linking degree. Furthermore, the conversion of the Schiff base within a PLL cross-linked shell to secondary amines provides additional functionalities such pH response and conjugation with bioactive molecules.

Conclusion

Figure 11. Cumulative drug releases from SCL micelles at different temperatures.

in each micelle is calculated as follows. The weight-averaged molar mass of the micelle (Mw,micelle) is determined to be 2.73 × 10-14 by SLS. Considering the amount of polymer used, the micelle number in the whole dispersion is around 1.1 × 1011. For 100% cross-linked micelles, the amount of drug loaded in each micelle is assumed to be 6.8 × 10-12 mg, while the value of 50% cross-linked micelles is 5.9 × 10-12 mg. As seen in Figure 11, the cumulative amount of released drug from targeted 50 and 100% cross-linked micelles at 38 °C in 170 h is 51.2 and 44.7%, respectively. When the drug release is performed at 25 °C, the cumulative amount of released drug from the micelles with 50 and 100% targeted cross-linking within 170 h is 22.8 and 17.1%, respectively. For the two SCL micelles, both release profiles at 38 °C exhibit faster cumulative release behavior. According to previous studies, for un-cross-linked micelles, the drug would diffuse out quickly when the core and the shell all turn hydrophilic.53 However, even though the core and shell of SCL micelles become hydrophilic simultaneously, the shell cross-linking would maintain the micelle structure, which could retard the drug diffusion out of the micelles.33 In our case, both core and shell of SCL drug-loaded micelles are hydrophilic at 25 °C, while PNIPAM chains turn hydrophobic to some extent at 38

Well-defined AB2 Y-shaped miktoarm star copolymer, PNIPAM-b-(PZLL)2, was synthesized via the combination of radical polymerization of NIPAM with AECPA as ATRP initiator, polymerization of ZLL NCA with propargyl amine as ROP initiator and click reaction between PNIPAM-N(CH2CH2N3)2 and alkynyl-PZLL. Removal of benzyloxycarbonyl group from PNIPAM-b-(PZLL)2 resulted in totally hydrophilic PNIPAM-b-(PLL)2. Above LCST of PNIPAM block, PNIPAMb-(PLL)2 self-assembled into the micelles with PLL as shell and PNIPAM as core. SCL micelles were then fabricated with glutaraldehyde as the cross-linking reagent and offered themselves with pH and thermal endurance. The micelles acted as drug nanocarriers through loading PNIPAM cores with a hydrophobic drug. The drug release from PNIPAM-b-(PLL)2 SCL micelles was dually controlled with the temperature and cross-linking degree. Acknowledgment. The financial support of NSFC (20934005), Ministry of Science and Technology of China (2007CB936401), and the HKSAR Earmarked Research Grant (403706) are greatly acknowledged.

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