Thermo-Responsive Porous Membranes of Controllable Porous

Biomacromolecules 2008, 9, 331–339

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Thermo-Responsive Porous Membranes of Controllable Porous Morphology from Triblock Copolymers of Polycaprolactone and Poly(N-isopropylacrylamide) Prepared by Atom Transfer Radical Polymerization F. J. Xu,†,‡ J. Li,‡,§ S. J. Yuan,† Z. X. Zhang,‡ E. T. Kang,*,† and K. G. Neoh† Department of Chemical and Biomolecular Engineering, National University of Singapore, Kent Ridge, Singapore 119260, Division of Bioengineering, National University of Singapore, 9 Engineering Drive 1, Singapore 117576, and Institute of Materials and Research and Engineering (IMRE), 3 Research Link, Singapore 117602 Received August 10, 2007; Revised Manuscript Received September 14, 2007

Stimuli-responsive polymers are of crucial importance in the design of smart biomaterials. The thermo-responsive triblock copolymers of polycaprolactone (PCL) and poly(N-isopropylacrylamide) (P(NIPAAm)), or P(NIPAAm)b-PCL-b- P(NIPAAm) copolymers, were synthesized in this work via atom transfer radical polymerization (ATRP). The P(NIPAAm)-b-PCL-b-P(NIPAAm) copolymers were cast by phase inversion in water into porous membranes with well-defined and uniformly distributed pores. The P(NIPAAm) content in the P(NIPAAm)-b-PCL-bP(NIPAAm) copolymers and the temperature of the aqueous medium for phase inversion could be used to control the pore size and porosity of the membranes. The thermo-responsive characteristics of the membranes were illustrated in the controlled water uptake and temperature-dependent glucose transport through the membranes. These temperature-sensitive membranes with controllable morphology have potential applications in biomedical engineering, drug delivery, and tissue engineering.

1. Introduction Polycaprolactone (PCL) is a biodegradable and biocompatible linear polyester with good mechanical and thermoplastic properties.1–6 It has been widely adopted for biomaterials and biomedical applications.4–10 PCL-based membranes are potentially useful as scaffolds in tissue engineering,11–13 filtration media in biochemical processes,14,15 and transport materials in drug or nutrient delivery systems.13,16,17 Intelligent or smart membranes have been developed from stimuli-responsive polymeric materials, which can respond to changes in temperature, pH, light, and ionic strength.18–26 These unique stimuliresponsive characteristics are of great interest in the drug delivery system, sensors, and biomaterials.18–23 Poly(N-isopropylacrylamide), or P(NIPAAm), is a well-known thermoresponsive polymer. It exhibits a lower critical solution temperature (LCST) of about 32 °C in an aqueous medium. It assumes a random coil structure (hydrophilic state) below the LCST and a collapsed globular structure (hydrophobic state) above the LCST.27,28 Because of this reversible phase transition, P(NIPAAm) has been widely used in the preparation of stimuliresponsive systems for biomedical applications, such as in the controlled release of drugs and in tissue engineering.27–30 PCL and related polymers from ring-opening polymerizations usually possess hydroxyl-terminated chains.31,32 In the present work, the terminal hydroxyl groups of commercial PCL are reacted with 2-bromoisobutyryl bromide29 to produce the 2-bromoisobutyryl-terminated PCL macroinitiators (Br-PCL-Br) * To whom correspondence should be addressed. Tel.: +65-6874-2189. Fax: +65-6779-1936. E-mail: [email protected]. † Department of Chemical and Biomolecular Engineering, National University of Singapore. ‡ Division of Bioengineering, National University of Singapore. § Institute of Materials and Research and Engineering (IMRE).

for the subsequent atom transfer radical polymerization (ATRP; Scheme 1). Triblock copolymers of PCL and P(NIPAAm), or P(NIPAAm)-b-PCL-b-P(NIPAAm), are prepared via ATRP from the Br-PCL-Br macroinitiators. Solutions of P(NIPAAm)b-PCL-b-P(NIPAAm) copolymers can be cast into porous membranes (PCL-PNP membranes) by phase inversion in water. The PCL-PNP membranes with well-defined pores in the micrometer range result from self-assembly of the triblock copolymers. The PCL-PNP membranes consist of biodegradable PCL and nondegradable P(NIPAAm) materials. The nonbiodegradable P(NIPAAm) blocks may enhance the stability of the membranes and reduce their systematic susceptibility to degradative enzymes in practical biomedical applications. The pore size and porosity of the membrane can be regulated by changing the length of the P(NIPAAm) block in the P(NIPAAm)-b-PCLb-P(NIPAAm) copolymers and the temperature of the aqueous medium used for phase inversion. In addition, permeation of glucose (one of the nutrients for cells) through the thermoresponsive porous PCL-PNP membranes is investigated. These stimuli-responsive PCL-PNP membranes have potential applications in biomedical engineering, drug delivery, and tissue engineering.

2. Experimental Section 2.1. Materials. Dihydroxyl-terminated polycaprolactone pellets (PCL, Sigma-Aldrich, cat. 181609: number of average molecular weight (Mn) ) 42500 and polydispersity index (PDI) ) 1.53; from gel permeation chromatography (GPC) measurements: Mn ) 41300; PDI ) 1.54), 2-bromoisobutyryl bromide (98%), N-isopropylacrylamide (NIPAAm, >99%), 1,1,4,7,10,10-hexamethyltriethylenetetramine (HMTETA, 99%), copper(I) bromide (CuBr, 99%) and 1,4-dioxane (>99%) were obtained from Aldrich Chemical Co. of Milwaukee, WI. D-(+)glucose (>99.5%), glucose oxidase/peroxidase (PGO) enzymes, and

10.1021/bm7008922 CCC: $40.75  2008 American Chemical Society Published on Web 12/08/2007

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Scheme 1. Schematic Diagram Illustrating the ATRP of NIPAAm from the Br-PCL-Br Macroinitiator To Produce the P$NIPAAm$-b-PCL-b-P$NIPAAm$ Triblock Copolymer and the Subsequent Preparation of the PCL-PNP Membrane from the Triblock Copolymer by Phase Inversion and Self-Assembly

o-dianisidine dihydrochloride were purchased from Sigma Chemical Co., St. Louis, MO. Purified argon was used in all reactions. 2.2. Synthesis of the P(NIPAAm)-b-PCL-b-P(NIPAAm) Triblock Copolymers via Atom Transfer Radical Polymerization (ATRP). The starting ATRP macroinitiator, 2-bromoisobutyrylterminated PCL (Br-PCL-Br; Scheme 1), was synthesized following the procedures similar to those reported earlier.27 About 4 g of PCL powders and 40 mL of methylene chloride containing 0.6 mol triethylamine were introduced into a 200 mL flask. The reaction flask, equipped with a magnetic stirrer, was kept in an ice bath. After the PCL powders had completely dissolved, 0.3 moles of 2-bromoisobutyryl bromide was added into the flask dropwise through an equalizing funnel. After the addition, the flask was sealed, and the reaction was allowed to proceed at room temperature for 24 h. The resulting Br-PCL-Br macroinitiator was precipitated in excess methanol. Two additional cycles of tetrahydrofuran (THF) dissolution and methanol reprecipitation were performed to remove the reactant residues. Finally, the Br-PCLBr macroinitiator for the subsequent ATRP was dried under reduced pressure. The P(NIPAAm)-b-PCL-b-P(NIPAAm) triblock copolymers were synthesized using a molar feed ratio [NIPAAm (2.2 g)]/[Br-PCL-Br (1.0 g, Mn ) 4.1 × 104 g/mol)]/[CuBr (6.9 mg)]/[HMTETA (28.1 µL)] of 800:1:2:4. The reaction was performed in a 50 mL flask equipped with a magnetic stirrer and under the typical conditions for ATRP.27 NIPAAm, Br-PCL-Br, and HMTETA were introduced into the flask containing 15 mL of dioxane. After Br-PCL-Br and NIPAAm had dissolved completely, the reaction mixture was degassed by bubbling argon through the reaction mixture for 40 min. Then CuBr was added into the mixture under an argon atmosphere. The reaction mixture was purged with argon for another 10 min. The flask was then sealed with a rubber stopper under an argon atmosphere. The polymerization was allowed to proceed under continuous stirring at 60 °C for 2–6 h. The reaction was stopped by diluting with THF. The catalyst complex was removed, bypassing the blue dilute polymer solution through a short aluminum oxide column. A colorless solution was obtained. After removal of THF in a rotary evaporator, the P(NIPAAm)-b-PCL-bP(NIPAAm) triblock copolymers were precipitated in excess methanol. The crude polymer was purified by reprecipitation twice to remove the reactant residues prior to being dried under reduced pressure. The

triblock copolymer yields (and the conversion of NIPAAm) from ATRP time of 2, 4, and 6 h are 1.10 g (4.4%), 1.19 g (8.6%), and 1.25 g (11.4 %), respectively. 2.3. Polymer Characterization. The polymers were characterized by gel permeation chromatography (GPC), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and differential scanning calorimetry (DSC). GPC measurements were performed on a Waters GPC system equipped with a set of Waters Styragel columns, a Waters2487 dual λ absorbance detector, and a Waters-2414 refractive index detector. THF was used as the eluent at a low flow rate of 1.0 mL/ min. Monodispersed polystyrene standards were used to generate the calibration curve. The XPS measurements were performed on a Kratos AXIS HSi spectrometer equipped with a monochromatized Al KR X-ray source (1486.6 eV photons), using the same procedures as those described earlier.27 After the samples were dispersed in KBr pellets, the FTIR spectra were measured on a Bio-Rad FTS 135 FT-IR spectrophotometer. Each spectrum was obtained by cumulating 64 scans. 1H NMR spectra were measured by accumulation of 1000 scans at a relaxation time of 2 s on a Bruker ARX 300 MHz spectrometer, using CCl3D as the solvent. The lower critical solution temperature (LCST) of the P(NIPAAm) blocks in the P(NIPAAm)-b-PCL-bP(NIPAAm) triblock copolymers was determined by DSC (TA 2920 Modulated DSC, TA Instruments). All samples were immersed in deionized water at room temperature for at least 24 h to reach the equilibrium state. The wet samples were placed in individual hermetic sample pans and then sealed. Thermal analyses of the copolymers were performed at a heating rate of 3 °C/min in the temperature range of 5–45 °C under a nitrogen flow rate of 40 mL/min, using deionized water as the reference. 2.4. Preparation of the Membranes. The membranes (denoted as the PCL-PNP membrane shown in Scheme 1) were prepared by phase inversion of the 15 wt% dioxane solution of the P(NIPAAm)-b-PCLb-P(NIPAAm) copolymer in water. The copolymer solution was cast on a glass plate, which was subsequently immersed into a bath of doubly distilled water at a predetermined temperature. Each membrane was left in the water bath for about 20 min. After the membrane had detached from the glass plate, it was extracted in a second water bath

Thermo-Responsive Porous Membranes from Copolymers

Figure 1. Sketch of the temperature-regulated glucose diffusion cell.

at 70 °C for 30 min. Dry membranes with a thickness of about 30–35 µm were obtained after pumping under reduced pressure. 2.5. Membrane Characterization. The surface and internal (crosssectional) morphologies of the membranes were imaged using a JEOL scanning electron microscope (SEM, Model 5600LV). Prior to the SEM measurements, specimens of the membranes and their cross sections were fixed on the metal holders and sputtered with a thin Pt layer. For the temperature-dependent water uptake experiments, the membrane samples were equilibrated in doubly distilled water for 24 h at 20 and 37 °C. The water uptake was calculated from the following formula: water uptake ) (Ww –- Wd)/Wd, where Ww and Wd are the weights of the wet and dried samples, respectively. The thermo-responsive characteristics of the porous membranes were illustrated in the temperature-dependent permeation of glucose. The glucose permeation experiments were carried out in a jacketed twocompartment diffusion cell (10 mL/compartment) shown in Figure 1. In each permeation experiment the donor compartment contained 10 mL of 10 g/L solution of D-(+)-glucose, while the acceptor compartment contained pure water. The membrane was placed between the two compartments. The area of the membrane disk was about 0.8 cm2. All the membrane samples were prewetted for 12 h prior to the diffusion experiments. During the experiments the temperature of the cells and the glucose solutions was controlled by the thermostatted water flowing through the jacket of each compartment. Due to the concentration gradient, glucose diffused through the membrane from the donor to the acceptor compartment. The diffusion was studied both at 20 and 37 °C. Vigorous stirring at 700 rpm on both sides of the membrane was carried out to eliminate the thermo-convective effect on permeation.24 At fixed time intervals, the glucose concentration in the acceptor compartment was determined using the glucose oxidase/peroxidase (PGO) enzymatic assay.33 Glucose in the solution reacted with the enzyme to produce an orange-colored solution. The absorbance of the colored solution was measured at the wavelength of 450 nm. The glucose concentration was then calculated with reference to the standard glucose solutions.

3. Results and Discussion The thermo-responsive PCL-PNP membranes were prepared according to the reaction sequence shown in Scheme 1: (i) the Br-PCL-Br macroinitiator for ATRP was prepared from commercial PCL by reaction of the hydroxyl end groups with 2-bromoisobutyryl bromide, (ii) the P(NIPAAm)-b-PCL-bP(NIPAAm) was synthesized via ATRP of NIPAAm from the Br-PCL-Br macroinitiator, and (iii) the porous PCL-PNP membrane was obtained by phase inversion in water of the dioxane solution of the triblock copolymer. Details of each reaction and material characterization are discussed below. 3.1. Synthesis of the P(NIPAAm)-b-PCL-b-P(NIPAAm) Triblock Copolymers via ATRP. The starting Br-PCL-Br macroinitiator for ATRP was prepared via reaction of the terminal hydroxyl groups of PCL with 2-bromoisobutyrl bro-

Biomacromolecules, Vol. 9, No. 1, 2008 333

mide. The GPC results show that Mn and PDI of the dibromoterminated PCL, or Br-PCL-Br, are about 4.1 × 104 g/mol and 1.54, respectively. These values are comparable to those of the pristine PCL. The P(NIPAAm)-b-PCL-b-P(NIPAAm) triblock copolymers were subsequently synthesized in dioxane at 60 °C via ATRP of NIPAAm from the Br-PCL-Br macroinitiator units. Triblock copolymers with different contents of the P(NIPAAm) block were synthesized by varying the ATRP time. Table 1 summarizes the GPC results of the copolymers as a function of polymerization time. With the increase in reaction time from 2 to 6 h, the Mn of the copolymer increases from 4.5 × 104 to 5.1 × 104 g/mol and the P(NIPAAm) content increases accordingly from 9.0 to 19.7 mol%. In addition, the PDIs of the triblock copolymers are comparable to that of the starting Br-PCL-Br, indicating that the ATRP of NIPAAm from BrPCL-Br is controlled. 3.2. Polymer Characterization. 3.2.1. XPS Analysis. The chemical composition of the polymers was determined by XPS. Figure 2 shows the wide scan and C 1s core-level spectra of the (a,b) Br-PCL-Br, (c,d) P(NIPAAm)-b-PCL-b-P(NIPAAm)1 (from 2 h of ATRP), (e,f) P(NIPAAm)-b-PCL-b-P(NIPAAm)2 (from 4 h of ATRP), and (g,h) P(NIPAAm)-b-PCL-bP(NIPAAm)3 (from 6 h of ATRP) polymers. A weak Br 3d signal at the binding energy (BE) of about 70 eV, characteristic of covalently bonded bromine,34 has appeared in the wide scan spectrum of the Br-PCL-Br macroinitiator. The corresponding Br 3d core-level spectrum is shown in Figure 2a′. The weak Br signal is consistent with the nature of the bromo groups (as terminal groups) in the Br-PCL-Br macroinitiator with a Mn of 4.1 × 104 g/mol. The C 1s core-level spectrum of the Br-PCLBr macroinitiator (Figure 2b) is similar to that of the pristine PCL (Figure 2b′) and can be curve-fitted into three peak components with BEs at about 284.6, 286.2, and 288.7 eV, attributable to the C–H, C–O (and C–Br), and OdC–O species, respectively.34 In comparison with the wide scan spectrum of the Br-PCL-Br macroinitiator (Figure 2a), a relatively strong N 1s signal at the BE of about 399 eV34 has appeared in the wide scan spectra of the P(NIPAAm)-b-PCL-b-P(NIPAAm)1, P(NIPAAm)-b-PCL-b-P(NIPAAm)2, and P(NIPAAm)-b-PCLb-P(NIPAAm)3 triblock copolymers, while the intensity of the O 1s signal at the BE of about 530 eV34 has decreased significantly. The corresponding C 1s core-level spectra of the copolymers can be curve-fitted into five peak components with BEs at about 284.6, 285.7, 286.2, 287.4, and 288.7 eV, attributable to the C–H, C–N, C–O, OdC–N, and OdC–O species, respectively.34 The C–N and OdC–N species are associated with the P(NIPAAm) blocks. The increase in intensities of the C–N and OdC–N species with ATRP time is consistent with the increase in P(NIPAAm) content in the P(NIPAAm)-b-PCL-b-P(NIPAAm) copolymer. From the XPSderived [N]/[C] ratio, the P(NIPAAm) content in each block polymer can also be estimated. It is in fairly good agreement with that obtained from the GPC results (Table 1). 3.2.2. FTIR Spectroscopy Studies. The chemical structure of the polymers was also verified by FTIR spectroscopy. Figure 3 shows the FTIR spectra of the (a) Br-PCL-Br, (b) P(NIPAAm)b-PCL-b-P(NIPAAm)1, (c) P(NIPAAm)-b-PCL-b-P(NIPAAm)2, and (d) P(NIPAAm)-b-PCL-b-P(NIPAAm)3 polymers. In all the FTIR spectra, the characteristic band at about 1736 cm-1 (peak 1, υOdC–O)27 is associated with PCL. Its relative intensity decreases with the increase in P(NIPAAm) content in the P(NIPAAm)-b-PCL-b-P(NIPAAm) triblock copolymers. The typical amide absorption bands of P(NIPAAm) at about 1650 (υOdC–NH, peak 2) and 1540 cm-1 (υN–H, peak 3)27 are observed


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Table 1. Characterization of the P(NIPAAm)-b-PCL-b-P(NIPAAm) Copolymers and the Resulting Porous Membranes membranes cast at different temperatureh 20 °C

sample PCL (or Br-PCL-Br) P(NIPAAm)-b-PCL-b-P (NIPAAm)1a P(NIPAAm)-b-PCL-b-P (NIPAAm)2a P(NIPAAm)-b-PCL-b-P (NIPAAm)3a

30 °C

40 °C

reaction Mn time (h) (g/mol)b PDIb [N]/[C]c

P(NIPAAm) content (mol%)d

2

4.1 × 104 1.54 4.5 × 104 1.49

0.014

9.0e 8.4f 7.3g

28

PCL PCL-PNP1

0.8

31

0.7

26

0.5

15

4

4.8 × 104 1.51

0.022 14.7e 13.2f 12.4g

51

PCL-PNP2

1.3

56

1.1

51

0.6

19

6

5.1 × 10 1.47

e

78

PCL-PNP3

2.7

76

1.8

62

1.0

34

4

f

0.031 19.7 18.5 17.8

degree of mean polymerization diameter porosity Dm porosity Dm porosity (DP)g sample (Dm, µm)j (%)k (µm)j (%)k (µm)j (%)k

g

a Synthesized using a molar feed ratio [monomer (2.2 g)]/[Br-PCL-Br (1.0 g)]/[CuBr (6.9 mg)]/[HMTETA (28.1µL] of 800:1:2:4 at 60 °C in 15 mL of dioxane. b Determined from GPC results. PDI ) weight average molecular weight/number average molecular weight or Mw/Mn. c Determined from XPS N 1s and C 1s core-level spectral area ratio. d Calculated from n[NIPAAm]/(n[NIPAAm] + n[CL]). e Determined from Mn and the molecular weights of NIPAAm (M[NIPAAm] ) µg/mol) and CL (M[CL] ) 114 g/mol), where n[NIPAAm] ) (Mn[P(NIPAAm)-b-PCL-b-P(NIPAAm)]-Mn[PCL])/MNIPAAm and n[CL] ) Mn[PCL]/M[CL]. f Determined from [N]/[C] ratio, where [N]/[C] ≈ n[NIPAAm]/(6n[NIPAAm] + 6n[CL]). g Determined from 1H NMR data. h Cast in doubly distilled water of a prescribed temperature from 15 wt% dioxane solutions of the respective P(NIPAAm)-b-PCL-b-P(NIPAAm) block copolymers. j 100 D )⁄n k Estimated using the Determined from 100 pores (n ) 100) of diameters Di in the SEM images by using the relationship Dm ) (∑ i)1 i relationship porosity(%) ) (bulk volume of membrane (Vb, m3) – skeletal volume of membrane (Vs, m3))/Vb, where Vb ) width × length × thickness and Vs ) weight of membrane (g)/mass density (g/m3). The thickness of the membrane was estimated from the SEM cross-sectional image, and the mass density of all the polymers was assumed to be 1.0 g/cm3.

Figure 3. FTIR spectra of the (a) Br-PCL-Br macroinitiator, and the (b) P(NIPAAm)-b-PCL-b-P(NIPAAm)1, (c) P(NIPAAm)-b-PCL-bP(NIPAAm)2, and (d) P(NIPAAm)-b-PCL-b-P(NIPAAm)3 triblock copolymers (peak 1, 1736 cm-1 (υOdC–O); peak 2, 1650 cm-1 (υOdC–NH); and peak 3, 1540 cm-1 (υN–H)).

Figure 2. XPS wide scan and C 1s core-level spectra of the (a,b) Br-PCL-Br macroinitiator and the (c,d) P(NIPAAm)-b-PCL-bP(NIPAAm)1,(e,f)P(NIPAAm)-b-PCL-b-P(NIPAAm)2,and(g,h)P(NIPAAm)b-PCL-b-P(NIPAAm)3 triblock copolymers. Insets (a′,b′) are the Br 3d core-level spectrum of the Br-PCL-Br macroinitiator and the C 1s core-level spectrum of pristine PCL.

only in the FTIR spectra of the P(NIPAAm)-b-PCL-bP(NIPAAm) triblock copolymers. The relative intensities of both

peaks 2 and 3 increase significantly with the increase in P(NIPAAm) content in the triblock copolymers. The above FTIR results are thus consistent with the XPS results of Figure 2. 3.2.3. 1H NMR Analysis. The chemical structures of the BrPCL-Br macroinitiator and the P(NIPAAm)-b-PCL-bP(NIPAAm) triblock copolymer were characterized by 1H NMR spectroscopy. Figure 4a shows the 1H NMR spectrum of the Br-PCL-Br macroinitiator. The chemical shifts at δ ) 1.3–1.8 ppm are attributable to the inner methylene protons (a, CH2–CH2). The chemical shift at δ ) 1.92 ppm is associated with the methyl protons (b, C(Br)-CH3) of the 2-bromoisobutyryl groups.20 The chemical shifts in the region of 2.2–2.4 ppm are associated with the methylene protons adjacent to the carbonyl group (c, CH2–CdO). The chemical shifts at δ ) 4.0–4.25 ppm correspond to the methylene protons adjacent to the oxygen moieties of the ester linkages (d,d′, CH2–O–CdO). From the area ratio of peak b and peak c, the extent of halogenation in the Br-PCL-Br macroinitiator is determined to be about 85%. Figure 4b shows the 1H NMR spectrum of the P(NIPAAm)-bPCL-b-P(NIPAAm)1 copolymer. In comparison with the spectrum of the Br-PCL-Br macroinitiator in Figure 4a, the new chemical shifts at δ ) 1.13 ppm are mainly associated with the methyl protons (d, CH(NH)–CH3) of the P(NIPAAm) blocks. The chemical shifts in the region of 1.3–1.8 ppm are attributable to the methylene protons (a, CH2–CH2 and a′, CH–CH2). The chemical shifts at δ ) 2.2–2.4 ppm are associated with the methylene (c, CH2–CdO) and methylidyne (c′, CH–CdO)



Figure 5. Typical DSC thermograms of the (a) Br-PCL-Br macroinitiator and the (b) P(NIPAAm)-b-PCL-b-P(NIPAAm)1, (c) P(NIPAAm)b-PCL-b-P(NIPAAm)2, and (d) P(NIPAAm)-b-PCL-b-P(NIPAAm)3 triblock copolymers.

Figure 4. 1H NMR spectra (300 MHz) of the (a) Br-PCL-Br macroinitiator and (b) P(NIPAAm)-b-PCL-b-P(NIPAAm)1 triblock copolymer in CCl3D.

protons adjacent to the carbonyl group. The chemical shifts at δ ) 4.0–4.25 ppm correspond to the methylene protons (d,d’, CH2–O–CdO) and the methylidyne protons adjacent to the amine moiety (d”, CH–NH).20 The composition of the block polymers was also calculated from the area ratio of peaks e and c and c′ and is summarized in Table 1. The results are in fairly good agreement with those obtained from XPS and GPC. 3.2.4. Lower Critical Solution Temperature (LCST). The LCST of thermo-responsive P(NIPAAm) is the consequence of hydrophobic (associated with the isopropyl groups) and hydrophilic (associated with the amide moiety in the pendent groups) interactions of P(NIPAAm) in aqueous enviroment.27 The LCSTs can be determined by DSC. Figure 5 shows the DSC thermograms of (a) PCL, (b) P(NIPAAm)-b-PCL-bP(NIPAAm)1, (c) P(NIPAAm)-b-PCL-b- P(NIPAAm)2, and (d) P(NIPAAm)-b-PCL-b-P(NIPAAm)3. The temperature at the minimum point of the endotherm is referred to as the LCST of each sample.27 No LCST is observed for PCL, as expected. On the other hand, the P(NIPAAm)-b-PCL-b-P(NIPAAm) triblock polymers exhibit LCSTs at about 31 °C (near that of the P(NIPAAm) homopolymer at about 32 °C),27 indicating that

the PCL segments do not have a significant effect on the LCST of the P(NIPAAm) blocks in an aqueous medium. Earlier studies had indicated that incorporation of hydrophobic moieties in the bulk may decrease the LCST of P(NIPAAm).27,35 When a hydrophobic component was incorporated into P(NIPAAm), the hydrophilic–hydrophobic balance will shift toward a more hydrophobic nature and the LCST will shift to a lower temperature. Below the LCST, the P(NIPAAm) blocks in the P(NIPAAm)-b-PCL-b-P(NIPAAm) triblock copolymers are solvated and remain fully extended as a hydrophilic phase in an aqueous medium, while the PLC blocks (with a molar content >80%, Table 1) associate hydrophobically and precipitate out from the aqueous medium. The strong repulsion arising from incompatability of the P(NIPAAm) blocks with the PCL blocks will lead to phase separation in an aqueous medium. The phase separation probably has limited the interaction of the hydrophobic PCL blocks with the P(NIPAAm) blocks. Thus, the LCST of the P(NIPAAm) blocks is not significantly affected by the hydrophobic PCL blocks 3.3. Membranes from P(NIPAAm)-b-PCL-b-P(NIPAAm) Copolymers (PCL-PNP Membranes). 3.3.1. Morphology of the PCL-PNP Membranes. The P(NIPAAm)-b-PCL-bP(NIPAAm) triblock copolymers were cast into the respective membranes (PCL-PNP membranes) from a 15 wt% dioxane solution of each copolymer by phase inversion in water at a predetermined temperature. Figure 6 shows the scanning electron microscope (SEM) images of top and cross-sectional views of the (a) PCL, (b) PCL-PNP1 (from the P(NIPAAm)-b-PCL-bP(NIPAAm)1 copolymer), (c) PCL-PNP2 (from the P(NIPAAm)b-PCL-b-P(NIPAAm)2 copolymer), and (d) PCL-PNP3 (from the P(NIPAAm)-b-PCL-b-P(NIPAAm)3 copolymer) membranes cast in 20 °C water. A microporous morphology is not discernible in the PCL membrane, while well-defined pores in the micrometer range were observed in the PCL-PNP membranes. The plausible process of formation of uniform and welldefined pores from the triblock copolymer is shown in Scheme 1. In dioxane, the triblock copolymer molecules remain well extended due to good solubility of both the PCL and P(NIPAAm) blocks. During the process of phase inversion in the 20 °C aqueous medium, the PCL blocks associate hydrophobically and precipitate out from the aqueous medium, while the hydrophilic P(NIPAAm) blocks remain fully extended in the medium. The strong repulsion arising from incompatability of the PCL blocks with the P(NIPAAm) blocks forces the triblock copolymers to undergo partial phase separation in the aqueous medium. Due to the persistence of good solubility of the P(NIPAAm) blocks in the medium, a large amount of water is trapped within the hydrodynamic volume of the self-assembled P(NIPAAm) chains in the reverse micells forming the initial membrane matrix. After drying, the hydrophilic P(NIPAAm) blocks collapse on the


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Figure 6. SEM images of the top view and cross-sectional view of the (a,a′) PCL, (b,b′) PCL-PNP1, (c,c′) PCL-PNP2, and (d,d′) PCLPNP3 membranes cast in a 20 °C water bath.

Figure 7. SEM images of the top view of the respective (a,a′) PCL, (b,b′) PCL-PNP1, (c,c′) PCL-PNP2, and (d,d′) PCL-PNP3 membranes cast in a 30 and 40 °C water bath.

hydrophobic PCL surfaces to leave behind well-structured or well-defined pores. The pore size of the membrane is determined by the size of the micelles and, thus, by the lengths of the amphiphilic blocks. Figure 6 also shows the corresponding crosssectional SEM images of the (a′) PCL, (b′) PCL-PNP1, (c′) PCLPNP2, and (d) PCL-PNP3 membranes. The interconnected and spherical pores in the micrometer range in the bulk of the membranes are similar to those on the surface, indicating the presence of a well-defined porous structure throughout the membranes and consistent with their origin from the reverse micelles. With the increase in P(NIPAAm) content of the P(NIPAAm)b-PCL-b- P(NIPAAm) copolymers from 9.0 to 19.7 mol% (Table 1, Figure 6), the mean pore diameter (Dm) of the corresponding PCL-PNP membranes cast in the 20 °C aqueous medium increases from 0.8 to 2.7 µm. The porosity increases correspondingly from 31 to 76%. The increase in pore size and porosity with the increase in P(NIPAAm) content is consistent with the reverse micelle formation during membrane casting by phase inversion. With longer hydrophilic blocks, the increase in hydrodynamic core radii of the reverse micelle will lead to the formation of larger reverse micelles and bigger pores. As shown in Figure 5, the P(NIPAAm) blocks in the P(NIPAAm)-b-PCL-b- P(NIPAAm) copolymers possess thermoresponsive characteristics. Thus, the temperature of the aqueous

media used for membrane casting will also affect the pore size of the PCL-PNP membranes. Figure 7 shows the SEM images of the (a,a′) PCL, (b,b′) PCL-PNP1, (c,c′) PCL-PNP2, and (d,d′) PCL-PNP3 membranes cast in the respective 30 and 40 °C water baths. As expected, no pores were observed for the PCL membrane. With the increase in casting temperature, the Dm of well-defined and uniformly distributed pores in the PCL-PNP membranes decreases gradually, in comparison with those of the corresponding PCL-PNP membranes cast at 20 °C (Figure 6). The variations in Dm and porosity are summarized in Table 1. The Dm values (or porosities) for the PCL-PNP membranes cast at 40 °C have decreased to about 0.5 (or 15%; for the PCLPNP1 membrane), 0.6 (or 19%; for the PCL-PNP2 membrane), and 1.0 µm (or 34%; for the PCL-PNP3 membrane) from the respective values of 0.8 (or 31%), 1.3 (or 56%), and 2.7 µm (or 76%) for the PCL-PNP membranes cast at 20 °C. The decrease in pore size and porosity with the increase in casting temperature is consistent with the hydrophilic to hydrophobic transition of the P(NIPAAm) blocks above the LCSTs. As mentioned earlier, the P(NIPAAm) blocks in the P(NIPAAm)b-PCL-b-P(NIPAAm) polymers exhibit LCSTs at about 31 °C in water (Figure 5). At the casting temperature (for example, 20 °C) below the LCSTs, the P(NIPAAm) blocks assume a random coil structure (hydrophilic state)36–39 and remain fully extended in the aqueous medium, giving rise to larger pore sizes, as mentioned earlier (Figure 6). However, with the increase in



Figure 9. Water uptake by the (a) PCL (cast at 20 °C), and the (b) PCL-PNP1 (cast at 20 °C), (c) PCL-PNP2 (cast at 20 °C), (c′) PCLPNP2 (cast at 40 °C), and (d) PCL-PNP3 (cast at 20 °C) membranes after immersion in doubly distilled water at 20 and 37 °C for 24 h (Ww and Wd are the weights of the wet and dried membranes, respectively).

Figure 8. XPS wide scan spectra of the respective (a,a′) PCL-PNP1, (b,b′) PCL-PNP2, and (c,c′) PCL-PNP3 membranes cast in 20 and 40 °C water baths.

casting temperature from 30 to 40 °C, the P(NIPAAm) blocks assume a collapsed globular structure (hydrophobic state) above the LCSTs. The transitions of the P(NIPAAm) blocks from the hydrophilic to the hydrophobic state will reduce their interaction with the aqueous environment and give rise to smaller hydrodynamic radii of the reverse micelles, leading to a reduction in pore size and porosity of the resulting PCL-PNP membranes. 3.3.2. Surface Composition Analysis. The surface compositions of the PCL-PNP membranes are determined from the XPSderived [N]/[C] ratios. Figure 8 shows the XPS wide scan spectra and corresponding [N]/[C] ratios of the (a,a′) PCL-PNP1, (b,b′) PCL-PNP2, and (c,c′) PCL-PNP3 membranes cast in the respective 20 and 40 °C water baths. Comparison of the surface [N]/[C] ratio of the PCL-PNP membrane with the corresponding bulk [N]/[C] ratio of the P(NIPAAm)-b-PCL-b-P(NIPAAm) triblock copolymer (Figure 2) reveals a higher surface [N]/[C] ratio. The phenomenon is consistent with the enrichment of the P(NIPAAm) block at the outermost surface during the course of membrane formation by phase inversion in an aqueous environment. The lack of miscibility between the PCL and P(NIPAAm) blocks probably plays a significant role in the surface enrichment of the P(NIPAAm), especially in the presence of the strong interaction of the hydrophilic P(NIPAAm) blocks with the aqueous medium. With the increase in casting temperature, the surface [N]/[C] ratios decreases (Figure 8). When the membrane is cast below the LCST, the P(NIPAAm) blocks are hydrated and assume a highly extended conformation to distribute more favorably than the hydrophobic PCL blocks on the membrane and pore surfaces. With the increase in casting temperature from 20 to 40 °C, or above the LCST, the transition

from the hydrophilic to the hydrophobic state of the P(NIPAAm) blocks gives rise to a lower concentration of P(NIPAAm) blocks on the membrane and pore surfaces, leading to a decrease in surface [N]/[C] ratio of the PCL-PNP membrane. 3.3.3. Water Uptake by the Membranes. Figure 9 shows the typical water uptake by the (a) PCL (cast at 20 °C), (b) PCLPNP1 (cast at 20 °C), (c) PCL-PNP2 (cast at 20 °C), (c′) PCLPNP2 (cast at 40 °C), and (d) PCL-PNP3 (cast at 20 °C) membranes. The water uptake was affected by the P(NIPAAm) content, porosity, and casting temperature. Compared to the PCL membrane (cast at 20 °C), the PCL-PNP membranes (cast at 20 °C) exhibit higher degree of water uptakes. The water uptake increases with increasing P(NIPAAm) content, consistent with the presence of more hydrophilic moieties (associated with P(NIPAAm)) and the correspondingly larger porous structures (Figure 6). The degree of water uptake by each PCL-PNP membrane (cast at 20 °C) is larger at 20 °C (below the LCST) than at 37 °C (above the LCST). During the phase inversion process at 20 °C, the membrane and pore surfaces are enriched with the P(NIPAAm) blocks, as mentioned earlier. At temperatures below the LCST, the hydrophilic groups (CONH) of P(NIPAAm) form hydrogen bonds with water molecules. These bonds act cooperatively to form a stable shell of hydration around the hydrophobic groups (CH(CH3)2), resulting in a greater extent of water uptake. On the other hand, as the external temperature increases above the LCST, the P(NIPAAm) blocks shrink and associate hydrophobically. The extent of hydrogen bonding interaction is reduced, and the associative interaction among the hydrophobic groups grows stronger, resulting in reduced water uptake. In addition, in comparison with the PCLPNP2 membrane cast at 20 °C, the PCL-PNP2 membrane cast at 40 °C (above the LCST) exhibits substantially lower water uptake and thermo-responsiveness, as expected, probably because of the lower porosity and the lower concentration of the P(NIPAAm) blocks on the membrane and pore surfaces. 3.3.4. Thermo-ResponsiVe Transport of Nutrient through the Membranes. The thermo-responsive characteristics of the porous PCL-PNP membranes are illustrated in the temperaturedependent transport of glucose (one of the nutrients for cells)13 through the membrane in a two-compartment liquid cell (Figure 1). Our earlier study on the commercial hydrophilic poly(vinylidene fluoride) membrane (d ) 0.65 µm, Millipore Corp.) in the same liquid cell has confirmed that the thermo-convective effect on permeation is negligible under the vigorously stirred condition.24 Thus, vigorous stirring of each cell compartment on both sides of the membrane was carried out to eliminate the thermo-convective effect on permeation. Figure 10 shows the


Figure 10. Typical glucose diffusion behavior through the (a) PCLPNP1 (cast at 20 °C) and (b) PCL-PNP3 (cast at 20 °C) membranes as a function of time at 20 and 37 °C. The ratios (Dr) of relative diffusivity at 37 and 20 °C for the PCL-PNP1 (cast at 20 °C) and PCL-PNP3 (cast at 20 °C) membranes are 1.44 and 1.56, respectively. Dr was estimated from the equation derived from Fick’s first law of diffusion:24,40 Dr ) D37°C/D20°C ) ln[(C1)0/((C1)t – (C2)t)]37°C/ ln[(C1)0/((C1)t – (C2)t)]20°C, where (C1)0 and (C1)t are the initial and intermediate glucose concentrations, respectively, at time t in the donor compartment at 37 °C (or 20 °C) and (C2)t) is the intermediate glucose concentration at time t in the acceptor compartment at 37 °C (or 20 °C).

time-dependent glucose diffusion through the (a) PCL-PNP1 (cast at 20 °C) and (b) PCL-PNP3 (cast at 20 °C) membranes at 20 and 37 °C. During the 24 h period of permeation study, the glucose transport through the membranes exhibits a rather linear dependence on time. The permeation rate of glucose through the PCL-PNP3 membrane (cast at 20 °C) is higher than that through the PCL-PNP1 membrane (cast at 20 °C). The observation is consistent with the difference in pore morphology between the two membranes (Figure 6). The larger pore size and higher porosity of the PCL-PNP3 membrane (cast at 20 °C) facilitate the P(NIPAAm) brushes-regulated diffusion of the glucose molecules across the membrane into the acceptor compartment. On the other hand, both the PCL-PNP1 and PCLPNP3 membranes (cast at 20 °C) exhibit higher permeation rates for glucose at 37 °C than at 20 °C. Because the thermally induced convective effect has been eliminated by vigorous stirring, the observed phenomenon can be attributed to the change in the effective dimension of the pores of the membrane, caused by the hydrophilic-hydrophobic transition of the P(NIPAAm) blocks (brushes) on the pore surfaces. At 20 °C (below the LCST), the hydrophilic P(NIPAAm) blocks on the pore surfaces are hydrated and fully extended to hinder the glucose diffusion. As the temperature of the medium is increased to 37 °C (above the LCST), the P(NIPAAm) blocks associate hydrophobically on the pore surfaces and all the pores become

Xu et al.

Figure 11. Typical glucose diffusion behavior through the (a) PCLPNP2 (cast at 20 °C) and (b) PCL-PNP2 (cast at 40 °C) membranes as a function of time at 20 and 37 °C. The ratios (Dr) of relative diffusivity at 37 and 20 °C for the PCL-PNP2 (cast at 20 °C) and PCL-PNP2 (cast at 40 °C) membranes are 1.49 and 1.04, respectively.

less hindered to the transport of the nutrient molecules through the membrane. The relative diffusivity ratio of solutes through the membrane, Dr, under two different conditions (37 and 20 °C) can be calculated using the following equation derived from Fick’s first law of diffusion:24,40 Dr ) D37°C/D20°C ) ln[(C1)0/ ((C1)t – (C2)t)]37°C/ln[(C1)0/((C1)t – (C2)t)]20°C, where (C1)0 and (C1)t are the initial and intermediate glucose concentrations at time t in the donor compartment at 37 °C (or 20 °C), and (C2)t) is the intermediate glucose concentration at time t in the acceptor compartment at 37 °C (or 20 °C). The Dr values for the PCLPNP1 and PCL-PNP3 membranes (cast at 20 °C) are 1.44 and 1.56, respectively, indicating that glucose diffusion across the thermo-responsive porous membranes increases with the surrounding temperature. Figure 11 shows the typical results on glucose diffusion at 20 and 37 °C through the (a) PCL-PNP2 (cast at 20 °C) and (b) PCL-PNP2 (cast at 40 °C) membranes. As expected, the glucose transport through the PCL-PNP2 membrane cast at 20 °C is similar to those of the PCL-PNP1 and PCL-PNP3 membranes cast at 20 °C (Figure 10). The Dr for glucose diffusion at 37 and 20 °C through the PCL-PNP2 membrane cast at 20 °C is 1.49. However, the temperature does not have an obvious effect on the nutrient transport through the PCLPNP2 membrane cast at 40 °C. The Dr for glucose diffusion at 37 and 20 °C through the PCL-PNP2 membrane cast at 40 °C is only 1.04, indicating that glucose diffusion across the PCLPNP2 membrane cast at 40 °C is temperature-insensitive. This phenomenon is consistent with the lower temperature-dependent water uptake of the PCL-PNP2 membrane cast at 40 °C (Figure 8). With the increase in casting temperature from 20 to 40 °C


(above the LCST), transition from hydrophilic to hydrophobic state of the P(NIPAAm) blocks gives rise to a lower concentration of P(NIPAAm) blocks on the surface, leading to reduced porosity and lower thermal sensitivity. The low Dr of 1.04 also indicates that the thermo-convective effect on permeation was almost eliminated by vigorous stirring in the present work. The diffusion coefficient of glucose and other compounds in open aqueous solutions is expected to be strongly dependent on temperature. However, the temperature-insensitive Dr for the present PCL-PNP2 membrane cast at 40 °C indicates that the thermo-convective effect on permeation through the membrane, or pore diffusion, is insignificant in the present experimental setup under vigorous stirring and that the P(NIPAAm) components play a direct role in regulating permeation through the pores.

4. Conclusions Thermo-responsive P(NIPAAm)-b-PCL-b-P(NIPAAm) triblock copolymers have been successfully prepared via ATRP from the dibromo-terminated PCL, or Br-PCL-Br, macroinitiator. The P(NIPAAm) blocks in the triblock copolymers exhibit LCSTs at about 31 °C (near that of the P(NIPAAm) homopolymer of about 32 °C) in water. The thermo-responsive triblock copolymers can be cast into microporous PCL-PNP membranes with well-defined and uniformly distributed pores by phase inversion in an aqueous medium. The pore size and porosity of the PCL-PNP membranes can be regulated by controlling the P(NIPAAm) content (block chain length) and the temperature employed for phase inversion. The thermo-responsive characteristics of the PCL-PNP membranes are illustrated by the temperature-sensitive glucose transport across the membranes. The thermo-responsive membranes, obtained via the present approach, have potential applications in biomedical areas, such as drug delivery and tissue engineering.

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BM7008922

Thermo-Responsive Porous Membranes of Controllable Porous

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