Comb-Shaped Conjugates Comprising Hydroxypropyl Cellulose

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Bioconjugate Chem. 2010, 21, 456–464

Comb-Shaped Conjugates Comprising Hydroxypropyl Cellulose Backbones and Low-Molecular-Weight Poly(N-isopropylacryamide) Side Chains for Smart Hydrogels: Synthesis, Characterization, and Biomedical Applications F. J. Xu,*,† Y. Zhu,† F. S. Liu,‡ J. Nie,† J. Ma,§ and W. T. Yang*,† State Key Laboratory of Chemical Resource Engineering, Key Laboratory of Carbon Fiber and Functional Polymers, Ministry of Education, College of Materials Science & Engineering, Beijing University of Chemical Technology, Beijing, China 100029, Brain Tumor Research Center, Beijing Neurosurgical Institute, Capital University of Medical Science, Beijing, China 100050, and State Key Laboratory of Molecular Oncology, Cancer Institute & Hospital, Chinese Academy of Medical Sciences, Beijing, China 100021. Received July 30, 2009; Revised Manuscript Received February 11, 2010

Hydroxypropyl cellulose (HPC) possesses a lower critical solution temperature (LCST) above 40 °C, while the poly(N-isopropylacrylamide) (P(NIPAAm)) exhibits a LCST of about 32 °C. Herein, comb-shaped copolymer conjugates of HPC backbones and low-molecular-weight P(NIPAAm) side chains (HPC-g-P(NIPAAm) or HPN) were prepared via atom transfer radical polymerization (ATRP) from the bromoisobutyryl-functionalized HPC biopolymers. By changing the composition ratio of HPC and P(NIPAAm), the LCSTs of HPNs can be adjusted to be lower than the body temperature. The MTT assay from the HEK293 cell line indicated that HPNs possess reduced cytotoxicity. Some of the hydroxyl groups of HPNs were used as cross-linking sites for the preparation of stable HPN hydrogels. In comparison with the HPC hydrogels, the cross-linked HPN hydrogels possess interconnected pore structures and higher swelling ratios. The in Vitro release kinetics of fluorescein isothiocyanatelabeled dextran and BSA (or dextran-FITC and BSA-FITC) as model drugs from the hydrogels showed that the HPN hydrogels are suitable for long-term sustained release of macromolecular drugs at body temperature.

INTRODUCTION Cross-linked hydrogels are of interest in biomedical applications, because of their tunable chemical and three-dimensional physical network structures, high water content in an aqueous medium without dissolution, and good mechanical properties (1-3). Intelligent or smart hydrogels, which can undergo volume changes in response to changes in temperature, pH, and antigen, are of great interest in drug delivery, cell encapsulation, and tissue engineering (4-6). Among the temperature-sensitive hydrogels, poly(N-isopropylacrylamide) (P(NIPAAm))-based hydrogels are the most widely studied. P(NIPAAm) is a wellknown thermoresponsive polymer and exhibits a lower critical solution temperature (LCST) of about 32 °C in an aqueous medium. P(NIPAAm)-based hydrogels absorb water and exist in swollen states below the LCST. On the other hand, they undergo an abrupt and drastic shrinkage in volume as the medium temperature is raised above the LCST. These characteristics make P(NIPAAm)-based hydrogels specially useful in biomedical applications, such as in controlled release of drugs and in tissue engineering (7-12). Natural polysaccharides are very suitable candidates for the design of novel biomaterials, because they are renewable and nontoxic materials. It has been reported that P(NIPAAm) can be grafted to polysaccharide biopolymers, including chitosan (13, 14), dextran (3, 15), and methylcellulose (16), for the preparation of thermosensitive biomaterials. Hydroxypropyl cellulose (HPC) is a derivative of natural polysaccharide cellulose, where some of the hydroxyl groups of cellulose have

been hydroxypropylated to form hydrophobic propylene oxide groups (1, 17). HPC materials have been approved by the USFDA and widely used in food and drug formulations (1). The hydrohphobic and hydrophilic groups impart HPC with a LCST of >40 °C (1, 17, 18). However, such LCST is far higher than the body temperature of 37 °C. In the present work, the body temperature-responsive combshaped copolymer conjugates (HPC-g-P(NIPAAm) or HPN) composed of HPC backbones and low-molecular-weight P(NIPAAm) side chains are prepared via atom transfer radical polymerization (ATRP) (19-21) from the bromoisobutyrylfunctionalized HPC (HPC-Br) backbones (Scheme 1). Some of the unreacted hydroxyl groups of HPNs are used as cross-linking sites for the preparation of stable HPN hydrogels. The resultant cross-linked HPN hydrogels with controllable LCSTs possess interconnected pore structures. In this work, the in vitro controlled release properties of the smart hydrogels were studied by using fluorescein isothiocyanate-labeled dextran and BSA (or dextran-FITC and BSA-FITC) as model macromolecular hydrophilic drugs. Dextran and BSA labeled or not labeled with FITC have been widely used as models for macromolecular drugs, such as proteins and peptides, in the studies of in vitro release kinetics of drug delivery systems (22-25). In comparison with HPC hydrogels, the HPN hydrogels can provide the longterm sustained release of macromolecular drugs at body temperature. Such porous HPN hydrogels with body-temperature responsive properties will have potential applications as biomedical materials.

EXPERIMENTAL PROCEDURES * To whom all correspondence should be addressed. E-mail: [email protected], [email protected]. † Beijing University of Chemical Technology. ‡ Capital University of Medical Science. § Chinese Academy of Medical Sciences.

Materials. Hydroxypropyl cellulose powder (HPC, SigmaAldrich cat #435007: Mn ) 10 000 g/mol and Mw ) 80 000 g/mol; from gel permeation chromatography (GPC) with THF as mobile phase: Mn ) 28 015 g/mol and Mw ) 64 655 g/mol;

10.1021/bc900337p  2010 American Chemical Society Published on Web 02/23/2010

Comb-Shaped Conjugates

Bioconjugate Chem., Vol. 21, No. 3, 2010 457

Scheme 1. Schematic Diagram Illustrating the Processes for the Preparation of the HPC-g-P(NIPAAm) (HPN) Copolymers via ATRP of NIPAAm from the Alkyl Bromide-Functionalized HPC Macroinitiator and the Formation of Stimuli-Responsive HPN Hydrogel via Cross-Linking

molar substitution of propylene oxide groups (MS) of about 3 per glucose unit (19), 2-bromoisobutyryl bromide (BIBB, 98%), N-isopropylacrylamide (NIPAAm, >99%), poly(N-isopropylacrylamide) (P(NIPAAm), Mn ) 20 000-25 000), 1,1,4,7,10,10hexamethyltriethyenetetramine (HMTETA, 99%), copper(I) bromide (CuBr, 99%), and divinyl sulfone (DVS, >98%), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), penicillin, streptomycin, fluorescein isothiocynate (FITC)conjugated BSA (BSA-FITC), and FITC-conjugated dextran (dextran-FITC, Mw ∼40 000) were obtained from Sigma-Aldrich Chemical Co., St. Louis, MO. The HEK293 cell lines were purchased from the American Type Culture Collection (ATCC, Rockville, MD). Dulbecco’s phosphate buffered saline (PBS, pH 7.4), used for the drug delivery experiments, was prepared freshly according to the recipes. Synthesis of HPC-g-P(NIPAAm) Comb-Shaped Copolymers (HPNs) via Atom Transfer Radical Polymerization (ATRP). The starting bromoisobutyryl-functionalized HPC (HPC-Br) was synthesized according to the following procedures. About 2.0 g of HPC was dissolved completely in 100 mL of anhydrous methylene chloride with stirring and then kept in an ice bath. About 0.3 mL of BIBB in 5 mL of methylene chloride was added dropwise into the flask through an equalizing funnel over a period of 10 min at 0 °C. After this addition, the flask was sealed. The reaction was allowed to proceed at room temperature for another 5 h to produce the HPC-Br macroinitiator. The final reaction mixture was precipitated with 1000 mL of diethyl ether. The crude polymer was purified by

reprecipitation thrice in diethyl ether. Finally, the HPC-Br macroinitiator for the subsequent ATRP was dried under reduced pressure. The HPC-g-P(NIPAAm) comb-like copolymers (HPNs, Scheme 1) were synthesized using a [monomer (2.0 g)]:[CuBr (25.4 mg)]:[HMTETA (77.6 µL)] molar feed ratio of 100:1:1.5 in 15 mL of dioxane containing 0.4 g of HPC-Br at 60 °C. The reaction was performed in a 25 mL flask equipped with a magnetic stirrer and under the typical conditions for ATRP (20). NIPAAm, HPC-Br, and HMTETA were introduced into the flask containing 10 mL of dioxane. After HPC-Br and NIPAAm had dissolved completely, the reaction mixture was degassed by bubbling argon for 30 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 room temperature for 8 to 24 h. The reaction was stopped by exposing to air. The reaction mixture was diluted with about 150 mL of THF. The catalyst complex was removed by passing the blue dilute polymer solution through a short aluminum oxide column where neutral aluminum oxide 90 (Activity I, 0.0630.200 mm, pH 7.3, Merck & Co., Inc.) was used. A colorless solution was obtained. After removal of THF in a rotary evaporator, the HPNs were precipitated in excess n-hexane. The crude polymer was purified by reprecipitation cycles to remove the reactant residues (21), prior to being dried under reduced pressure.

458 Bioconjugate Chem., Vol. 21, No. 3, 2010

Preparation of HPN Hydrogels. The temperature-responsive hydrogels were prepared from the HPN comb-shaped copolymers. Some of the unreacted hydroxyl groups of HPNs were cross-linked using the similar procedures reported earlier (1, 17). A homogeneous polymer solution was obtained by adding 1.0 g of the HPN to 10 mL of deionized water. The solution was stirred slowly overnight to ensure uniform mixing while avoiding bubble formation. Then, 40 wt % NaOH was used to adjust the pH of solution to about 13. Subsequently, about 0.1 mL of divinylsulfone (DVS) was added to the copolymer solution. The mixture was stirred for 2 min at room temperature. The mixture was then centrifuged to remove the air bubbles trapped during the stirring process. The air bubble-free sticky solution was kept at 30 °C for 12 h. After the cross-linking reaction, the sample was removed and immersed into distilled water (about 500 mL) at room temperature for 72 h to remove the reactant residues. The water was changed at a 6 h interval. The cross-linked HPN hydrogel was finally freeze-dried. Polymer Characterization and Cytotoxicity Assay. The temperature-responsive 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 Waters Styragel columns, a Waters-2487 dual wavelength (λ) UV detector, and a Waters-2414 refractive index detector. THF with 2 vol % triethylamine 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 (20). After the samples were pressed into KBr pellets, the FTIR spectra were measured on a Bio-Rad FTS 135 FT-IR spectrophotometer. Each spectrum was collected by cumulating 64 scans. 1H NMR spectra were measured on a Bruker ARX 300 MHz spectrometer, using d-chloroform (CCl3D) as the solvent with 1000 scans at a relaxation time of 2 s. The LCST of the HPN samples was determined by DSC (TA 2920 modulated DSC, TA Instruments). All samples were immersed in deionized water at room temperature for 24 h to reach the equilibrium state. These wetted samples were placed in individual hermetic sample pans and then sealed. The thermal analysis of the swollen samples was performed at a heating rate of 3 °C/min in the temperature range 10-60 °C under a nitrogen flow rate of 40 mL/min, using deionized water as the reference. The cytotoxicity of the HPNs was evaluated using the MTT assay in HEK293 cell lines. Before cell culture, the control commercial P(NIPAAm) was purified by reprecipitation cycles in excess n-hexane to remove the potential reactant residues (21). The cell lines were cultured in Dulbecco’s modified eagle medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/mL of penicillin and 100 µg/ mL of streptomycin at 37 °C, under 5% CO2, and 95% relative humidity atmosphere. The cells were seeded in a 96-well microtiter plate (Nunc Co., Wiesbaden, Germany) at a density of 104 cells/well and incubated in 100 µL of DMEM/well for 24 h. The culture media were replaced with fresh culture media containing serial dilutions of polymers, and the cells were incubated for 24 h. The culture media were replaced with fresh media. Then, 10 µL of sterile-filtered MTT stock solution in PBS (5 mg/mL) was added to each well, reaching a final MTT concentration of 0.5 mg/mL. After 5 h, the unreacted dye was removed by aspiration. The produced formazan crystals were dissolved in DMSO (100 µL/well). The absorbance was measured using a microplate reader (Spectra Plus, Tecan, Zurich,

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Switzerland) at a wavelength of 570 nm. The cell viability (%) relative to control cells cultured in media without polymers was calculated from [A]test/[A]control × 100%, where [A]test and [A]control are the absorbance values of the wells (with the polymers) and control wells (without the polymers), respectively. For each sample, the final absorbance was the average value measured from six wells in parallel. Hydrogel Characterization. The morphology of the HPN hydrogels was imaged using a JEOL scanning electron microscope (SEM, model 5600LV). Prior to the SEM measurements, specimens of the hydrogels were fixed on the metal holders and sputtered with a thin Pt layer. The dry weight (Wd) of each sample was determined after drying the gel to a constant weight under reduced pressure overnight. For the study of temperaturedependent equilibrium swelling ratios, the dry hydrogel samples were equilibrated in deionized water for at least 48 h at a predetermined temperature between 25 and 60 °C. The swelling ratios of the samples were measured gravimetrically. After the excess water on the sample surfaces was wiped off with moist filter papers, the samples were weighed (Ww) (20). The weights from three measurements were averaged, and the swelling ratio was calculated from the following formula: swelling ratio ) Ws /Wd where Ws () Ww - Wd) is the weight of water in the swollen sample at a specific temperature. Drug Loading and in Vitro Release. Dextran-FITC and BSA-FITC were selected as model hydrophilic macromolecular drugs to study the release behaviors of the HPN hydrogels. Drug loadings were achieved by immersing about 60 mg of the freeze-dried hydrogels in about 3 mL of 30 mg/mL PBS solution of dextran-FITC or BSA-FITC at 4 °C for 3 days. The total drug uptake was calculated from weight differences between the freeze-dried hydrogels with and without the drug loading. In vitro protein release analyses of the drug-loaded hydrogels were carried out in 5 mL of PBS buffer at 37 or 20 °C. At a specified sample collection time, 5 mL of the solution was removed and the medium in the test tube was replenished with 5 mL of fresh PBS buffer. The concentrations of the released dextran-FITC and BSA-FITC were determined using a Shimadzu RF 5301PC luminescence spectrophotometer (excitation at 495 nm, emission at 515 nm) with reference to the standard calibration curve. In addition, the circular dichroism (CD) spectra of the released BSA proteins were measured using a CD spectrometer (J-810, JASCO Co., Japan) in the wavelength range 200-250 nm. A JASCO cell of path length of 0.1 cm was used. The R-helix content corresponds to the ellipticity at 208 nm. Statistical Analysis. All experimental data used for the statistical analysis were obtained from at least three repeated runs. The data were collected in triplicate and expressed as mean ( standard deviations. Error bars represent the standard deviation. The statistical assay was performed using the Student’s t-test and the differences were considered statistically significant with p < 0.05.

RESULTS AND DISCUSSION Synthesis of Comb-Shaped Copolymers (HPC-gP(NIPAAm) or HPN) Composed of HPC Backbones and P(NIPAAm) Side Chains via ATRP. The preparation of functional polymers is usually carried out in organic solvent. Because of its unique characteristics of both water and organic solubility, HPC is an ideal substrate for developing novel polysaccharide-based biomaterials. The molecular weight (Mn) of the HPC used in this work, as determined from gel permeation chromatography (GPC), is about 2.80 × 104 g/mol, with the



Table 1. Characterization of HPC-g-P(NIPAAm) (HPN) Copolymers polymers samples HPC HPC-g-P(NIPAAm)1, or HPN1a HPC-g-P(NIPAAm)1, or HPN2a HPC-g-P(NIPAAm)1, or HPN3a

reaction time (h) -8 16 24

[N]/[C]b

Mn (g/mol)c

PDIc

-0.062 0.101 0.113

2.80 × 10 4.06 × 104 5.57 × 104 7.18 × 104

2.3 2.1 1.9 1.8

4

P(NIPAAm) content (wt/wt %) -31c 50c 61c

-35d 56d 64d

LCSTe (°C) 47.7 33.3-40.5 34.1 33.7

a Synthesized using a [monomer (2.0 g)]:[CuBr (25.4 mg)]:[HMTETA (77.6 µL)] molar feed ratio of 100:1:1.5 in 15 mL of dioxane containing 0.4 g of HPC-Br at 60 °C. b Determined from XPS N 1s and C 1s core-level spectral area ratios. c Determined from the GPC results. PDI ) Weight average molecular weight/Number average molecular weight, or Mw/Mn. d Determined from the XPS [N]/[C] ratios and the molecular weights of NIPAAm (M[NIPAAm] ) 113 g/mol) and HPC unit (C15H27O8; M[HPC] ) 335 g/mol), where [N]/[C] ≈ n[NIPAAm]/(6n[NIPAAm] + 15 m[HPC]). e The temperatures at the minimum points of the endotherms are referred to as the lower critical solution temperatures (LCSTs).

corresponding polydispersity index (PDI) of about 2.3. In order to prepare comb-like copolymers using HPC as a backbone via ATRP, it is essential to introduce alkyl halide initiators onto HPC. In this work, the bromoisobutyryl-functionalized HPC (HPC-Br) (19) was prepared via the reaction of hydroxyl groups of HPC with 2-bromoisobutyryl bromide (BIBB) (Scheme 1). Well-defined comb-shaped copolymer conjugates of HPC and P(NIPAAm) (HPC-g-P(NIPAAm) or HPN) were subsequently synthesized via ATRP of NIPAAm from HPC-Br. The HPNs with different contents of P(NIPAAm) can be synthesized by varying the ATRP time. Table 1 summarizes the GPC results of HPN1 (from 8 h of ATRP), HPN2 (from 16 h of ATRP), and HPN3 (from 24 h of ATRP). With the increase in reaction time from 8 to 24 h, the Mn of HPN increases from 4.06 × 104 to 7.18 × 104 g/mol, and the P(NIPAAm) content in HPN increases accordingly from 31 to 61 wt %. In addition, the PDIs of HPNs are slightly lower than that of the pristine HPC (Table 1), indicating that the ATRP of NIPAAm is well-controlled. XPS Analysis. The chemical compositions of the polymers were first determined by XPS. The C 1s core-level spectra of the pristine HPC and HPC-Br samples are shown in Figure 1a,b, respectively. The C 1s core-level spectrum of the pristine HPC can be curve-fitted into three peak components with binding energies (BE’s) at about 284.6, 286.2, and 287.6 eV, attributable to the C-H/C-C, C-O, and O-C-O species, respectively (19). From the [C-O]/[O-C-O] ratio of 11, the average number of propylene oxide groups (-OCH2CH(OH)CH3) per anhydroglucose unit of HPC is determined to be about 3, as reported earlier (19). When ATRP is carried out from a multifunctional backbone with a high local concentration of initiation sites, the radical-radical coupling of the propagating chains will occur and result in a gel. In order to avoid potential gelation, the starting HPC-Br with moderate concentration of initiation sites was prepared (19). The XPS C 1s core-level spectrum of HPC-Br can be curve-fitted into four peak components with BE’s at about 284.6, 286.2, 287.6, and 288.4 eV, attributable to the C-H/C-C, C-O/C-Br, O-C-O, and OdC-O species, respectively (19, 20). The new O-CdO peak component is associated with the bromide-capped ester groups of HPC-Br. The corresponding Br 3d core-level spectrum is shown in Figure 1b′. From the [Br]/[C] ratio of 0.011 (determined from the sensitivity factor-corrected Br 3d and C 1s corelevel spectral area ratio), it can be deduced that about 6 glucose units of HPC-Br possess one ATRP initiation site, or every HPC chain contains about 13 ATRP initiation sites on the average. No gelation was found during subsequent ATRP from such HPC-Br multifunctional initiators. Figure 1 also shows the C 1s core-level spectra of (c) HPN1 (from 8 h of ATRP), (e) HPN2 (from 16 h of ATRP), and (g) (from 24 h of ATRP). The C 1s core-level spectra of HPNs can be curve-fitted into four peak components with BE’s at about 284.6, 285.5, 286.2, and 287.6 eV, attributable to the C-H/ C-C, C-N, C-O and O-C-O/OdC-N species, respectively (22). The C-N and OdC-N peak components are associated with the P(NIPAAm) side chains. The increase (and decrease)

Figure 1. C 1s core-level spectrum of (a) HPC, C 1s, and Br 3d corelevel spectra of (b,b′) HPC-Br, and C 1s and N 1s core-level spectra of (c,d) HPN1, (e,f) HPN2, and (g,h) HPN3.

in intensities of the C-N and OdC-N (and C-O) species with ATRP time is consistent with the increase in P(NIPAAm) contents. The corresponding N 1s core-level spectra of HPN1, HPN2, and HPN3 are shown in Figure 1d,f, and h, respectively. From the [N]/[C] ratios determined by XPS, the P(NIPAAm) contents of HPNs can be calculated and are summarized in Table 1. The results are in fairly good agreement with those obtained from the GPC measurements. FTIR and 1H NMR Studies. Figure 2 shows the FTIR spectra of the (a) pristine HPC, (b) HPC-Br, (c) HPN1, (d) HPN2, and (e) HPN3 polymers. In comparison with that of HPC, the new characteristic band at about 1736 cm-1 (peak 1, υOdC-O) is associated with the bromide-capped ester groups of HPC-Br. In all the IR spectra of HPNs, the two strong amide


Figure 2. FTIR spectra of (a) HPC, (b) HPC-Br, (c) HPN1, (d) HPN2, and (e) HPN3. (peak 1, 1736 cm-1 (υOdC-O); peak 2, 1650 cm-1 (υOdC-NH); and peak 3, 1540 cm-1 (υN-H).)

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Figure 4. Typical DSC thermograms of HPC, P(NIPAAm), HPN1, HPN2, and HPN3 (the temperatures at the minimum points of the endotherms are referred as LCSTs).

Figure 5. Cell viability assay in HEK293 cell lines with various concentrations of HPC, P(NIPAAm), HPN1, HPN2, and HPN3. Cell viability was determined by the MTT assay and expressed as a percentage of the control cell culture.

Figure 3. 300 MHz 1H NMR spectra of (a) HPC-Br and (b) HPN3 in CCl3D.

absorption bands at about 1650 (υOdC-NH, peak 2) and 1540 cm-1 (υN-H, peak 3) are associated with the P(NIPAAm) side chains (20). The relative intensity of peaks 2 and 3 increase significantly with the increases in P(NIPAAm) content. The FTIR results are thus consistent with the XPS results of Figure 1. The chemical structures of HPC-Br and HPN are also characterized by 1H NMR spectroscopy. Figure 3a shows the 1 H NMR spectrum of the HPC-Br macroinitiator. The chemical shift at δ ) 1.12 ppm is attributable to the methyl protons of propylene oxide groups (a, C-CH3). The chemical shift at δ ) 1.92 ppm is associated with the methyl protons (b, C(Br)-CH3) of 2-bromoisobutyryl. The broad chemical shifts in the region 2.9-4.3 ppm are mainly associated with the inner methylidyne and methylene protons (c, c′, O-CH and O-CH2) on the glucose units and propylene oxide groups. From the area ratio of peak a and peak b, the initiator density of HPC-Br corresponds to one initiator site for every 6.8 glucose units (19),

fairly consistent with that determined by XPS. Figure 3b shows the 1H NMR spectrum of HPN3. The chemical shifts (a′, C-CH; a′′, C-CH2; a′′′, C-CH3) associated with the P(NIPAAm) side chains overlapped completely with the methyl protons of propylene oxide groups (a, C-CH3) of the HPC backbone. The new chemical shift at δ ) 4.15 ppm corresponds to the (c′′) N-CHMe2 methylidyne protons of P(NIPAAm) (24). The signal intensities associated with the HPC backbone (c, c′′) have decreased substantially. Due to the serious overlaps in the characteristic shifts of the components, it is difficult to quantify the P(NIPAAm) segments by 1H NMR. Lower Critical Solution Temperature (LCST). The LCSTs of HPNs were determined by differential scanning calorimetry (DSC). Figure 4 shows the typical DSC thermograms of the HPC, commercial P(NIPAAm), HPN1, HPN2, and HPN3 polymers. The temperature at the minimum point of the endotherm is referred to as the LCST of the sample. The pristine HPC used in this work exhibits a LCST of about 47.7 °C, similar to that reported earlier (18). The LCST of HPC is the consequence of hydrophobic (associated with propylene oxide groups) and hydrophilic (associated with hydroxyl groups) interaction (17, 18). HPCs from different batches may have obviously different LCSTs, due to their difference in substituent distribution along the polymer chains (20). Nevertheless, their general LCST is significantly higher than body temperature (37 °C). The control commercial P(NIPAAm) with average Mn of 20 000-25 000 has a LCST of about 32 °C, due to the hydrophobic (associated with isopropyl groups) and hydrophilic (associated with amide moiety in the pendant groups) interaction (20). As shown in Figure 4, the LCST of HPN decreases with



Figure 7. Typical DSC thermograms of HPC, HPN2, and HPN3 hydrogels.

Figure 6. SEM images of (a) HPC, (b) HPN2, and (c) HPN3 hydrogels.

the increase in P(NIPAAm) content (Table 1). The HPN1 with average P(NIPAAm) content of about 33 wt % exhibits a very broad LCST range of 33 to 41 °C. When average the P(NIPAAm) content in HPN2 or HPN3 reaches above 53 wt %, only one minimum point in the DSC endotherm is observed and the corresponding LCST becomes lower than body temperature of 37 °C. The LCST of HPN in this work is the consequence of synergistic hydrophobic and hydrophilic interactions arising from HPC and P(NIPAAm) individual components. Cytotoxicity Assay. Cytotoxicity is one of the most important factors to be considered in selecting biomaterials. Figure 5 shows the in vitro MTT assay results of cytotoxicity of HPC, commercial P(NIPAAm), HPN1, HPN2, and HPN3 at various concentrations in HEK293 cell lines. HPC does not show any

cytotoxicity at polymer concentrations up to 2.0 mg/mL. No acute cytotoxicity associated with the commercial P(NIPAAm) was found, consistent with that reported earlier (6). However, the MTT assay suggested that the HPNs at higher polymer concentrations are better tolerated by the cell lines than those of the P(NIPAAm) homopolymer, probably because of the introduction of HPC biopolymer. Stimuli-Responsive HPN Hydrogels. Because of their lower LCSTs than the body temperature (Figure 4), HPN2 and HPN3 were selected for the preparation of the temperature-responsive HPN hydrogels. Some of the remaining hydroxyl groups of HPN2 and HPN3 can be cross-linked by divinylsulfone (DVS) to produce the corresponding stable HPN2 and HPN3 hydrogels. DVS introduces cross-linking by an addition reaction with the hydroxyl groups on either the HPC backbone or the substituent groups, saturating the DVS carbon-carbon double bond in alkaline solution (1, 17). Figure 6 shows the SEM images of the freeze-dried (a) HPC, (b) HPN2, and (c) HPN3 hydrogels. The HPN2 and HPN3 hydrogels possess interconnected pore structures, while no obvious pore structures were observed on the HPC hydrogel under similar conditions. The P(NIPAAm) components may possess some different physical properties (such as solubility) from the HPC counterparts in aqueous medium. The resultant poor incompatibility or phase separation during the hydrogel preparation probably has led the formation of pore structures. In comparison with those in Figure 4, the DSC thermographs of the hydrogels in Figure 7 suggest that the presence of DVS in the cross-linked matrix do not have a serious effect on the LCSTs (Table 1 and Table 2). The temperature dependence of the swelling ratios of HPC, HPN2, and HPN3 hydrogels was also investigated. Figure 8 shows the typical temperaturedependent swelling ratio of the three hydrogels over the temperature range of 25 to 60 °C in deionized water. The equilibrium swelling ratios of three hydrogels exhibit a similar trend of temperature dependence. As the temperature was increased, the swelling ratio decreases, with the most drastic decrease being observed at around the respective LCSTs of HPC, HPN2, and HPN3 hydrogels (Figure 7). Generally, the main reason for this distinctive characteristic of the hydrogels can be attributed to the unique and rapid transformation between the hydrophilic and hydrophobic states (20). At temperatures below the LCST, the hydrophilic groups of the hydrogels form hydrogen bonds with water molecules. These bonds act cooperatively to form a stable shell of hydration around the hydrophobic groups, resulting in greater water uptake and producing a larger swelling ratio. As the external temperature increases, the associative interactions among the hydrophobic groups release the entrapped water molecules from the hydrogel networks. In addition, HPN2 and HPN3 hydrogels possess higher initial swelling ratios than HPC hydrogels at starting


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Table 2. Characterization of HPC-g-P(NIPAAm) (HPN) Hydrogels hydrogelsb cumulative drug releasedd (%) c

dextran

drug loading (mg/mg dry gel)

BSA

samples

LCSTa (°C)

dextran

BSA

20 °C

37 °C

20 °C

37 °C

HPC HPC-g-P(NIPAAm)1, or HPN1a HPC-g-P(NIPAAm)1, or HPN2a HPC-g-P(NIPAAm)1, or HPN3a

47.8 -35.0 34.4

0.25 -0.47 --

0.34 --0.59

80.3 -89.4 --

82.1 -83.8 --

75.2 --85.6

76.4 --79.5

a The temperatures at the minimum points of the endotherms are referred to as the lower critical solution temperatures (LCSTs). b Prepared in about 10 mL of pH 13 water containing 1.0 g of polymers and 0.1 mL of DVS at 30 °C. c Obtained by immersing 60 mg of dry gels in 3 mL of the drug-loaded solution of 30 mg/mL at 4 °C for 3 days. d After 10-day in vitro release.

Figure 8. Temperature-dependent equilibrium swelling ratio of HPC, HPN2, and HPN3 hydrogels.

temperature of 25 °C (far lower than their LCSTs), arising probably from the interconnected pore structures of HPN2 and HPN3 hydrogels (Figure 6). In Vitro Controlled Drug Release. In this work, the in vitro controlled release properties of the smart hydrogels were studied by using fluorescein isothiocyanate-labeled dextran and BSA (or dextran-FITC and BSA-FITC) as model macromolecular hydrophilic drugs. The drug loadings were achieved by immersing the dry HPC, HPN2, and HPN3 gels in 30 mg/mL solution of dextran-FITC (or BSA-FITC) at 4 °C for 3 days. From the data in Table 2, the amount of loaded drug per unit mass of HPN2 and HPN3 gels is higher than that in the corresponding HPC gels, probably due to the interconnected pore structures of HPN2 and HPN3 hydrogels (Figure 6). The release behavior of the loaded drugs was studied at 37 and 20 °C. Figure 9 shows the release profiles of (a) dextran from the HPC and HPN2 hydrogels and (b) BSA from the HPC and HPN3 hydrogels. The release profile was characterized by an initial burst, followed by a slower and sustained release. This release behavior is a typical biphasic release pattern, similar to those reported (26, 27). The initial burst may have been caused by the release of drug molecules located on the outer surfaces, while the sustained release was probably due to the slow macromolecular diffusion from the interior of the hydrogels. There is no obvious difference between the release profiles of HPC hydrogels at 20 and 37 °C (lower than the LCST of HPC of about 48 °C). However, the sustained drug release from the HPN2 and HPN3 hydrogels was slower at 37 °C than at 20 °C. At 37 °C above the LCST of HPN2 and HPN3 (Figure 7), their hydrogel mesh size has decreased upon hydrogel collapse, which in turn decreases the diffusivity of the macromolecules through the hydrogel network and leads to the slower drug release. The slow and sustained release could provide a continuous drug delivery, prevent the problems of cyclic variations in the

Figure 9. Release profiles of (a) dextran-FITC from HPC and HPN2 hydrogels and (b) BSA-FITC from HPC and HPN3 hydrogels at 20 and 37 °C.

drug concentrations in blood, and produce the maximum pharmacological efficiency (27, 28). In addition, the drug release at 20 °C for the HPN2 and HPN3 hydrogels was slightly faster than that of the corresponding HPC hydrogel. The pore structures of the HPN2 and HPN3 hydrogels probably have facilitated the diffusion of drug molecules into the in vitro medium. After 10-day in vitro release (Table 2), the loaded drugs were still not released completely. This phenomenon is very common in drug delivery systems. The presence of physical interactions between the drug and hydrogels probably has hindered the drug diffusion (28, 29). The functions and structures of proteins are inherently correlated. Here, UV-circular dichroism (CD) was used to examine whether the released BSA has retained its native secondary structures. In general, proteins possess three common secondary structures of R-helix, β-sheets, and random coil. The quantitative structural changes of proteins can be evaluated by analyzing the preserved content of R-helix. The R-helix content of proteins can be estimated from the following formula (29, 30): % R-helix ) (θmrd - 4000)/(33 000 - 4000)



LITERATURE CITED

Figure 10. Circular dichroism (CD) spectra of the native BSA-FITC and released BSA-FITC from HPN3 hydrogel.

where θmrd is the mean molar ellipticity per amino acid residue at 208 nm (deg.cm2. dmol-1). θmrd can be calculated using the following formula θmrd ) (θdM0)/(10CL) where, θd is the measured ellipticity in the unit of mdeg from the CD spectrometer, M0 is the average molecular weight (Da) per amino acid residue, C is the BSA concentration (mg.mL-1), and L is the sample cell path length (cm). BSA is an ellipsoidal protein with the dimensions of 14 × 4 × 4 nm3. Its average molecular weight (Da) per amino acid residue is about 118 Da (31). Figure 10 shows the typical CD spectra of BSA-FITC released from the HPN3 hydrogel and native BSA-FITC. The CD spectrum of the released BSA is similar to that of the native BSA. The calculated R-helix contents in the released and native BSA are 53% and 57%, respectively, indicating that the conformation of BSA has not been severely or irreversibly altered by adsorption in the hydrogels.

CONCLUSIONS The comb-shaped copolymer conjugates composed of HPC backbones and short P(NIPAAm) side chains (HPC-gP(NIPAAm) or HPN) have been successfully prepared via ATRP from the bromoisobutyryl-functionalized HPC backbones. The HPN with average P(NIPAAm) content of above 53 wt % exhibited a LCST below the body temperature of 37 °C. The MTT assay from the HEK293 cell line indicated that HPNs possess reduced cytotoxicity. Some of the remaining unreacted hydroxyl groups of HPNs were used as cross-linking sites for the preparation of stable HPN hydrogels. In comparison with the HPC hydrogels, the resultant cross-linked HPN hydrogels possess interconnected pore structures and higher swelling ratios. The HPN hydrogels can also provide the long-term sustained release of macromolecular drugs at body temperature. Such porous smart hydrogels of HPN with controllable LCSTs are potentially useful for biomedical applications. However, it should be noted that such hydrogels may be not suitable as implanted or injected devices due to their poor degradability.

ACKNOWLEDGMENT This work was supported by Research Fund for the Doctoral Program of Higher Education of China (project no. 20090010120007), National Natural Science Foundation of China (grant no. 50903007), Chinese Universities Scientific Fund (project no. ZZ0904), and Program for New Century Excellent Talents in University.

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Comb-Shaped Conjugates Comprising Hydroxypropyl Cellulose

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