Thermosensitive Transparent Semi-Interpenetrating Polymer Networks

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Biomacromolecules 2008, 9, 1313–1321

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Thermosensitive Transparent Semi-Interpenetrating Polymer Networks for Wound Dressing and Cell Adhesion Control T. Thimma Reddy, Arihiro Kano, Atsushi Maruyama, Michiko Hadano, and Atsushi Takahara* Institute for Materials Chemistry and Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan Received December 14, 2007; Revised Manuscript Received February 12, 2008

Thermosensitive, transparent, and flexible semi-interpenetrating polymer networks (semi-IPNs) composed of segmented polyurethane urea/poly(N-isopropylacrylamide) (SPUU/ PNiPAAm) were new class of materials, which holds promise for its potential use as wound dressings. A series of semi-IPNs, obtained via thermal initiated polymerization of NiPAAm, were characterized by infrared spectroscopy (IR), nuclear magnetic resonance (NMR), dynamic viscoelastic measurements, wide-angle X-ray diffraction (WAXD), and mechanical properties. The resulting semi-IPNs were also investigated for their dynamic water contact angles, thermodynamic interaction parameters, in vitro drug release, and cell adhesion and detachment. The semi-IPNs with differing compositions possess good mechanical properties in both dry and hydrated states. In addition, NIH3T3 fibroblasts can attach to and detach from these semi-IPN films with varying temperature. In addition, these film extracts do not show significant cytotoxicity. Therefore, these materials have great potential for the construction of a new generation of dressings and cell transplantation for wound healing.

Introduction Wound healing is a dynamic process, and the primary objective in wound care is the promotion of rapid wound healing with the best functional and cosmetic results.1,2 The objective of using a wound dressing is to accelerate wound healing by preventing bacterial infection and the acceleration of tissue regeneration.3 According to modern insights, a wound dressing should have flexibility, gas permeability, durability, and the ability to control water loss. Numerous wound dressing materials based on calcium alginates, hydrogels, and hydrocolloids have been developed for moist wound treatment. However, no single dressing will be useful for all kinds of wounds; many dressings will be tried during the healing process of a single wound. For the treatment of burns and chronic wounds, tissue engineered skin products using cultured fibroblasts have been used for clinical practice for many years. These products rapidly and effectively enhance the wound healing process by producing various growth factors. However, wide use of these products is still limited due to cost and handling difficulties. An alternative approach to targeting cells to wound sites is a challenging task. It is well-known that poly(N-isopropylacrylamide) (PNiPAAm) is a temperature-responsive polymer and has been successfully used in cell culture, cell delivery, and cell sheet engineering.4–8 Moreover, Lin et al. and Yang et al. recently exploited the temperature-responsive property of PNiPAAm, for easy peeling and cell adhesion control in wound dressing applications.9–11 However, such systems suffer from the disadvantage of poor flexibility in both dry and hydrated state. Flexibility is one of the important properties for wound dressing, which would be useful to retain their shape during application as well as to graft the harvested cells to the target place. It is known that a number of polymer surface parameters such as composition and morphology are responsible for the cell adhesion and prolifera* To whom correspondence should be addressed. E-mail: takahara@ cstf.kyushu-u.ac.jp. Fax: +81-92-802-2518.

tion. An increasing number of publications have been devoted to the preparation of interpenetrating polymer networks (IPNs) as a means of tailoring the bulk and the surface properties of polymeric materials for biomedical applications.12–15 Due to the excellent mechanical properties and tissue compatibility, polyurethane has been widely used as a biomedical material. Previously, we have studied the fatigue behavior of polyurethane urea after absorption of blood components.16 The purpose of our basic strategy in this study is to synthesize semi-IPNs based on SPUU and PNiPAAm and their properties were thoroughly investigated with a view to develop an ideal wound dressing by utilizing the thermosensitivity and flexibility of PNiPAAm and SPUU, respectively. This was done in the belief that the resultant semi-IPNs may provide a better approach for cell grafting. Because of their flexibility and thermosensitivity, cells can be grafted to a target place by the simple application of the cooling process. It is also to be expected that the resultant films would be transparent in the dry state and thermosensitive in swelling ratio, which would facilitate the easy removal of the film from the wound surface and, thus, the chance of damage to the newly formed tissue can be reduced. Furthermore, the transparency of these films permits constant observation of the wound during healing process so that the dressing would not be prematurely removed.

Experimental Section Materials. The synthesis of segmented polyurethane urea (SPUU) and its characterization has been reported recently.17,18 The sample code of SPUU was designated as PCL(PCL Mn)(PCL fraction)LDI-BDA (Table 1), Mn is molecular weight, and PCL, LDI, and BDA are polycaprolactone, lysine methyl ester diisocyante, and 1,4-butanediamine, respectively. N-Isopropylacrylamide (NiPAAm), obtained from Wako Pure Chem. (Japan) was recrystallized from hexane. N,N′Methylenebisacrylamide (MBAm; Kishida Co., Japan), R,R′-azobisisobutyronitrile (AIBN; Kanto Chemical Co., Inc. Japan), sulfamethaxazole, and phosphate buffer solution (PBS; pH ) 7.4; Aldrich chemical)

10.1021/bm701390f CCC: $40.75  2008 American Chemical Society Published on Web 03/21/2008

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Table 1. Composition Used To Make Semi-IPNs compositionsa [wt %] sample code

semi-IPNs

PCL(530) (50)LDI-BDA

NiPAAm

SPUU SPUU-20 SPUU-30 SPUU-40 SPUU-50

PCL(530)(50)LDI-BDA PCL(530)(50)LDI-BDA-20 PCL(530)(50)LDI-BDA-30 PCL(530)(50)LDI-BDA-40 PCL(530)(50)LDI-BDA-50

100 80 70 60 50

0 20 30 40 50

a In addition, 0.1 wt % cross-linker (MBAm) and 1 wt % (AIBN) thermal initiator based on the total polymerizable group were added into the above composition. Dimethylacetamide (DMAc) is used as a solvent (15 wt % solids are used for all compositions).

were used directly as received. Dimethylacetamide (DMAc) was purchased from Wako Co. (Japan). Synthesis of SPUU/NiPAAm Semi-IPNs. SPUU/PNiPAAm semiIPNs, containing 20–50 wt % of NiPAAm, were synthesized by thermal radical initiated polymerization with AIBN initiator and MBAm crosslinker for the NiPAAm network. The feed compositions of semi-IPNs are shown in Table 1. The reaction mixture consisting of PCL(530)(50)LDI-BDA and NiPAAm of approximately 15 wt % of total solids were taken in a glass Petri dishes and were kept in a separable flask. The flask was heated to 80 °C under controlled vacuum over 6 h for polymerization of NiPAAm and evaporation of solvent simultaneously. Obtained semi-IPNs were purified by soaking in water at 22 °C for 2 days. Disk-shaped samples (8 mm in diameter and 0.2–0.3 mm thick) for swelling and drug absorption studies were cut from water equilibrated semi-IPN films at room temperature and then dried. For cell adhesion and detachment study, semi-IPN films prepared in Petri dishes (35 mm diameter) were directly used after purification without removing the films from the glass Petri dishes. Polymer Characterization. 1H (400 MHz) NMR spectra were recorded in DMSO-d6 with a JEOL JNM-EX400 spectrometer using tetramethylsilane (TMS) as an internal standard. FTIR spectra were obtained from 64 scans at 2 cm-1 resolution at room temperature using a Perkin-Elmer Model 029452 spectrophotometer. The samples for infrared analysis were prepared as thin films by dropping (50 µL) semiIPN solution of 5% (w/v) in DMAc directly onto NaCl plates and dried. The films used in this study were sufficiently thin to be within the absorbance range. None of the spectra showed residual solvent. The temperature dependence of the dynamic mechanical properties were obtained using a microprocessor-controlled Rheovibron DDV 01-FP under a dry nitrogen purge in tensile mode in a temperature range of -150 to 170 °C. Samples were cooled to -150 °C and data were subsequently taken at a test frequency of 1 Hz and a heating rate of 2 °C/min. Wide angle X-ray diffraction (WAXD) measurements were performed by using Rigaku RINT 2500V system (Rigaku Denki Co., Ltd., Japan) at room temperature. The tube was operated at 40 kV and 200 mA. Nickel filtered Cu KR radiation (λ ) 0.154 nm) was applied on the sample in a conventional horizontal axis configuration, with a scan rate of 3°/min between 2θ ) 5–40°. The advancing and receding water contact angles of each sample at 20 and 37 °C were measured by sensile drop method using a Drop Shape Analysis system DSA-10 (Kruss Co. Ltd.) with a thermostatted water bath. Dry films were used for all measurements. Contact angle hysteresis, the difference (θa – θr), was also recorded. Five repetitions were performed and the average values were presented. Tensile-Stress Tests and Network Parameters. Tensile strength of the parent PCL(530)(50)LDI-BDA network and the dry and swollen state of semi-IPNs were measured using a tensile testing machine EZ graph (Shimazu, Co., Ltd.) with a cross head speed of 10 mm/min. The samples were molded into dumbbell shape with a gauge length of 12 mm, a width of 2 mm, and a thickness of 0.3 mm using a dumbbell shape die SDL-100 (Dumbbell Ltd., Japan). Tests were performed in a temperature (23 °C) and humidity-controlled environment (70%). For each sample, five specimens were tested. In the swollen state, the component of semi-IPN shows elastomer-like behavior. Apparent

polymer network parameters were obtained using the modulus of elasticity, E, derived from tensile experiments and the following equations:19

νe )

G E ) 0.33 RTφ2 3RTφ20.33

(1)

F νe

(2)

Mc )

where G is the shear modulus, νe is the effective cross-link density, R is the universal gas constant, T is the temperature, F is the dry polymer density (calculated from dry polymer mass and geometric volume), Mc is the molecular weight between cross-links, and φ2 is the polymer volume fraction at equilibrium swelling of semi-IPNs, calculated from20

φ2 )

( ) D0 D1

3

where D0 is the diameter of the dry sample and D1 is the diameter of swollen sample. Drug Loading. SolVent Casting Method. A total of 5 wt % of sulfamethaxazole (SF) was dispersed in PCL(530)(50)LDI-BDA solution for 2 h separately. After complete dispersion, the homogeneous drug containing solution was poured into a PTFE casting dish. The cast films were air-dried, followed by vacuum-drying at 60 °C for 12 h, to remove residual solvent, the films were released from casting dishes and then trimmed to 8 mm in diameter discs, and the resulting film discs were used for in vitro drug release studies. The drug loaded film was designated as PCL(530)(50)LDI-BDA-SF. SF represents sulfamethaxazole. Absorption Method. Sulfamethaxazole was loaded into PCL(530)(50)LDI-BDA-50 and PNiPAAm using solution sorption method. SF solution (1.6 wt %) was prepared in ethanol. The drug was loaded by keeping the samples in the above solution at 4 °C for 10 min. The drug loaded films were used for in vitro drug release studies. Before immersion in the release medium, the samples were briefly rinsed with PBS (same temperature as the loading solution) and the surface was blotted with a damp filter paper. The drug-loaded samples were coded as PCL (530)(50)LDI-BDA-50-SF and PNiPAAm-SF. The % of drug loading in both the cases (solvent casting and absorption methods) was determined spectrophotometrically at 264 nm after extensive extraction in PBS (pH ) 7.4) solution. Loading percent was calculated as:

%loading ) (Wdrug ⁄ (Wpolymer + Wdrug)) × 100 Drug Release. Drug release from disk-shaped dry samples was conducted at 32 °C in PBS at pH ) 7.4. The dried, drug-loaded samples were immersed directly in the 10 mL fresh PBS, pre-equilibrated at 32 °C, at predetermined times. Aliquots (0.5 mL) were withdrawn periodically to determine drug concentration and, in all cases, equal volumes of dissolution medium were immediately added to maintain a constant volume. SF concentration was determined spectrophotometrically at 264 nm. Absorbance from blank (polymer films without drug) as a function of time was systematically measured and substracted from the drug-loaded films absorbance value. This measurement ensured to take into account any unreacted material that leached into external solvent that might occur during the time of release experiments. Samples were withdrawn until two successive aliquots showed no increase in absorbance. The amount of SF released from the polymer films in a dissolution medium, at a given time, was calculated using standard curves of SF in corresponding buffer and expressed as percentage of total drug content of the investigated films. Experiments were performed in triplicate, and the average value was considered during data treatment and plotting. Cytotoxicity Study of Semi-IPN Extracts. The polymer samples (8 mm diameter) for cytotoxicity tests in cell culture were UV sterilized. The cytotoxicity test essentially followed the method of Guidoin et al.21 To test the cytotoxicity of possible substances that could leach from the semi-IPN films, first the films were cut into 8 mm in diameter and three samples of each composition were immersed separately in 2

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Scheme 1. Schematic Diagram Showing Components Used for the Synthesis, and the Resulting Structure of Semi-IPNs

mL of complete Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS; Wako Pure Chemical Industries Ltd., Japan) for 72 h at 37 °C. The medium, containing the extracts from the films, was diluted at volume ratio of 1:1 using the culture medium. NIH3T3 cells were seeded at a density of 5000 cells/ mL into wells containing 100 µL of the respective IPN extracts, and the wells containing only the cells and the culture medium served as controls. The cells were incubated for 24 h at 37 °C with 5% CO2. At the end of the exposure time, cell viability was measured (n ) 4) using (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) (MTT) assay. Absorbance was measured at 405 nm and values relative to control were reported. Cell Attachment. PCL(530)(50)LDI-BDA and semi-IPN films prepared on a glass Petri dish (35 mm diameter) were UV sterilized before being used for cell attachment. Pure PCL(530)(50)LDI-BDA was used as a control. The NIH3T3 fibroblasts were seeded at a density of 2.5 × 106 cells on each sample in the culture medium and incubated at 37 °C with 5% CO2. Attached cells were observed with a phase contrast microscope after 24 and 72 h. Cell Detachment. For this test, the cells were seeded onto a glass Petri dish (35 mm diameter) containing PCL(530)(50)LDI-BDA and semi-IPN films of all compositions. NIH3T3 fibroblasts were seeded onto the each sample at a density of 5 × 106 cells in the culture medium and incubated at 37 °C with 5% CO2. After 24 h, the samples were transferred to an incubator at 15 °C and incubated 15 min, and the detached cells were monitored though phase contrast microscope. A cell detachment movie was recorded only in the case of PCL(530)(50)LDI-BDA-30, and the recorded movie was converted into images by using Pinnacle studio9 movie editing software. Subsequently, the

contents of each well were aspirated and transferred to a fresh culture plate, which was then returned to the incubator to allow the detached cells to reattach onto the plate and resume cell growth. Statistical Analysis. For every test, the data are expressed as means plus or minus the standard deviation (n ) 3). The statistical analysis was peroformed with a Students’ t-test at a 0.05 level.

Results and Discussion Synthesis of Semi-IPNs. In the semi-IPN synthesis, a little amount of methylene bisacrylamide has been used as the crosslinker to lightly cross-link the PNiPAAm. Table 1 summarizes the composition of the semi-IPN films. All the reaction mixtures taken in the dimethylacetamide (DMAc) solvent are transformed into transparent solid polymer films after free radical initiated polymerization under thermal conditions, as shown in Scheme 1. Earlier semi-IPN films based on polyurethane and PNiPAAm were prepared by photopolymersiation followed by thermal heating to remove the solvent.22 Depending on the feed composition and thickness of the solution, thermal effects take place during photopolymerization, which causes nonhomogeneity and defects in the final material. These defects greatly alter the physical and bioresponsive properties of the final products.23 We synthesized semi-IPN films by thermal free radical polymerization even at low feed compostion of NiPAAm. Semi-IPNs exhibited swelling in water and were soluble in DMAc, which suggested that these IPNs are physically cross-linked. The semi-IPN structure allowed for

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Figure 1. FTIR spectra of (a) PCL(530)(50)LDI-BDA, PNiPAAm, and semi-IPNs; (b) carbonyl stretching region of PCL(530)(50)LDI-BDA, PNiPAAm, and semi-IPNs.

Figure 3. Wide angle X-ray diffraction pattern of (a) PCL(530)(50)LDIBDA, (b) PCL(530)(50)LDI-BDA-20, (c) PCL(530)(50)LDI-BDA-30, (d) PCL(530)(50)LDI-BDA-40, and (e) PCL(530)(50)LDI-BDA-50.

Figure 2. Dynamic mechanical analysis of SPUU and semi-IPNs (1) PCL(530)(50)LDI-BDA, (2) PCL(530)(50)LDI-BDA-20, (3) PCL(530)(50)LDI-BDA-30, (4) PCL(530)(50)LDI-BDA-40, (5) PCL(530)(50)LDIBDA-50.

the mixing of two incompatible polymers to get an enhanced mechanical strength. Bulk Polymer Characterization. The FTIR spectrum confirmed the presence of PNiPAAm in semi-IPNs. Figure 1a illustrates the FTIR spectra of PNiPAAm (A), semi-IPNs with 20–50 wt% of NiPPAm (B-E), and PCL(530)(50)LDI-BDA (F). In both polymers of PCL(530)(50)LDI-BDA and PNiPAAm contain the -CONH- group. The characteristic peaks corresponding to PCL(530)(50)LDI-BDA were reported elsewhere.18 From Figure 1b (carbonyl region) it is found that the peak at about 1731 cm-1 is assigned to the CdO stretching. The peak at about 1650 cm-1 for N-H bending vibration of primary amino group is known as the amide II band.24–26 From Figure 1a and b, it is found that the relative intensity ratio values of N-H bending over CdO stretching for PCL(530)(50)LDI-BDA, PCL(530)(50)LDI-BDA-20, PCL(530)(50)LDI-BDA-30, PCL(530)(50)LDI-BDA-40, and PCL(530)(50)LDI-BDA-50 are 0.005, 0.54, 0.65, 0.76, and 1.29, respectively. These values increase with increasing the PNiPAAm content in semi-IPNs. Dynamic Mechanical Analysis. Figure 2 shows the storage modulus (E′) and loss modulus (E′′) versus temperature of PCL(530)(50)LDI-BDA and semi-IPNs. The damping peak is associated with the partial loosening of the polymer structure so that small chain segments can move, which occurs near Tg at low frequencies. The maximum in the loss modulus (E′′) at low frequencies is, however, very close to Tg. From Figure 2, the temperature corresponding to E′′, very close to Tg as obtained from DSC reported elsewhere,18 was found to be ∼-19 °C for the parent polymer. In the semi-IPN systems, the effect of PNiPAAm content on the PCL(530)(50)LDI-BDA network is observed as variation of Tg compared to the parent system. With an addition of PNiPAAm of 20 wt % in the semi-IPNs, the Tg is first observed to increase, and further addition of PNiPAAm

in the matrix does not have much influence on Tg. The increase in Tg observed due to addition of PNiPAAm could be attributed to the more interaction of PNiPAAm chains with PCL(530)(50)LDI-BDA soft segments. It is also observed from Figure 2 that the damping peak height decreases with the addition of PNiPAAm (curves 2-5). This suggests greater limitations on freedom of chain mobility in the soft segment, which may be explained by physical interactions between PNiPAAm and PCL(530)(50)LDI-BDA soft segments, which reduces free volume in soft segment domain. The E′′ curves of PCL(530)(50)LDI-BDA-30, PCL(530)(50)LDI-BDA-40 and PCL(530)(50)LDI-BDA-50 samples display smaller transition at higher temperatures. This transition may corresponded to the Tg of LDI-BDA hard segments or secondary transition of PNiPAAm chains. However, all E′′ curves show a low temperature transition (below -50 °C). This may be due to rotation or local motion of smaller groups in the main chain. This relaxation at similar temperature was observed in other polyurethanes.27 Figure 2 also shows dynamic storage modulus (E′) as a function of temperature. As can be seen a sharp decrease of E′ in case of parent polymer above glassy region has been observed. However, in the case of semi-IPNs, a systematic decrease of rubbery plateau region or high rubbery plateau modulus (E′) has been observed with an increase of PNiPAAm content implies that elastic behavior predominates rather than rubbery behavior with PNiPAAm, which may be due to the increased number of interactions between PCL(530)(50)LDI-BDA and NiPAAm. Polymer samples were further characterized by WAXD. Figure 3a-e presents and compares the WAXD of SPUU and semi-IPNs with 20–50 wt % of PNiPAAm. WAXD of these polymer samples show only a broad diffusion scattering (amorphous halo) with maximum at 20 2θ and 21.8 2θ. The positive shift in the 2θ value from 20 to 21.8 with the insertion of PNiPAAm may be due to a decrease in the average d-spacing between amorphous chains as a result of the increased intermolecular interaction. It is also observed that a certain degree of orientation corresponding to PNiPAAm appeared at 7.5 2θ when the

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Figure 4. Uniaxial stress–strain curves of polymer films (a) in the dry state and (b) in the hydrated state. Table 2. Tensile Properties of Polymer Films in the Dry State (Mean ( SD, n ) 5)

sample code

Young’s modulus (MPa)

tensile strength (MPa)

elongation at break (%)

PCL(530)(50)LDI-BDA PCL(530)(50)LDI-BDA-20d PCL(530)(50)LDI-BDA-30d PCL(530)(50)LDI-BDA-40d PCL(530)(50)LDI-BDA-50d

24 ( 1.5 26 ( 3.9 43 ( 4.2 63 ( 2.8 86 ( 5.2

21.7 ( 1.7 22.7 ( 2.7 18.5 ( 1.4 12.2 ( 1.8 9.8 ( 1.2

1798 ( 10 1600 ( 10 1123 ( 10 600 ( 10 412 ( 10

Table 3. Tensile Properties of Polymer Films in the Hydrated State (Mean ( SD, n ) 5) sample code PCL(530)(50)LDI-BDA-20 PCL(530)(50)LDI-BDA-30 PCL(530)(50)LDI-BDA-40 PCL(530)(50)LDI-BDA-50

h h h h

tensile strength (MPa)

elongation at break (%)

12 ( 1.5 9.2 ( 1.6 5.6 ( 1.9 3.3 ( 1.6

1650 ( 10 1520 ( 10 1215 ( 10 1028 ( 10

concentration of NiPAAm was 40 and 50 wt % in semi-IPNs (Figure 3d,e). This peak appears to be strange, but a similar diffraction peak was observed in the case of pure PNiPAAm at 7.5 2θ.28 Mechanical Properties of Semi-IPNs. The synthesized SPUU/ PNiPAAm semi-IPNs were tested for their dimensional and mechanical properties to reveal the effect of PNiPAAm content. The stress–strain curves shown in Figure 4a,b, obtained from tensile studies, provided information on the strength, modulus, and elongation of the parent system (SPUU) and the semi-IPNs (dry and hydrated state), as listed in Tables 2 and 3. The data reported is the average of five measurements, and the maximum error associated with the determination of the elastic modulus, ultimate tensile strength, and % elongation are within the confidence limit of (10%. The synthesized semi-IPNs are highly extensible, with breaking strains from 804 to 1600% in the dry state and 1050 to 1650% in the hydrated state. Increasing the PNiPAAm content in semi-IPNs has the effect of increasing the initial modulus and decrease in tensile strength in both the dry and the hydrated states compared to parent polymer (Tables 2 and 3). From the engineering perspective, good mechanical properties would allow materials used for wound dressings to maintain their shape during application. In this study, three parameters, tensile strength, elongation at break, and Young’s modulus, were measured to determine the mechanical properties of these semi-IPNs. Polymer films with different compositions were found to differ in mechanical behavior. Their tensile strengths varied from 9 to 22 MPa in dry state and 4 to 12 MPa in hydrated state. In comparison, the tensile strength and Young’s modulus of skin are normally 2.5–16 MPa and 6–40 MPa, respectively. Polymers with similar strengths and slightly higher Young’s moduli are most frequently

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used to replace skin tissue.29 The fact that the tensile strength of the semi-IPNs ranges from 3 to 22 MPa and that the Young’s modulus ranges from 20 to 80 MPa suggests that the polymers have sufficient mechanical durability to be used as wound dressings and excellent flexibility for grafting cells to any part of the body. Recently, PNiPAAm-based nanostructured membranes have been reported10,11 for wound dressing applications and their tensile strength 4.8 to 6.9 MPa and Young’s modulus 140 to 380 MPa, respectively. However, their elongation at break was below 90%. Water Contact Angle. To understand the surface wettability of parent polymer and semi-IPNs, water contact angles are measured at different temperatures. The semi-IPNs exhibited temperature-dependent surface properties, that is, all semi-IPNs show lower water contact angle at 20 °C and increase with an increase of temperature (Table 4). The contact angles for all semi-IPNs at 20 °C are in the range of 35 to 50°, whereas the contact angles at 37 °C vary from 60 to 75°, which is an optimum for cell culture.30 The change in contact angle with temperature is mainly due to conformational change of PNiPAAm molecules below and above lower critical solution temperature (LCST). PNiPAAm is fully hydrated with an extended conformation in aqueous solution below 32 °C and is extensively dehydrated and compact above this temperature. Change in water contact angle at 37 °C indicates that the surface has become hydrophobic due to collapse and compact nature of PNiPAAm above LCST. However, the contact angle is almost the same for the parent polymer at all temperatures. However, little increase in ∆θ is observed at high PNiPAAm content and high temperature, which indicates that the surface roughness is due to the compact nature of PNiPAAm above LCST. This conclusion of varying contact angles with temperature in semiIPNs is not fully supported by the cell attachment study shown below, which depends on several factors. However, the contact angle data is well-supported on cell detachment study. Network and Interaction Parameters for Physically CrossLinked Semi-IPNs. Effective cross-link density (νe) is an important aspect for the dimensional stability of hydrogel biomaterials. Because SPUU Tg is below room temperature (RT) and Tg of PNiPAAm in the hydrated state is assumed to be below RT, it can be assumed that the effective cross-link density for the semi-IPNs were obtained from stress–strain experiments using eqs 1 and 2. From the Young’s modulus (E), νe for each sample was calculated using eq 1. The apparent polymer–water interaction parameter (χ) in a swollen sample was determined from the eq 331

χ)

- [ln(1 - φ2) + φ2 + νeV1(φ0.3 2 - 0.5φ2)] φ22

(3)

where V1 is the molar volume of water (1.8 × 10-5 m3/mol). The network and interaction parameters of the current samples are presented in Table 5. For all of the samples, the values of E, νe, and χ decreased with the increasing of PNiPAAm content. Thus, water becomes a poor “solvent” at lower PNiPAAm contents and physical interactions assume more importance and contribute to the effective cross-link density, which is an elastic contribution.31 A decrease in χ with increased amounts of PNiPAAm (hydrophilic) polymer meaning that water becomes a better “solvent”. This should be the case because the hydrophobic polyurethane block is reduced when PNiPAAm content was increased. This can also be explained in terms of polymer volume fraction, the polymer volume fraction (φ2) of the semi-IPNs decreased with an increased PNiPAAm content, as presented in Table

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Table 4. Water Contact Angles of the Polymer Films at Two Different Temperatures (Mean ( SD, n ) 5) 20 °C

37 °C

sample code

θa/°

θr /°

∆θ/°

θa/°

θr /°

∆θ/°

PCL(530)(50)LDI-BDA PCL(530)(50)LDI-BDA-20 PCL(530)(50)LDI-BDA-30 PCL(530)(50)LDI-BDA-40 PCL(530)(50)LDI-BDA-50

72 ( 1.4 65 ( 1.9 47 ( 2.1 40 ( 1.6 38 ( 2.3

39 ( 1.9 36 ( 1.3 24 ( 1.6 20 ( 2.4 17 ( 2.3

33 ( 2.2 29 ( 1.5 23 ( 1.6 20 ( 1.9 21 ( 1.3

70 ( 1.3 68 ( 2.1 67 ( 2.2 70 ( 1.7 70 ( 1.5

30 ( 1.7 39 ( 1.3 44 ( 2.3 39 ( 1.8 40 ( 1.6

30 ( 1.9 29 ( 1.5 23 ( 1.6 31 ( 1.4 30 ( 1.3

Table 5. Mechanical Properties and Network Parameters of Hydrated Semi-IPNs at Equilibrium Swelling sample

composition (%)

φ2

E, MPa

G, MPa

νe (mol/m3)

Mc Kg/mol

χ

PCL(530)(50)LDI-BDA-20 PCL(530)(50)LDI-BDA-30 PCL(530)(50)LDI-BDA-40 PCL(530)(50)LDI-BDA-50

80:20 70:30 60:40 50:50

0.86 0.77 0.61 0.51

5.67 3.43 2.3 1.3

1.89 1.14 0.76 0.43

0.8 × 103 0.5 × 103 0.3 × 103 0.2 × 103

1.45 2.32 3.21 5.35

1.48 1.17 0.88 0.76

Table 6. Drug Loading and Release Kinetics Analysis from Eq 4 sample PCL(530)(50)BDA-SFa PCL(530)(50)BDA50-SFb PNiPAAm-SFb

% drug diffusion loading exponent, n 4 3.5 6.3

0.55 0.5 0.52

k

correlation coefficient, r

0.036 0.035 0.03

0.9952 0.9932 0.9951

a Drug was loaded by solvent cast method. b Drug was loaded by absorption method in saturated drug solution in ethanol.

5. This can be attributed to an increased PNiPAAm content, which led to hydrophilic polymers with higher equilibrium water content (EWC) and, hence, lower φ2. Physically crosslinked hydrogels are arising from secondary molecular forces such as hydrogen bonding, van der Waals forces, and hydrophobic interactions.32 Therefore, in the current system it is possible to assume that there would be physical crosslinking between SPUU and PNiPAAm, which makes the gels mechanically strong. All of the current semi-IPNs are soluble in DMAc solvent though PNiPAAm is slightly chemically cross-linked with MBAm. However, when films are placed in water or PBS, they swell without dissolving. Furthermore, for the aqueous environment experimental conditions described, we did not observe any mass loss of the hydrogels, indicating the absence of soluble fractions. Drug Loading. Solvent casting and absorption methods were used to load the drug into polymer samples. The results of % drug loading in PCL(530)(50)LDI-BDA, PCL(530)(50)LDIBDA-50, and PNiPAAm are shown in Table 6. Pure PNiPAAm has higher loading than semi-IPN, which is due to higher equilibrium swelling of PNiPAAm in drug solution than semiIPN. Thus, the drug loading is influenced by the solubility of the drug as well as the extent of equilibrium solvent uptake of the matrices. Drug Release. Maceration and bacterial overgrowth at the wound site is very common due to accumulation of wound exudates. To prevent bacterial infection, it is necessary to load antibacterial agent and study its release kinetics. Here, drug release kinetics are conducted in pH 7.4 PBS; to practically apply this semi-IPN films on the wound site, the surface skin temperature of 32 °C is taken into account for the drug release.33,34 Figure 5 indicates the release behavior of SF from the PCL(530)(50)LDI-BDA, PCL(530)(50)LDI-BDA-50, and PNiPAAm in pH 7.4 PBS solution at 32 °C. For comparison, PCL(530)(50)LDI-BDA and PNiPAAm are used for release experiments along with the PCL(530)(50)LDI-BDA-50. It is observed that more than 90% of the drug is released from pure PCL(530)(50)LDI-BDA within 10 h, which may be attributed to segmental motion of soft

Figure 5. Drug release versus time curves of polymer films. The error bars represent standard deviation based on triplicate analysis.

Figure 6. MTT assay of fibroblasts on semi-IPN film extracts.

segments at 32 °C. However, a two-stage process is observed from PCL(530)(50)LDI-BDA-50 and PNiPAAm. The faster release at the initial stage and slower release at the second stage. The dramatic increase in drug dissolution at the initial period might be due to the rapid swelling of PNiPAAm, which increased the surface area of PNiPAAm blocks for dissolution. The decrease of drug release from PCL(530)(50)LDIBDA-50 compared to pure PCL(530)(50)LDI-BDA may be due to interpenetrating network formation between polyurethane and PNiPAAm which restricts the mobility of chains or free volume in the network and hence decrease the release rate.

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Figure 7. Typical phase contrast microscopy images of NIH3T3 fibroblast attached to the surface of PCL(530)(50)LDI-BDA (A), PCL(530)(50)LDIBDA-20 (B), PCL(530)(50)LDI-BDA-30 (C), PCL(530)(50)LDI-BDA-40 (D), and PCL(530)(50)LDI-BDA-50 (E); after 24 and 72 h, seeding of 2.5 × 106 cells onto each of the samples (scale bar 200 µm).

To understand the type of transport phenomenon, we have analyzed the release data, that is, Mt/M∞ (before 60% release), using an empirical relationship, as in eq 4.35

( )

Mt ) ktn M∞

(4)

Here, k is a kinetic constant related to the drug-polymer interaction, and n is the exponent parameter, which gives an indication about the type of transport phenomenon. The values of k and n have been calculated by the least-squares method at the 95% confidence limit. The results of the above calculations are included in Table 6. Equation 4 is valid up to the initial 60% release of the drug, and Fickian diffusion is defined for the value of n ) 0.5; for the non-Fickian diffusion, n > 0.5. The values of n calculated for all the systems varied between 0.51 and 0.55, indicating a slight variation from Fickian transport.36 The lower k values for all the systems indicate a lesser interaction between the polymer and the drug. Cytotoxicity. Wound dressing materials should not release any agent that may be cytotoxic. To know whether the semi-IPN

film extracts are harmful to cells, the fibroblast cells were cultured in the presence of the extractables of these films over 24 h at 37 °C. MTT assays were carried out to evaluate the potential cytotoxicity of semi-IPN extracts. Aqueous extracts of all the IPNs, at concentrations of 50% (by volume), are used. The results of this study, shown in Figure 6, revealed that the cells not only remained viable but also proliferated similar to control. Cell Attachment and Proliferation. As seen in Figure 7, the films show no significant cytotoxicity to NIH3T3 cells, which are securely attached and successfully grow to confluence, depending on the number of cells, time, and composition of semi-IPN films. In other words, cell adhesion, proliferation, depends on the composition of semi-IPNs and time of incubation. It is clearly shown in Figure 7 that when 2.5 × 106 number of cells/sample are used for culture studies, and the cells are just attached to all compositions of the semi-IPN films in a 24 h incubation. However, after 72 h, incubation attached cells are successfully grown to confluence in all cases except in the case of PCL(530)(50)LDI-BDA-40 and PCL(530)(50)LDI-BDA-50.

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Figure 8. Typical phase contrast microscopy images of NIH3T3 fibroblast detachment from the PCL(530)(50)LDI-BDA-30 surface after 15 min incubation at 15 °C (scale bar 200 µm).

The decrease of cell spreading with increase of PNiPAAm content can be explained by increased water uptake that happened during cell culture experiments because we have not taken any precaution to add the culture media above LCST temperature of PNiPAAm, which lead to increased hydrophilicity (more water absorption) with increased PNiPAAm content and, thereby, reduced cell adhesion. Though we have not given enough time to swell in the culture medium, its crucial when high PNiPAAm content was used. To increase the cell adhesion and spreading on these surfaces, further incubation time may be needed or still more cells need to be used. Most importantly, all culture experiments and media used should be maintained above LCST of PNiPAAm. Surface topography may also affect many cellular processes such as cellular differentiation, cell metabolism, protein production, and phenotypic expression.37 Apart from this, it may also be possible to improve the cell adhesion by adding extracelluar matrix. More recently, thermosensitive methylcellulose (MC) hydrogels have been developed for cell sheet harvesting where the cell adhesion on MC gels have been improved by collagen coating.38 Cell Sheet Detachment. After cells reach confluence, a monolayer cell sheet formed on the surfaces of PCL(530)(50)LDIBDA, PCL(530)(50)LDI-BDA-20, and PCL(530)(50)LDI-BDA30, whereas in the case of PCL(530)(50)LDI-BDA-40 and PCL(530)(50)LDI-BDA-50, cells are not reached to confluence. However, to evaluate the detachment property of the cells from these film samples, sample dishes were placed outside of the incubator at 15 °C for 15 min, and cells were detached gradually from all the thermoreversible semi-IPN films spontaneously. For example, the time-dependence of cell detachment from the PCL(530)(50)LDI-BDA-30 is shown in Figure 8. However, no apparent cell detachment is observed in either culture dish or in PCL(530)(50)LDI-BDA (SPUU; controls, Figure not shown). Earlier it was reported that cell-detachment efficiency of NiPAAm based copolymers depends on the temperature, the highest detachment efficiency had been observed at 15 °C, the detachment of cells at low temperature is not only due to conformational change of PNiPAAm but also due to changes in morphology of the cells from a spread form to more compact, and rounded shape at low temperature leads to detachment from the substrate.10 Further decrease of temperature, for example below 15 °C, does not enhance cell detachment, as cell metabolism is suppressed at lower temperatures. It was also reported by Okano et al.4–7 that cells, or cell sheets, cultured on TCPS dishes modified by grafted PNiPAAm by irradiation method, can be detached just by decreasing temperature below LCST of PNiPAAm. However, cell sheets were transferred from PNiPAAm grafted surfaces to target place by utilizing poly(vinylidene difluoride) (PVDF) membranes as supporting materials during the transfer procedure.39 It was also stated previously that hydration changes of grafted NIPAAm at the

Figure 9. Cells detached from the thermosensitive semi-IPN films reattached and grown on the culture plate at 37 °C (scale bar 200 µm).

cell/material interface is an important initial stimulus to induce active cell detachment mediated by cellular processes.40 Because semi-IPNs exhibit temperature sensitivity and segmental mobility due to the presence of PNiPAAm and low Tg SPUU, respectively. Therefore, it is expected that cells cultured on semi-IPN films could be detached simply by decreasing the temperature from 37 °C (hydrophobic) to 15 °C (hydrophilic). The time dependence of cell sheet detachment by decreasing the temperature to 15 °C is shown in Figure 8. In the present study, cell sheets were recovered from the semi-IPN film surface within 15 min of starting the decrease in temperature. Therefore, this procedure could be useful in culturing cells for transplantation to wound sites directly without support of PVDF membrane support. The key advantages of the present system is flexible (with good mechanical properties) and thermosensitive (toward cell adhesion and detachment) even up to 0.3 mm film thickness may lead to the transfer of cells directly onto the wound site. Finally, we examined the reculture of cells, such as cell adhesion, survival, and proliferation, obtained by temperatureregulated detachment from the PCL(530)(50)LDI-BDA-30, are assessed by transferring the cells onto new tissue-culture plates. These cells are observed to be well attached to the new surface and undergo significant spreading, exhibiting viability, normal growth, and a healthy morphology of the NIH3T3 fibroblasts (Figure 9). This unique property of the membranes makes them promising substrates for cell grafting to promote wound healing. Conclusion. Transparent and flexible semi-IPN films have been prepared successfully by thermal free radical initiated polymerization. The resultant IPNs exhibit mechanical and thermal endurance and characteristics of thermosensitivity in wetting, swelling ratio, and cell adhesion, making them ideal candidates for wound dressing and cell grafting. More importantly, the cells detach from all the semi-IPN surfaces when environmental temperature reduces to 15 °C. No significant thermosensitivity in cell attachment is found in the pure PCL(530)(50)LDI-BDA. The low rates of hydration of PNiPAAm

Polymer Networks for Wound Dressing

chains due to SPUU domains in the vicinity of PNiPAAm was determined to be the cause of such temperature-dependent cell attachment and detachment behavior. Cell adhesion and detachment also depends on the composition of semi-IPNs, cell type, cell number, and time of incubation. In the near future, a cell quantification study will be conducted to optimize the conditions for effective cell adhesion and delivery. Acknowledgment. The present work was supported by a Grant in aid for the Global COE program “Science for Future Molecular systems from the MEXT”, Japan. T.T.R gratefully acknowledges Japan Society for Promotion of Science (JSPS) for the financial support in the form of a fellowship. Supporting Information Available. 1H NMR data of SPUU, PNiPAAm, and various semi-IPNs. This material is available free of charge via the Internet at http://pubs.acs.org.

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