Article pubs.acs.org/Langmuir
Injectable and Thermoresponsive Self-Assembled Nanocomposite Hydrogel for Long-Term Anticancer Drug Delivery Ying-Yu Chen,†,# Hsi-Chin Wu,‡,# Jui-Sheng Sun,§ Guo-Chung Dong,∥ and Tzu-Wei Wang*,†,⊥ †
Department of Materials Science and Engineering, National Tsing Hua University, Hsinchu, Taiwan Department of Materials Engineering, Tatung University, Taipei, Taiwan § Department of Orthopedic Surgery, National Taiwan University Hospital-Hsinchu Branch, Hsinchu, Taiwan ∥ Division of Medical Engineering Research, National Health Research Institutes, Taipei, Taiwan ⊥ Institute of Biomedical Engineering, National Tsing Hua University, Hsinchu, Taiwan ‡
S Supporting Information *
ABSTRACT: The purpose of this study is to develop an injectable thermoresponsive hydrogel system that can undergo sol−gel phase transition by the stimulation of body temperature with improved mechanical stability and biocompatibility as a controlled drug delivery carrier for cancer therapy. Hexamethylene diisocyanate (HDI) was introduced into Pluronic F127 as a chain extender to improve the mechanical stability. HDI−Pluronic F127 copolymer was then incorporated with hyaluronic acid to develop a thermoresponsive nanocomposite hydrogel system. The physiochemical properties were characterized. The anticancer drug release profile and effect to inhibit tumor cells growth were analyzed in vitro and in vivo. The results showed that HDI−Pluronic F127/hyaluronic acid thermoresponsive hydrogel could undergo sol−gel transition as temperature increased to 37 °C. The nanocomposite polymer can spontaneously self-assemble into micellar structure with size of 100−200 nm. The release of doxorubicin (DOX) from HDI−PF127/HA composite hydrogel was a zero-order profile and maintained sustained release for over 28 days. The viability of tumor cells and size of tumor significantly decreased with incubation time, indicating the potential to have a therapeutic effect for cancer therapy. The injectable thermoresponsive nanocomposite hydrogel system was biocompatible and degradable and had the slow controlled release property for anticancer drugs with potential applications in the field of drug delivery.
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INTRODUCTION Hydrogel has been discovered to possess many advantages when used in drug delivery, one of which is its unique pharmacokinetic property.1 For example, hydrogel can be used as an encapsulation device from which slow elution of drugs originates. Therefore, it is able to maintain high local concentration of drugs in the surrounding tissue over an extended period of time. Particularly, in recent years there is a growing interest with in situ gel-forming hydrogel system as candidate for injectable drug and cell delivery.2,3 Injectable hydrogel has the ability to undergo reversible sol−gel phase transition in response to the external physical or chemical stimuli such as temperature,4−6 pH,7,8 ionic concentration, and light.9−11 For clinical applications, injectable delivery systems not only can take the shape of the wound cavity but also offer the advantage of minimally invasive surgery and minimize the possibility of scar formation, thus reducing the risk of infection and complications. Pluronic F127 (PF 127) is a triblock copolymer composed of hydrophobic propylene oxide (PPO) and hydrophilic ethylene oxide (PEO).12 Since PF127 has hydrophobic and hydrophilic property simultaneously, the polymer molecules can selfassemble into micelles in aqueous solutions above critical © 2013 American Chemical Society
micelle concentration. It has been found that the critical micelle concentration decreased with increasing temperature.13−15 As the temperature increases, more and more micelles appear and aggregate to gel form, indicating that PF127 has the ability to undergo sol−gel phase transition by the change of environmental temperature.16 Gelation temperature of PF127 can also be controlled to approach body temperature by modulating the concentration of the polymer solution. Although PF127 has thermosensitive property, there are still issues to be solved such as weak mechanical strength and stability due to the delicate network of Pluornic F127 hydrogel that is only held together by molecular entanglement and hydrophobic force.17 Hyaluronic acid (HA) is a high molecular weight polysaccharide found in the extracellular matrix, especially in soft connective tissue.18 Hyaluronic acid is a linear biopolymer composed of repeating disaccharides of D-glucuronic acid and N-acetyl-D-glucosamine. It is found in previous studies to be a good biocompatible natural polymer and also could up-regulate phenotype expression for certain cells.19−21 The properties of Received: June 2, 2012 Revised: February 22, 2013 Published: February 26, 2013 3721
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Micelle Formation and Size Distribution. Pluronic which has amphiphilic property can self-assemble into micelles in aqueous solutions. For comparing the difference of micelles before and after extending the PF127 polymer, TEM was used to observe the micellar morphology. The aqueous polymer solution (0.1 wt %) of PF127 or HDI−PF127 was dropped onto a carbon-coated copper grid and then incubated in oven for 2 min at 37 °C. Subsequently, the grid was dried under vacuum condition for 4 h, and then phosphatotungstic acid (0.5 wt %) was dropped onto the grid for 2 min at room temperature. The copper grid was finally air-dried overnight at room temperature before inspection under the microscope. The morphology of Pluronic copolymers (HDI−PF127) was examined using a high resolution transmission electron microscope (H-7500, Hitachi). Also, the micellar size of the HDI-PF127 was determined by dynamic light scattering (DLS) measurements. Preparation of Hexamethylene Diisocyanate−Pluronic F127/Hyaluronic Acid (HDI−PF127/HA) Composite Hydrogel. The HDI−PF127/HA composite was prepared by dissolving different amounts of HDI−PF127 in deionized water by mixing under continuous stirring at 4 °C until a clear solution was obtained, followed by the addition of HA (MW: 1.2 × 106 Da) into HDI− PF127 solution to obtain the final concentration of HA at 0.5% (w/w). Sol−Gel Phase Transition Diagram. Sol−gel transition of PF127 and HDI−PF127/HA hydrogel with temperature was determined by the test tube inverting method.26 Each sample was prepared in a 1.5 mL Eppendorf containing 0.5 mL of polymer solution. The prepared samples were placed into dry bath. The temperature was lowered to 4 °C and then gradually increased. The transition temperature was determined at the absence of visual solution fluidity by inverting the Eppendorf for 1 min. Sample was heated via stepwise temperature increment, and data were recorded for every 2 °C. Evaluation of Rheological Behavior. The rheological properties were examined by a rheometer (AR2000ex system, TA Instruments) equipped with parallel plate sensor. Samples were observed in oscillatory mode. The storage (elastic) modulus G′ and loss (viscous) modulus G″ versus temperature were measured at a gap of 0.1 mm and 0.005 shear strain. The heating rate was set at 2.25 °C/min, while the frequency was set at 1 Hz. Degradation in Vitro. The in vitro degradation of the HDI− PF127/HA composite hydrogel was examined as follows. Vials containing 1 mL of HDI−PF127/HA solutions were incubated at 37 °C for 5 min for gel formation. Subsequently, 2 mL of PBS at pH 7.4 was added into sample vials and incubated at 37 °C. At predetermined time points, 2 mL release content was taken out and replenished with equal volume of fresh PBS. The carbazole method proposed by Bitter and Muir was used to determine the degradation rate by measuring HA release concentration.27 In brief, 0.955 wt % sulfuric acid solution and 0.125 wt % carbazole solution were used in this assay. 0.5 mL release content was added into 2.5 mL of 95% ethanol and shaken vigorously by vortex. Then, the precipitation of hyaluronic acid was centrifuged at 2500 rpm for 25 min, and the supernatant was discarded. The precipitation of HA was redissolved in 1 mL of DI water. 3 mL of sulfuric acid solution was added into test tubes and cooled to 4 °C. 0.5 mL of sample solution was added into test tube and shaken vigorously for 1 min in an ice−water bath. The solution mixture was then heated to 95−100 °C in boiling water bath for 10 min and cooled down to 4 °C. Subsequently, 0.1 mL of carbazole solution was added into test tube and shaken vigorously by vortex for 1 min. 150 μL of solution mixture was added to the 96-well plate, and the optical density was detected by a microplate reader at a wavelength of 550 nm. In Vitro Release of Doxorubicin. Doxorubicin (DOX) is a cytotoxic drug widely used as the treatment for several solid tumors. In this study, DOX was selected to suppress the proliferation of tumor cells and to estimate the sustained release potency of HDI−PF127/ HA hydrogel. 500 μg of DOX was added into 1 mL of HDI−PF127/ HA solution. Subsequently, the solution was incubated at 37 °C for 5 min to form gel. After gel formation, 2 mL of PBS at pH 7.4 was added into sample vial and incubated at 37 °C. At predetermined time points, 2 mL of release media was taken out and replenished with equal
biological activity, nontoxicity, low immune response, biocompatibility, and biodegradability make HA to be a valuable material for biomedical applications. Of note is that HA has been largely investigated as a target-specific material because many malignant cancer cells overexpress HA receptors such as cluster determinant 44 (CD44),22,23 and thus incorporation or conjugation of HA can be used as targeting ligand. Park et al. have demonstrated thermoresponsive hydrogels based on HA and Pluronic by photopolymerization.24 In their study, although HA/Pluronic composite hydrogels exhibited temperature-dependent swelling and collapse behaviors, the ability to be used for cancer therapy was not in detail addressed. Cohn et al. also developed reverse thermoresponsive polymeric systems by polymerization of PEO and PPO segments with HDI.25 This study was focused on physiochemical properties investigation while the biological effect of HA was not much mentioned. In our current study, hexamethylene diisocyanate (HDI) was introduced into Pluronic F127 polymer for crosslinking the polymer chain to interconnect with each other. The introduction of HDI into Pluronic F127 is expected to improve the mechanical properties of hydrogel. The thermogelation behavior occurred at 37 °C, and the hydrogel structure was maintained for 30 days which could achieve a sustained release over a prolonged period. The thermosensitive HDI-PF127/HA composite hydrogel with adequate physiochemical and biological properties was used as a delivery carrier for sustained release of anticancer drugs in cancer therapy.
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EXPERIMENTAL SECTION
Materials. Pluronic F127, 1,6-diisocyanatohexane, stannous(II) 2ethylhexanoate, chloroform, petroleum ether, ethyl ether, and doxorubicin were purchased from Sigma-Aldrich (St. Louis, MO). The LIVE/DEAD Assay Kit was purchased from Invitrogen (Eugene, OR). The WST-1 Cell Proliferation Assay Kit was purchased from GBiosciences (St. Louis, MO). All other chemicals were used as received. Synthesis of Hexamethylene Diisocyanate−Pluronic F127 (HDI−PF127) Copolymer. For improving the strength and stability of Pluronic F127 hydrogel, hexamethylene diisocyanate (HDI) was chosen as a chain extender to connect the bulk Pluronic F127 polymer chains. The method was modified from Cohn et al.25 In brief, 15 g (0.0012 mol) of Pluronic F127 (M = 12 600) was added into a twonecked flask, dried under vacuum for 3 h, and then heated to 85 °C. Next, 200 μL of HDI and 100 μL of SnOct2 were added into the reaction mixture and reacted at 85 °C for 30 min under mechanical stirring and vacuum condition. The polymer produced was then dissolved in chloroform and precipitated in petroleum ether/ethyl ether mixture in the volume ratio of 1:1. Finally, the precipitate was filtered and washed repeatedly with petroleum ether followed by drying under vacuum at room temperature. Characterization of HDI−PF127 Copolymers. The fabricated HDI−PF127 sample was investigated with Fourier transform infrared spectroscopy (Spectrum 100, PerkinElmer) to confirm the appearance of urethane bond functional group formation. In brief, 5 mg of sample was filled in load cell, and the press was applied to sample. For each spectrum, four scans between the wavelength of 600 and 4000 cm−1 region were recorded in the transmission mode. On the other hand, the molecular weight of HDI−PF127 copolymers was determined by gel permeation chromatography (GPC). In brief, 20 μL of Pluronic copolymer solution dissolved in THF (1%) was injected into GPC column. The elution solvent was THF, and the flow rate was 1 mL/ min. The degree of polymerization (DOP) of PF127 was defined as
DOP of F127 =
mol wt of HDI−PF127 mol wt of PF127 3722
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volume of fresh media. 150 μL of release media was added to the 96well culture plate, and the optical density was read at a wavelength of 490 nm. Inhibition Effect on Tumor Cells Proliferation. Inhibition of tumor cells proliferation was evaluated with human breast cancer cells (MCF-7). MCF-7 cells were cultivated in DMEM/F12 with 10% FBS and 1% penicillin/streptomycin. Medium was changed every other day until the cells achieved 80% confluence. The cells were then seeded into 24-well cell culture plates with 1 mL of culture medium per well (5 × 104 cells/well). After 24 h of culture, Transwell with 200 μL of hydrogel encapsulating 100 μg of DOX was mounted on 24-well plates and cocultured with cells. Hydrogel without encapsulating DOX was used as control. The LIVE&DEAD kit (Gibco) is a two-color fluorescence cell viability assay. The LIVE/DEAD assay reagents was formulated according to manufacturer’s protocol. At the designated time points, the wells were washed twice with PBS and 100 μL of the combined LIVE/DEAD assay reagents was added. After the addition of LIVE/ DEAD assay reagents, the plate was incubated for 30 min in room temperature and observed with fluorescence microscope. WST-1 assay (G-Biosciences) which is a measurement of survival by metabolic activity was used to assess cell survival. At the designated time points, WST-1 solution was added to each well, and the plate was incubated for 4 h. 150 μL of the incubated medium was transferred to a 96-well culture plate, and the optical density was read using a microplate reader at a wavelength of 440 nm. The negative control was performed along with the experimental groups. In Vivo Animal Study. This study was conducted in accordance with the regulations of the Institutional Animal Care and Use Committee (IACUC) at Laboratory Animals Center in National Tsing Hua University. Tissue collection was performed according to a protocol approved by the National Tsing Hua University Institutional Review Board. In brief, 5 wt % HDI−PF127/HA hydrogel in PBS was administered by dorsal subcutaneous injections in male athymic nude mice to evaluate biocompatibility in vivo. Each injection was 0.25 mL in volume and performed through a hypodermic syringe. For histological evaluation, animals were sacrificed and excised gel implants were rinsed in PBS and fixed in 10% buffered formalin at 37 °C. The gel explants and surrounding tissues were dehydrated in a graded series of alcohols and embedded in paraffin. The transverse paraffin was sectioned through the center of gel implants at 5 μm thickness and histologically processed using hematoxylin and eosin (H&E) stains. The immunostain CD68 was selected as a marker for inflammatory macrophages. On the other hand, for antitumor efficacy study when tumor size reached 200−300 mm3, mice were given DOX (10 mg/kg) intratumorally either in 100 μL of PF127/HA or HDI-PF127/HA hydrogel solution. 100 μL of free DOX and PBS was used as control groups for comparison. At predetermined time points, mice were anesthetized. To evaluate antitumor efficacy, tumor size was measured and animals were monitored for survival or signs of toxicity for 4 weeks postdrug administration. Tumor size was determined using the formula: tumor volume = width2 × length/2. Animals were euthanized when they became moribund with severe weight loss, significant ascites, or any other sign of significant toxicity. Statistical Analysis. All data are expressed as mean standard error of the mean unless otherwise indicated. The significance of the effect of selected parameters on the outcome variables was analyzed by multifactor analysis of variance (ANOVA). Group comparisons were made by Fisher’s PLSD. Statistical significance was accepted at a level of p < 0.05.
into Pluronic F127 as the extender to interconnect the polymer chains. The specific ratio was used and determined because of the consideration of mechanical property of nanocomposite hydrogel system so that long-term drug release can be achieved with our modification approach. As shown in the Supporting Information (SI Figure 1), the FT-IR spectrum of HDI-PF127 indicates that peaks at 1733 cm−1 were assigned to CO stretching vibration of carbonyl group. The appearance of carbonyl group was belonged to the urethane linkage which was formed when the hexamethylene diisocyanate reacted with Pluronic F127. There was no obvious peak at 1733 cm−1 when comparing with the spectrum of PF127, demonstrating that the copolymerization of Pluronic F127 was successful in HDI−PF127. 1H NMR analysis was also performed to confirm the changes in chemical structure of HDI-PF127. As shown in Supporting Information (SI Figure 2), the spectrum of HDI−PF127 presented similar signals for the ethylene protons of PEO segments and methyl protons of PPO segment at 3.65 and 1.02 ppm, respectively, when compared with Pluronic F127. Of note is that three specific peaks of HDI−PF127 representing the methylene protons of aliphatic chain from hexamethylene diisocyanate were also observed at 1.3, 1.5, and 3.2 ppm from the NMR spectrum. This result indicates that the elongation of Pluronic F127 polymer chain was successfully cross-linked by HDI. The degree of polymerization was determined by GPC. It was found that the molecular weight increased from 10 742 (PDI = 1.21) of the native PF127 to 73 527 (PDI = 1.10) of the HDI− PF127. The degree of polymerization calculated from the ratio of Mw (HDI−PF127)/Mw (PF127) was 6.8. The degree of polymerization represented that every HDI−PF127 copolymer chain consisted of 6.8 units of cross-linked Pluronic F127. Micelle Formation and Size Distribution. TEM images of PF127 and HDI−PF127 are shown in Figure 1. Pluronic copolymer molecules self-assemble into micelles in aqueous solutions which consist of a hydrophobic PPO core and a
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RESULTS Characterization of HDI−PF127 Copolymers. In recent years, injectable biomaterials are widely studied and hold great promise in both the fields of drug delivery and tissue engineering due to the minimally invasive nature with which they can be delivered and for localized injection to an affected site.28−31 Hexamethylene diisocyanate (HDI) was introduced
Figure 1. TEM images of (a) PF127 and (b) HDI−PF127. (c) Sol− gel phase transition diagram of PF127 and HDI−PF127/HA. 3723
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hydrophilic PEO corona. From the TEM images in Figure 1a, PF127 polymers were observed to form spherical dark spots without any significant aggregation, and the micellar size of PF127 was about 30 nm. The spherical dark spots were also observed in the images of HDI−PF127, which proved that HDI−PF127 still formed micelles in the aqueous solutions. The spherical dark spots were about 100 nm in diameter (Figure 1b). This phenomenon might be resulting from the cross-linking of PF127 polymers by HDI and formed the close packing of HDI−PF127 micelles. The copolymer chains may be incorporated into the HDI−PF127 micelles, and then the multiblocks can form connections between separated micelles (Supporting Information, SI Figure 3). The nature of these connections may occur either through single chain spanning over two micelles or entangled chains of different micelles, generating interconnecting loops.32 From the result measured by dynamic light scattering (DLS), it was found that the size distribution of Pluronic F127 was ranged within 30−70 nm (PDI = 1.0), while HDI−PF127 copolymers were in diameter around 100−283 nm. The result was comparable with that shown in TEM images. The diameters of micelles observed by TEM were generally smaller than those obtained by DLS. This is because the diameter of micelles obtained by DLS reflected the hydrodynamic diameter of swelling micelles, while data observed by TEM was the diameter of dried micelles due to TEM sample processing procedure. Sol−Gel Phase Transition Diagram. Above the gelation concentration and temperature, the self-assembly of closely packed spherical PF127 or HDI−PF127 micelles has the ability to undergo thermosensitive sol−gel phase transition. The sol− gel transition of this HDI−PF127/HA thermoresponsive hydrogel can be achieved from seconds to minutes depending on the cross-linking degree and concentration of PF127. From Figure 1c, we can observe that HDI−PF127/HA hydrogel became solidified at 37 °C compared to the liquid form at 4 °C. From the diagram shown in Figure 1c, it was found that the HDI−PF127/HA hydrogel exhibited substantially different sol−gel transition behavior from Pluronic F127 with altered critical gelation concentration and critical gelation temperature. The sol−gel phase transition of HDI−PF127/HA showed lefthand shift compared to PF127. The gradual left-hand shift of the phase curve means that the critical gelation concentration of HDI−PF127/HA hydrogel at the specific temperature was decreased, and the gelation temperature was lower at the specific concentration when compared to PF127. The results described above can be related to the structural stability of individual spherical Pluronic micelles that self-assembled and were closely packed to form a three-dimensional gel structure at a critical gelation concentration and temperature. Thus, the structural stabilization of packed micelles might be achieved at lower critical gelation concentration and temperature.18,29 We also found that high temperature environment made HDI− PF127/HA micellar structure broken down, and the superhydrophobicity of block copolymers resulted in precipitation of HDI−PF127. Evaluation of Rheological Behavior. The mechanical properties of HDI−PF127/HA hydrogel were examined by oscillatory rheological experiments as a function of temperature in Figure 2. The storage modulus G′ provides information regarding the elasticity or energy stored in the material during deformation, whereas the loss modulus G″ describes the viscous character or energy dissipated as heat. For hydrogel
Figure 2. Storage modulus (G′) and loss modulus (G″) of the (a) 15%/0.5%, (b) 10%/0.5%, and (c) 5%/0.5% HDI−PF127/HA hydrogel in PBS.
with the property of sol−gel transition, we employ the value of tan δ (δ = G″/G′) to determine the state of solid hydrogel. When the value of tan δ is larger than 1, the hydrogel is in a solution form; when the value of tan δ is less than 1, the hydrogel is in a gel form. The intersection point of G′ and G″ is defined as the gel point. It could be found that the temperature of the gel point increased as the HDI−PF127 concentration decreased. The results were 30, 33, and 37 °C for 15, 10, and 5 wt %, respectively. This result was consistent with data provided by the phase diagram. The value of the intersection of G′ and G″ altered with the variation of HDI−PF127 concentration, indicating that the mechanical properties could be tuned by the concentration of HDI−PF127. Figure 3 depicts strain sweep data for PF127 and HDI− PF127. It could be found that the yield point of PF127 gel was noted at very low strain value (0.01). However, HDI−PF127/ HA hydrogel possessed more resistance for the external applied shear force; therefore, larger strain value (0.1) was required to be applied for making the gel yield when compared with PF127. In addition, the decrease of the HDI−PF127 concentration resulted in the right shift of yield point, meaning that the HDI− 3724
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Figure 3. Diagram of strain versus G′ value of PF127 and different concentrations of HDI−PF127/HA hydrogel system.
In Vitro Release of Doxorubicin. The In vitro release profile of doxorubicin in different HDI−PF127/HA compositions was investigated and is shown in Figure 4b. HDI−PF127/ HA hydrogel containing free doxorubicin could sustain release for at least 28 days. It was until 30 days that ∼100% of encapsulated doxorubicin was released from the hydrogel. The delivery rate of doxorubicin during this period was about 20 μg/day. The constant release profile of HDI−PF127/HA hydrogel approximated zero-order release dynamics which might prove to be beneficial in that accumulation level created from the slow controlled release elution of drugs. This preserves a high local concentration of anticancer drug in the surrounding tissues over an extended longer period of treatment time. The applicable release mechanism of DOX from HDI− PF127/HA hydrogel is that the entrapped DOX in the outer corona layer may diffuse out of the mesoporous pores in the swollen surface layer. As for the DOX entrapped in the inner core layers of the hydrogel, diffusion occurs at a much slower rate. The diffusion-controlled mechanism of DOX released from the inner layer was regarded as secondary release. Higher concentration HDI−PF127/HA hydrogel also had a denser internal network structure, and the release model was more similar to a zero-order release curve.35 Effect on the Inhibition of Tumor Cells Proliferation. A human breast cancer cell line (MCF-7) was employed to investigate the in vitro activities of doxorubicin released from HDI−PF127/HA hydrogel matrix. From the results of WST-1 assay (Figure 5), it could be found that MCF-7 cells cultured in the presence of hydrogel without DOX maintained their viability, indicating that the HDI−PF127/HA hydrogel had no inhibitory effect to the growth of cancer cells. However, MCF-7 cell survival rate was significantly decreased when they were exposed to the DOX released from hydrogel over 3 days and
PF127/HA hydrogel with lower HDI−PF127 concentration could endure higher deformation and sustain its gel state even though it was very soft. Degradation Behavior in Vitro. Degradation of HDI− PF127/HA hydrogel was examined by measuring the release amount of hyaluronic acid. Figure 4a shows the remaining percentage of composite HDI−PF127/HA and PF127 hydrogels. PF127 hydrogel was observed to be completely dissociated within 5 days, even with such high 15 wt % concentration. This was due to rapid hydrolytic dissolution and disintegration of the PF127 gel structure upon incubation in the PBS buffer solution. In contrast, HDI−PF127/HA hydrogel exhibited higher resistance against degradation. It could be observed that HDI−PF127/HA hydrogel had yet to completely decompose after 28 days, and the degradation process was gradually and could be modulated by changing the concentration of HDI− PF127, in this case 5 and 10 wt %. During the incubation period, a distinct swollen layer could be visually observed on the outer hydrogel surface. It was likely that this surface layer was preferentially degraded, so-called surface degradation, rather than the interior region of the hydrogel process. This was due to the fact that the surface layer was more directly exposed to a larger amount of buffer media and the incorporation of hydrophilic hyaluronan and PEO polymers in this hydrogel system, resulting in more susceptible to decrease in polymer concentration to below the critical gelation concentration. Therefore, modulating the concentration of HDI−PF127 influences the concentration on the surface layer and leads to changes in degradation resistance. Moreover, the biodegradation rate of hydrogel and drug release can be easily adjusted via altering the HDI−PF127 concentration, thus improving its application in the field of biomedicine.33,34 3725
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Figure 4. (a) Degradation rate of 15 wt % PF127 and different concentration of HDI−PF127/HA composite hydrogels in PBS at 37 °C (n = 3). (b) Release profile of DOX from 5 and 10 wt % HDI−PF127/HA composite hydrogels in PBS at 37 °C (n = 3).
continued to decrease until 14 days in HDI−PF127/HA with the DOX group. On the other hand, although the MCF-7 cell viability was inhibited in the first 7 days in PF127 with DOX group, these cancer cells became proliferative thereafter. This suggests that PF127 hydrogel without cross-linking by HDI may have been degraded, the effect on the growth inhibition of MCF-7 was maintained for only 7 days, and then no inhibitory effect due to lack of DOX. These findings demonstrated that the released DOX could preserve its function against tumor cells when encapsulated into the HDI cross-linked PF127/HA hydrogel. Inhibition of proliferative activity of tumor cells in vitro persisted for at least 14 days. The morphology and cell numbers of MCF-7 after exposing to the DOX were also fluorescently stained with LIVE&DEAD assay under optical microscope (Figure 6). The phenomenon of cell apoptosis was observed, and the number of MCF-7 cells cultured in the presence of hydrogel with DOX was significantly decreased with the incubation time. When using the LIVE& DEAD assay, live cells were fluorescently labeled in green. As we can see from Figure 6, the total number of live cells stained with green was observed to decrease with time
Figure 5. Inhibitory effect on MCF-7 cells proliferation by WST-1 assay in different culture conditions (n = 3, *p < 0.05).
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Figure 7. Histological pictures of subcutaneous implantation of HDI− PF127/HA hydrogel into nude mice at 1, 2, and 3 weeks ((a) 1 week, (c) 2 weeks, (d) 3 weeks) with H&E stain (scale bar: 200 μm). (b) Immunohistological stain of CD68 marker for the demonstration of macrophages in terms of inflammation response at week 1 (star: remaining hydrogel; scale bar: 50 μm).
Antitumor Efficacy of DOX after Intratumoral Administration. The antitumor efficacy of free DOX and DOXloaded hydrogels was examined with MCF-7 human breast tumor bearing nude mice. The tumor growth rates of mice treated with free DOX, DOX-loaded HDI−PF127/HA hydrogel, DOX-loaded PF127/HA hydrogel, and PBS are shown in Figure 8. The free DOX and DOX-loaded hydrogel groups exhibited similar effectiveness in preventing tumor growth in the first 7 days. However, after that the DOX-loaded HDI− PF127/HA hydrogel was the only group demonstrated better growth inhibition of tumor volume in comparison to free DOX and DOX-loaded PF127/HA hydrogel due to the loss of free DOX and fast degradation of un-cross-linked PF127/HA hydrogel. Tumor volumes were decreased up to 70.0% by DOX-loaded HDI-PF127/HA hydrogel, while they increased in size about 250% in PF127/HA hydrogel and about 210% in free DOX groups at the end of 28 days (Figure 8a). From the above results, it is suggested that DOX-loaded HDI−PF127/ HA hydrogel showed higher antitumor efficacy and therapeutic effects. Figure 8b shows the survival of the mice during the investigative period. As it can be seen, by day 28 the survival of the DOX-loaded HDI−PF127/HA group was significantly higher than other groups (p < 0.01). It was noted that the survival rate in free DOX group was still apparently decreased during the late stage of study period. This may be due to the systemic cytotoxic effect of free DOX.
Figure 6. MCF-7 cultivation with HDI−PF127/HA hydrogel incorporated with or without DOX treatment.
from day 1 to day 7 in the group of HDI-PF127/HA hydrogel containing DOX, while the group of cells in HDI−PF127/HA hydrogel without DOX was observed to increase in numbers. In Vivo Biocompatibility. The addition of HA in the hydrogel reduce the cytotoxicity of our Pluronic polymer and benefit with its hygroscopic property in water absorption which may help in swelling to fit the defect size when implanted in vivo.36 Furthermore, HA has been investigated as a targetspecific ligand since many malignant cancer cells overexpress HA receptors such as cluster determinant 44 (CD44), and thus incorporation or conjugation of HA can be used for targeting purpose in the future. In previous studies, it was found that PF127 reduced cell viability when the concentration reached the critical gelation concentration (15 wt %).37 In our study, the developed thermoresponsive HDI−PF127/HA composite hydrogel system has a lower critical gelation concentration (about 5 wt %) which decreases the cytotoxicity that may be originally resulted from PF127. HDI−PF127/HA hydrogels were implanted into the dorsal subcutaneous region of athymic nude mice for 1, 2, and 3 weeks to investigate in vivo biocompatibility (Figure 7). The animal experiments confirmed the availability of in situ gel formation after injection. The representative histological images of the HDI−PF127/HA hydrogel after implantation was imaged and displayed. The immunohistochemistry staining of the implanted hydrogel and nearby skin tissue is presented in Figure 7b. A significant amount of the hydrogel remained, and no severe macrophage infiltration was noticed in the subcutaneous tissue. After 2 and 3 weeks, there is also no sign of chronic inflammation phenomenon (Figure 7c,d).
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CONCLUSIONS In this study, HDI−PF127/HA nanocomposite hydrogel with improved mechanical strength and stability was successfully developed by introducing HDI as a chain extender cross-linking agent. The HDI−PF127/HA nanocomposite hydrogel possessed the thermoresponsive property to be gelationed at the temperature near to physiological condition, and the degradation time of hydrogel can be extended and modulated by varying the concentration of HDI−PF127. The results showed that HDI−PF127 polymer can spontaneously selfassemble into micellar structure with size of 100−200 nm. The release of anticancer drug DOX from HDI−PF127/HA 3727
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ACKNOWLEDGMENTS
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REFERENCES
Article
The authors thank the funding support from NSC 100-2628E007-002-MY3.
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Figure 8. Statistical analysis of in vivo study on the change of (a) tumor size and (b) mice survival rate after implantation for 28 days in different study groups (n = 4).
composite hydrogel was a zero-order profile and sustained release for over 28 days. When cocultured with MCF-7 human breast tumor cells, the results showed that the viability of tumor cells significantly decreased with incubation time, indicating the potential to have therapeutic effect for cancer therapy. The animal study was also demonstrated to reveal antitumor efficacy for 4 weeks. In brief, this thermoresponsive injectable nanocomposite hydrogel system might have considerable potential applications in the field of tissue engineering and drug delivery system and benefit in clinical surgery to take the irregular shape of the wound cavity and offer the advantage of avoiding complicated surgical procedures.
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ASSOCIATED CONTENT
* Supporting Information S
FT-IR spectra, 1H NMR spectra, and the thermoresponsive behavior of HDI−PF127/HA composite hydrogel. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel +886-3-5715131 ext 33856; Fax +886-3-5722366; e-mail
[email protected]. Author Contributions #
Y.-Y.C. and H.-C.W. contributed equally to this work.
Notes
The authors declare no competing financial interest. 3728
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