Doxorubicin Hybrid

Feb 2, 2015 - The purpose of this study was to fabricate a safe and effective doxorubicin (DOX)-delivery system for focal cancer chemotherapy. A novel...
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Self-Assembling Polymer Micelle/Clay Nanodisk/Doxorubicin Hybrid Injectable Gels for Safe and Efficient Focal Treatment of Cancer Koji Nagahama,* Daichi Kawano, Naho Oyama, Ayaka Takemoto, Takayuki Kumano, and Junji Kawakami Department of Nanobiochemistry, Frontiers of Innovative Research in Science and Technology, Konan University, 7-1-20 minatojima-minamimachi, Kobe 650-0047, Japan S Supporting Information *

ABSTRACT: The purpose of this study was to fabricate a safe and effective doxorubicin (DOX)-delivery system for focal cancer chemotherapy. A novel biodegradable injectable gel was developed through self-assembly of poly(D,Llactide-co-glycolide)-b-poly(ethylene glycol)-b-poly(D,L-lactide-co-glycolide) (PLGAPEG-PLGA) copolymer micelles, clay nanodisks (CNDs), and DOX. We discovered that DOX loaded in the hybrid gels acts as an anticancer drug and as a building block to organize new gel networks. Accordingly, long-term sustained release of DOX from hybrid injectable gels without initial burst release was achieved. Moreover, it was revealed that the DOX incorporated into gel networks controls its own release profile. This hybrid injectable gel is a self-controlled drug release system, which is a novel concept in controlled drug release. Importantly, a single injection of PLGA-PEGPLGA/CND/DOX hybrid gel provides long-term sustained antitumor activity in vivo against human xenograft tumors in mice, suggesting the potential of hybrid gels as a valuable local DOX-delivery platform for cancer focal therapy.

1. INTRODUCTION Among the various antitumor drugs used clinically, doxorubicin (DOX), an anthracycline antibiotic, is crucial in the treatment of a wide range of neoplasms such as breast, ovarian, and gastric cancers. DOX is therefore the most commonly used drug in cancer chemotherapy.1−3 In order to improve the therapeutic efficacy of DOX, numerous DOX-delivery systems using nanoparticles, including polymeric micelles and liposomes as blood circulating nanocarriers, have recently been widely studied.4−9 The use of these nanocarriers improved the antitumor activity of DOX because they prolonged both the blood circulation time and EPR (enhanced permeability and retention) effect. However, there are concerns regarding the short-term and long-term toxicity arising from the repeated administration of synthetic nanocarriers, in addition to the toxicity of DOX itself.10 Furthermore, there are inherent difficulties in achieving a high DOX loading per carrier.11−13 Hence, large dosages or repeated administration of DOXloaded nanocarriers are required, in order to achieve the desired therapeutic efficacy, while can cause severe side effects. Taken together, the development of an alternative DOX-delivery system remains urgent and challenging. Recently, injectable in situ-forming gels composed of biodegradable polymers have been proposed as carriers for direct local DOX delivery to the tumor environment.14−20 Focal DOX treatment of solid tumor using optimized biodegradable injectable gels is a promising method because it allows (i) targeted delivery of DOX to the tumor environment in a minimally invasive manner; (ii) rapid © XXXX American Chemical Society

achievement of an effective concentration of DOX at the tumor environment; (iii) reduced frequency of DOX administration; (iv) potential reduction of harmful side-effects; and (v) gradual decomposition and removal of the biodegradable gels. Long-term sustained release of DOX without an initial “burst release” following administration is required in order to ensure the efficacy of focal DOX treatment using injectable gels. However, most injectable gels reported previously resulted in DOX burst release, and long-term sustained release was not achieved. These polymer gels entrap nanosized water-soluble DOX hydrochloride salt in the microsized polymer gel network. Therefore, water diffuses across the gel network, causing gel swelling and uncontrolled diffusion of DOX from the gel network, resulting in burst release. Seib et al. recently reported DOX-loaded silk hydrogels and prolonged release of DOX over 4 weeks without burst release in vitro;19 and no swelling was observed for these silk gels (2 and 4 wt %) after a 24 h incubation in phosphatebuffered saline (PBS) at 37 °C. That the silk hydrogels do not swell appears to be a crucial factor for the lack of burst release, although this was not discussed in that paper. However, the formation of DOX-loaded silk hydrogel from precursor solution requires 60 min; consequently, incomplete gelation at the injection site would result in leakage of DOX to normal tissues. Lee et al. reported that injectable gels formed through the Received: December 8, 2014 Revised: January 28, 2015

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Figure 1. (A) Structures of PLGA-PEG-PLGA. (B) Schematic illustration of hydrogel networks of PLGA-PEG-PLGA copolymer gel, PLGA-PEGPLGA/CND gel, and PLGA-PEG-PLGA/CND/DOX hybrid gel.

hydrophobic interactions between DOX-loaded liposomes and hydrophobically modified chitosan.16 Long-term DOX release over 10 days without burst release was observed because the liposomal bilayers prevent uncontrolled DOX diffusion. However, these gel networks become disrupted under shear stress, resulting in the gels readily converting into a viscous solution, so liposomal DOX gels are unsatisfactory for in vivo use. Consequently, effective focal DOX treatment of solid tumor urgently requires an alternative injectable gel that exhibits no burst release of DOX and supports the long-term sustained release of the drug. This gel should exhibit the advantageous characteristics of injectable in situ-forming gels. We recently developed a new class of nanocomposite-type injectable in situ-forming gel that form through hierarchical self-assembly of biodegradable poly(D,L-lactide-co-glycolide)-bpoly(ethylene glycol)-b-poly(D,L-lactide-co-glycolide) copolymer micelles, PLGA-PEG-PLGA (Figure 1A), and clay nanodisks (CND).21 An aqueous solution of the nanocomposites instantaneously underwent an irreversible sol−gel transition in response to body temperature, and the obtained gel was fully cell compatible. Importantly, the nanocomposite injectable gels dis not swell, whereas PLGA-PEG-PLGA gels used as a negative control showed 150% swelling after 24 h incubation in PBS at 37 °C. Furthermore, the nanocomposite gel exhibited unique molecular adsorption ability, with the CNDs strongly adsorbing compounds such as including DOX, amoxicillin, pyrene, proteins, and DNA due to the large and highly charged plane surface area (>350 m2/g) of the CNDs. Consequently, these biodegradable nanocomposite injectable gels may allow long-term sustained release of DOX without burst release and, thus, could be effective as local DOX-delivery carriers for focal chemotherapy of solid tumor. Here, we examined the thermogelling properties of PLGA-PEG-PLGA/ CND/DOX hybrid aqueous solutions, and the physicochemical properties, gel network structures, degradation profiles, and DOX release profiles of hybrid injectable gels loaded with different DOX concentrations. Moreover, the long-term antitumor activities of the gels were examined by in vitro and in vivo experiments. Interestingly, the DOX incorporated into hydrogels aced as cross-linker to organize new gel networks by intercalating between CNDs (Figure 1B). Moreover, the DOX loaded in the gel controlled its own release profile. Therefore, here, we propose a novel concept regarding controlled drug release from injectable gels, which we term a self-controlled drug release system. To the best of our knowledge, this paper is the first to report the molecular design of injectable gels

utilizing drug molecules as building blocks to enhance its functionality of drug delivery gel.

2. EXPERIMENTAL SECTION 2.1. Materials. PEG (Mw: 1.5 kDa and 3.0 kDa), glycolide, and tin 2-ethylhexanoate were purchased from Sigma-Aldrich Japan (Tokyo, Japan). D,L-Lactide was purchased from Musashino Chemical Laboratory, Ltd. (Tokyo, Japan). These materials were used as received without further purification. DOX in the form of DOX·HCl and all solvents used in this study were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Laponite was kindly supplied by Wilbur-Ellis Co. Japan Ltd. (Tokyo, Japan). 2.2. Synthesis of PLGA-PEG-PLGA Copolymers. PLGA-PEGPLGA was synthesized through bulk ring-opening copolymerization of D,L-lactide and glycolide using tin 2-ethylhexanoate as a catalyst. Briefly, under a nitrogen atmosphere, PEG3k (2250 mg, 0.75 mmol), D,L-lactide (1944 mg, 13.5 mmol), glycolide (783 mg, 6.75 mmol), and tin 2-ethylhexanoate (12 mg, 30 μmol) were placed in a glass flask, and the mixture was dried under vacuum overnight. The flask was then purged with nitrogen and sealed in vacuo. The sealed flask was placed in an oil bath at 150 °C for 6 h. The reaction mixture was purified by reprecipitation using chloroform as a solvent and diethyl ether as a nonsolvent, and dried under vacuum overnight to give flakes of PLGAPEG3k-PLGA. PLGA-PEG1.5k-PLGA copolymer was synthesized by the similar polymerization and purification procedures. The average number molecular weight (Mn) and the polydispersity indexes (Mw/ Mn) of these copolymers were determined by gel permeation chromatography (GPC; detector: RI; standard: PEG; eluent: DMSO). The molecular compositions of PEG, D,L-lactide, and glycolide in the obtained copolymers were estimated from 1H NMR measurements (JEOL, ECA-500; solvent: CDCl3). 2.3. Characterization. The critical micelle concentration (CMC) values of the nanohybrids in water were determined using pyrene as a fluorescence probe at 20 °C. Pyrene partitioned preferentially into the hydrophobic core of the aggregates (micelles) and changed the photophysical properties of the micelles. Pyrene was dissolved in acetone and then added to water to a concentration of 740 μM. The acetone was subsequently removed by reducing the pressure and stirring for more than 12 h at 20 °C. The concentrations of the copolymers and CNDs in the sample solutions were varied from 0.00001 to 0.1 wt % and 0.0001 to 0.01 wt %, respectively. The fluorescence emission spectra of pyrene at 335 and 338 nm in the presence of copolymer and CNDs were measured at room temperature using a fluorescence spectrophotometer (JASCO, FP6200) at a fixed emission wavelength of 390 nm. The average diameters of the nanostructures in water were measured by dynamic light scattering (DLS, ZETASIZER NanoSeries ZEN-3600, Malvern). P3k/CND/DOX hybrid solutions were prepared by simply mixing P3k solution, CND solution, and DOX solution at room temperature. These solutions were heated at above their critical gelation temperature to prepare the gel samples. The gel samples were frozen B

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area was cleaned. A total of 3 × 106 cells (PC3, human prostate cancer cell line) in 50 μL of Matrigel (BD Bioscience) were injected subcutaneously into the back bilaterally using a disposable syringe and a 26-gauge needle. When the tumor size reached 300 mm3, the mice were treated with either 300 μL of hybrid gel precursor solutions and P1.5k gel precursor solutions containing 210 μg of DOX. This dose of DOX corresponds to 10 mg/kg. The DOX solution dissolved in PBS containing 210 μg of DOX and PBS were used as controls. These gels were injected in the vicinity of each tumor using a disposable syringe and a 26-gauge needle. The tumor volumes were measured using a caliper and calculated according to the formula: tumor volume = (shorter diameter)2 × (longer diameter)/2. After 21 days of DOX treatment, the animals were sacrificed and the tumors were carefully excised and weighted. The averaged data and standard deviation of three samples are reported.

carefully to maintain the nanostructures composed of PLGA-PEGPLGA, CND, DOX, and water. Then, the samples were lyophilized, and the dried samples were measured by scanning electron microscope (SEM). 2.4. Thermoresponsive Behavior. Specified amounts of polymers were dissolved in acetone, a predetermined volume of pure water was added, and then the acetone was completely evaporated to prepare aqueous solutions of polymers at designed concentrations. CND aqueous solution and DOX aqueous solution were added to the polymer solutions to prepare PLGA-PEG-PLGA/ CND/DOX hybrid solutions. The temperature-responsive sol−gel transitions of the sample solutions were determined by the test tube inverted method. The aqueous solutions were each placed in a glass vial and then the vial was immersed in a water bath at a designated temperature for 2 min. The transition temperatures were determined by the flow (sol)−no flow (gel) criterion when the vial was inverted using a temperature increment of 1 °C per 30 s. The sample was regarded as a gel when no flow was kept for 1 min after the vial was inverted by 180°. 2.5. Swelling and Degradation Properties. Gel samples (200 μL) with different concentrations of the three components were prepared in vials, the vials were weighted, 2 mL of PBS was placed on top of the gels, and then the vials were incubated at 37 °C. At regular intervals, the PBS was removed, the vials were weighted, and then 2 mL of fresh PBS was added to the vials. The weight change was calculated from the weight of the initial gel sample as prepared (W0) and the weight of the swollen and degraded gel samples after exposure to PBS (Wt): Weight change (%) = [Wt − W0/W0] × 100 + 100. 2.6. In Vitro DOX Release. DOX-loaded nanocomposite gels were prepared as described above. Gel samples (200 μL) were placed in the bottom of a 3.5 mL vial, PBS (3.0 mL, pH 7.4, 140 mM) was added on top, and the vials were incubated at 37 ± 0.1 °C. The supernatant was repeatedly monitored using a UV−vis microplate reader by measuring absorbance of DOX at 480 nm; following the measurement, the solution was returned to the vial. Cumulative release was plotted by converting the absorbance to the corresponding DOX concentration and calculating the ratio of the amount of DOX in the supernatant to the initial amount of DOX in the gel. The averaged data and standard deviation from three samples were reported. 2.7. In Vitro Antitumor Activity. The cytotoxicity of DOX diffusing through a Corning Transwell filter was evaluated using HeLa, a human cervical carcinoma cell line. HeLa cells were purchased from the American Type Culture Collection (ATCC). Initially, 3.0 × 104 cells were plated in each well (in 1 mL of DMEM) and 200 μL of gel sample (e.g., DOX-loaded hybrid gels, P1.5k gel as control sample) was added to the upper chamber above the polycarbonate membrane (pore size: 8.0 μm). Additional medium (0.4 mL) was added to the upper chamber. The cultures were maintained in a humidified incubator at 37 °C with 5% CO2. Every 2 days, the upper chamber containing the DOX-loaded gel sample was transferred to a new well plated with 3.0 × 104 cells. The cytotoxic effects of treating HeLa cells with gel samples were evaluated by the WST-assay. This procedure was repeated every 2 days for 22 days. The averaged data and standard deviation of three samples are reported. The cellular uptake behavior and intracellular localization of the DOX released from the hybrid gels after 12 h were followed using confocal laser scanning microscopy (CLSM). 2.8. In Vivo Gel Formation and Release Test. All the animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the NIH. The animal experiments were also carried out in strict accordance with the guidelines for animal experiments at Konan University. Hybrid aqueous solution (200 μL; DOX 70 μg/200 μL) was injected carefully into the back of mice (BALB/cCrSlc, 16 week old male) by syringe using a 26-gauge needle. After 5 h, the mouse was sacrificed and the injected site was carefully cut open and the gel was removed. 2.9. In Vivo Antitumor Activity. Female nude mice (BALB/cSlcnu/nu) at 6 weeks of age were purchased from Charles River. For surgery, the animals were anesthetized using isoflurane and the surgical

3. RESULTS AND DISCUSSION 3.1. Characteristics of PLGA-PEG-PLGA/CND Hybrid Micelles in the Presence of DOX. PLGA-PEG-PLGA copolymers using PEG with different molecular weights were called as P1.5k and P3k, respectively. The molecular composition, the molecular weight, and the molecular weight distribution of P1.5k and P3k were determined by 1H NMR spectroscopy in CDCl3 (Figure S1A in the Supporting Information, SI) and gel permeation chromatography (GPC). P1.5k and P3k copolymers each showed a unimodal GPC curve with a polydispersity of less than 1.2. These data are summarized in Table S1 (in the SI). 1H NMR showed that both the P1.5k and P3k amphiphilic copolymers formed micelles with PEG shells and a PLGA core in aqueous solution by 1H NMR technique. The signals corresponding to the methylene groups in the PEG segments (3.7 ppm) and the methyl groups attributed to the lactic acid unit in the PLGA segments (1.6 ppm) were observed as sharp peaks in CDCl3 because chloroform is a good solvent for PEG and PLGA segments. In contrast, the methyl proton signal of the PLGA segments was broadened and drastically weakened in D2O, whereas the methylene proton signal of the PEG segments was detected as a sharp peak (Figure S1B in the SI), indicating that both end PLGA segments form the micelle core and that the major part of the PEG middle segment extends out into the aqueous environment to form the micelle shell (Figure 2B). The presence of hydrophobic cores in the micelles was characterized using the usual hydrophobic dye (pyrene) method. DLS measurements showed that the hydrodynamic diameter of P3k micelles in water was 60 nm. Nanocomposite gels were prepared using laponite as a fully synthetic biocompatible CND. Laponite is a disk-shaped nanosheet approximately 25 nm in diameter and 1 nm thick with a negative face charge and a weak positive rim charge. We have reported that CNDs interact with PEG segments to form P3k micelle shells at the micelle surface through hydrogen bonding in aqueous environments following the mixing of CND solution with P3k micelle solution. These results suggest hierarchical, capped micelle structures (Figure 2C).21 The code numbers for the P3k/CND hybrid samples indicate the concentrations used; for example, P3k1/C0.1 solution means that the hybrid was made using 1 wt % P3k solution and 0.1 wt % CND solution. We examined the DOX-adsorbing ability of CNDs bound at the surface of P3k micelles by 1H NMR measurements of mixed solutions of P3k/CND hybrid micelles and DOX in D2O. Although characteristic peaks attributed to DOX were clear in the reference 1H NMR spectrum of a mixture of P3k micelles (0.5 wt %) and DOX (0.1 wt %), these peaks were completely C

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concentrations (0−1.0 wt %) in the presence of different amounts of DOX (10−70 μg/200 μL). P3k/CND solutions with certain composite ratios underwent thermoresponsive sol−gel transition in an irreversible manner. The lower critical gelation temperature (LCGT: sol-to-gel transition temperature) can be tuned by varying the concentrations of P3k and CND, as well as their composite ratios (Figure S4A in the SI). P3k/CND micelle solutions also underwent thermogelation in the presence of DOX (Figure S4B−D in the SI). The red transparent solutions of P3k/CND/DOX hybrids instantaneously became red transparent hydrogels at the LCGT (Figure S4E in the SI). This thermogelation was completely irreversible, and the resultant gels could support their own weight when cooled to below their LCGT. The LCGT of P3k/CND/DOX hybrid solutions can be tuned by varying the concentration of P3k, CND, and DOX as well as their composite ratios; consequently, the LCGT of P3k/CND/DOX hybrid solutions can be readily set between room temperature and body temperature. In contrast, free P3k and free CND solutions did not form hydrogels in the presence of any concentration of DOX. It should be noted that the LCGT of P3k/CND micelle solutions significantly decreased with an increase in DOX concentration (Figure S5 in the SI). Furthermore, the upper critical gelation temperature (UCGT: gel-to-precipitate transition temperature) drastically increased with an increase in DOX concentration. In comparison, no changes in LCGT and UCGT were observed for P1.5k reference thermogels in the presence of any concentration of DOX. These results indicate that the ability of P3k/CND hybrid micelles to form gel networks was enhanced and the resultant gel networks were thermodynamically stabilized in the presence of DOX. P3k/ CND hybrid gels prepared with higher CND concentrations showed lower LCGT and higher UCGT in the presence of the same amount of DOX, as shown in the phase diagrams, suggesting that the facilitated formation of gel networks and their stabilization resulted from interactions between CND and DOX. SEM measurements of freeze-dried P3k/CND/DOX hybrid gels were conducted to observe the gel network structures of P3k/CND/DOX hybrid gels prepared with different concentrations of DOX. Interestingly, the average pore size of the P3k/ CND/DOX hybrid gels clearly decreased as the DOX concentration increased (Figure 3), although the total amount of gel did not change. These results indicate that P3k/CND/ DOX hybrid gels with higher DOX content have a higher crosslinking density and concomitant smaller pore size. The hybrid gel network structures was studied in more detail using FTIR measurements of freeze-dried P3k/CND/DOX hybrid gels prepared with various DOX concentrations. Figure S6 (in the SI) shows FTIR spectra of CND, P3k copolymer, DOX, and P3k/CND/DOX hybrid gels. The peaks at 3440 and 980 cm−1 in the CND spectra can be attributed to −Si−O−H bending vibration and −Si−O− stretching vibration, respectively. The peaks at 2880, 1750, and 1100 cm−1 in the P3k spectra can be attributed to the −C−H stretching vibration of the PEG segments, −CO stretching vibration of the PLGA segments, and −C−O−C− stretching vibration of the PEG segments, respectively. The peaks at 3310 and 1590 cm−1 in the DOX spectra can be attributed to the −N−H stretching vibration and −CO stretching vibration, respectively. CND is known to form three-dimensional house-of-cards network structures through hydrogen bonding between silanol groups (−Si− OH) and electrostatic interactions between negative face

Figure 2. (A) Hydrodynamic diameters of P3k/LP hybrid micelles in the presence of DOX at different concentrations. Illustration of (B) P3k micelle, (C) P3k/CNDs hybrid micelle, and (D) assembled P3k/CNDs hybrid micelle through DOX binding to CND surfaces in an aqueous environment.

absent in the 1H NMR spectrum of a mixture of P3k/CND micelles (P3k0.5/C0.1) and DOX (Figure S2 in the SI). No precipitate was observed in the 1H NMR tube containing the P3k/CND micelles and DOX sample, indicating that DOX is insoluble at the nanoscale CND surface in the hybrid micelles due to the interactions between DOX and CND. Wang et al. reported that DOX intercalates within the CND interlayer space either through hydrogen bonding or electrostatic interaction when DOX is mixed with free CND in water.22 As this intercalation might result in intermicellar aggregation, we examined the effects of DOX intercalation on the size of P3k/CND hybrid micelles by DLS measurements of solutions comprising a fixed concentration of P3k/CND micelles (P3k0.5/ C0.1) and different concentrations of DOX. Figure 2A shows the averaged hydrodynamic diameters (Dh) of P3k/CND hybrid micelles in the presence of different concentrations of DOX measured in water. The Dh values gradually increased with increasing DOX concentration up to 0.001 mg/mL, then increased significantly at a DOX concentration of 0.01 mg/mL to approximately three times that of micelles formed in the absence of DOX. This result suggests the self-assembly of P3k/ CND hybrid micelles in the presence of sufficient DOX. Thus, DOX causes intermicellar assembly of P3k/CND hybrid micelles through their specific binding to CNDs located on the hybrid micelle surface (Figure 2D). 3.2. Characteristics of PLGA-PEG-PLGA/CND/DOX Hybrid Thermogels. Next, we investigated the influence of the intermicellar assembly of P3k/CND hybrid micelles in the presence of DOX on their thermoresponsive sol−gel transition behavior. Aqueous solution of P3k copolymer showed relatively high phase transition temperature at around 80 °C, but gelation did not occur at any concentration and temperature (Figure S3 in the SI). In contrast, P1.5k solution exhibited a thermoresponsive sol−gel transition at between room temperature and body temperature. So, P1.5k copolymer gel was used as control samples in this study. Figure S4 (in the SI) shows the phase diagrams of P3k/CND hybrid micelle solutions at three fixed P3k concentrations (1.0, 2.0, and 3.0 wt %) with various CND D

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groups in DOX with CND. The main interactions between CND and DOX would be hydrogen bonding between silanol groups of CND and ketone and amino groups in DOX. Thus, it is likely that hybrid gel networks are composed of PLGA-PEGPLGA micelle networks and stacking networks of DOX and CND located on PLGA-PEG-PLGA micelle surfaces (Figure 1B). Taken together, it appears that DOX in P3k/CND hybrid gels acts as a cross-linker and causes intermicellar assembly of P3k/CND hybrid micelles, thus, forming hybrid gel networks consisting of P3k/CND micelles and DOX. 3.3. Swelling and Degradation Profiles of PLGA-PEGPLGA/CND/DOX Hybrid Gels. Uncontrolled water penetration and diffusion into gels causes gel swelling, resulting in drugs readily diffusing out of the swollen gels (initial burst). This is likely the main mechanism of drug release from most gel.24 Moreover, gel erosion arising from polymer degradation is another major mechanism for drug release from degradable polymer gels. P3k/CND hybrid gels are biodegradable, so both their swelling and degradation properties could be major factors determining DOX release. Therefore, we examined the weight change of P3k/CND/DOX hybrid gels in PBS at 37 °C. Code names were used to refer to the P3k/CND/DOX hybrid gels: for example, P3k3/C0.8/D10 hydrogel indicates the hybrid prepared from 3 wt % P3k, 0.8 wt % CND, and 10 μg DOX. Figure 4 shows the weight change over time of P1.5k/DOXx gels and P3k/CND/DOXx hybrid gels prepared using various concentrations of DOX and P3k, where x means the amount of DOX (μg) loaded in 200 μL of gel. P1.5k gel swelled rapidly to 150% of its original volume within 24 h and then gradually to approximately 200% of its original volume after 192 h. P1.5k/ DOXx gels also swelled rapidly, more so than P1.5k gels (Figure 4A), and then completely collapsed (gel-to-precipitate transition) after 72 h. Interestingly, no significant differences in the molecular weight reduction of P1.5k were observed during the same period between P1.5k gel and P1.5k/DOXx gels. These results indicate that DOX loaded in the P1.5k gels disrupts the

Figure 3. SEM images of freeze-dried P3k/CND/DOX hybrid gels loaded with different amounts of DOX. Scale bars indicate 20 μm.

charges and positive rim charges. Therefore, the −Si−O−H bending vibration of CND appeared as a weak peak.23 The peak intensity of the −Si−O−H bending vibration increased with an increase in DOX concentration in the P3k/CND/DOX hybrid gels (in the spectra of P3k3/C0.8/Dx gels). No changes in the peak intensity of the −C−H stretching vibration and −C−O− C− stretching vibration of the PEG segments, or in the −C O stretching vibration of the PLGA segments, were observed in P3k/CND/DOX hybrid gels compared to the P3k copolymer spectra. In contrast, the peaks due to the −N−H stretching vibration and −CO stretching vibration in DOX were completely absent in the spectra of P3k/CND/DOX hybrid gels. These results indicate the weakened interaction between CND forming house-of-cards network structures as well as newly formed interactions between the −N−H and −CO

Figure 4. Weight change over time of P3k/CND/DOX hybrid gels, P3k/CND gels, and P1.5k gels immersed in PBS at 37 °C. (A) P1.5k15/Dx gels; (B) P3k3/C0.8/Dx gels; (C) P3k4/C0.8/Dx gels; (D) P3k5/C0.8/Dx gels. * indicates collapsed gels (gel-to-precipitate transition). E

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Figure 5. (A) Photograph of in vivo formed P3k3/C0.8/D70 gel (200 μL) at 5 h after subcutaneous injection into the back of mouse. (B) P3k3/ C0.8/D70 gel strongly adhered to subcutaneous tissue at the injection site. (C) P3k3/C0.8/D70 gel excised from the mouse. Scale bar indicates 10 mm. (D) Stretchable characteristics of P3k3/C0.8/D70 gel excised from the mouse. Scale bar indicates 10 mm.

Figure 6. In vitro release of DOX over time from P3k/CND/DOX hybrid gels and P1.5k gels with different amounts of loaded DOX in PBS at 37 °C. (A) P1.5k gels; (B) P3k3/C0.8/Dx gels; (C) P3k4/C0.8/Dx gels; (D) P3k5/C0.8/Dx gels. *Collapsed gels (gel-to-precipitate transition).

differences in molecular weight reduction of P3k between hybrid gels with the same P3k concentration and various DOX concentrations, suggesting that the slower degradation rate of hybrid gels containing higher DOX concentrations is due to higher cross-linking density, as revealed by SEM observations. The lack of swelling and the gradual degradation of P3k/CND/ DOX hybrid gels would be advantageous for the sustained slow release of DOX without an initial burst. 3.4. Characteristics of In Vivo-Formed PLGA-PEGPLGA/CND/DOX Hybrid Gels. For antitumor therapeutic application, DOX-loaded thermogels should form immediately in situ upon injection of the precursor solution and not migrate from the injection site. Moreover, the shape and volume of the gels should be retained during DOX release under physiological

gel network structures formed in response to an increase in temperature, weakening the three-dimensional structure of the gel, but not its chemical integrity. In contrast, P3k/CND hybrid gels (3−5 wt %) did not swell and gradually degraded (Figure 5B−D), with the degradation rate decreasing with an increase in P3k concentration. Importantly, P3k/CND/DOXx hybrid gels also did not swell, and these gels gradually degraded without a gel-to-precipitate transition. Since the PLGA segment is the only hydrolytic cleavage site in P3k, it was expected that all P3k/ CND/DOXx hybrid gels with the same P3k concentration would exhibit similar degradation rates. Unexpectedly, however, significantly different degradation profiles were seen with hybrid gels containing a higher DOX concentration showing a slower degradation rate. GPC measurements showed no F

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concentration, and that this was more pronounced during early phase than late phase, suggesting that DOX loaded in the hybrid gels acts as a cross-linker and a building block of the gel network. Consequently, P3k/CND/DOX hybrid gels with a higher DOX concentration have a significantly higher crosslinking density than hybrid gels with a lower DOX concentration, as described above (Figure 3). Moreover, the degradation kinetics of hybrid gels significantly decreased with an increase in DOX concentration (Figure 4B−D). This suggests that slow release of DOX without burst release at early phase observed for hybrid gels with a higher DOX concentration is due to the incorporation of DOX into the gel network and subsequent DOX-CND interactions resulting in high cross-linking density. Thus, DOX release kinetics can be controlled by adjusting the DOX concentration in P3k/CND/ DOX hybrid gels. In previous biodegradable injectable gel systems, the only way to change DOX release kinetics was to reduce pore size by increasing the polymer concentration. However, this approach is inadequate because the gel pore size is significantly larger than the size of a DOX molecule, even at high polymer concentration. In contrast, the P3k/CND/DOX hybrid gel is a “self-controlled drug release system”, which is a novel concept in controlled drug release. Previously, some studies on “self-regulated drug release system” have been reported.26−30 Such systems aim to adjust release rates of drug in response to a physiological need. In these self-regulated drug release systems, drug release from matrices can be caused in the presence of specific molecule with high concentration, such as glucose and matrix metalloproteinase. In contrast, the P3k/ CND/DOX hybrid gel regulates drug release rate by drug itself, independent of external changes in concentrations of specific molecules. To the best of our knowledge, this concept, selfcontrolled drug release system, is proposed for the first time in this paper. The interactions between DOX and CND have been reported to dissociate at acidic pH (pH < 6.0),18 suggesting that P3k/CND/DOX hybrid gels may exhibit faster DOX release kinetics at acidic conditions as compared to physiological conditions (pH = 7.4). Therefore, we studied the pH dependence (pH = 5.5, 6.0, 6.5, 7.0, 7.4) of DOX release kinetics from P3k/CND/DOX hybrid gels containing various DOX concentrations. Unexpectedly, no pH dependence was observed (Figure S8 in the SI). DLS measurements of the medium containing the released DOX were conducted to investigate the structure of DOX released from the hybrid gels. A unimodal distribution with a peak top at Dh = 180 nm was detected, but no peak corresponding to free DOX (ca. Dh = 4 nm) was observed, nor were peaks corresponding to free CND (ca. Dh = 21 nm) or P3k/CND hybrid micelles (ca. Dh = 55 nm) detected (data not shown). These results suggest that DOX was released from the hybrid gels as assembled nanostructures with P3k/CND hybrid micelles, associated through DOX−CND interactions. The in vivo DOX release profiles from hybrid gels were investigated next, using P1.5k gel as the control. DOX is a fluorescent molecule, so its distribution can be directly observed by fluorescence microscopy. Figure 7 shows the distribution of DOX in the subcutaneous tissue of mice after injection of P3k/CND/DOX hybrid solution (P3k3/C0.8/D70) and P1.5k(15 wt %)/DOX solution. The blue and green squares indicate the area containing the DOX-loaded gels formed in situ and the neighboring area, respectively. P1.5k gels exhibited initial burst release within 5 h after injection, and DOX diffused

conditions and physical stress. Consequently, the in vitro and in vivo gel-forming characteristics of the P3k/CND/DOX hybrids and the resulting thermogels were investigated. P3k3/C0.8/D70 hybrid precursor solution instantly formed DOX-loaded hydrogel after injection to heated PBS (37 °C, pH 7.4, 140 mM; Figure S7A in the SI). Moreover, P3k3/C0.8/D70 hybrid precursor solution can be readily injected into the subcutaneous layer of mice through a 26-gauge needle and formed DOXloaded hydrogel within 5 min in situ without any loss of gel volume (Figure S7B in the SI). Interestingly, the formed P3k3/ C0.8/D70 gels strongly adhered to subcutaneous tissue at the injection site (Figure 5A,B). Similar tissue-adhering properties were observed for all P3k/CND/DOX hybrid gels with an LCGT between room temperature and body temperature; this property is clearly advantageous for securing the gel close to the target tumor. Wu et al. reported the synthesis of tissue-adhesive hydrogels by photopolymerization of PEG-diacrylate and methoxy-PEG-acrylate in the presence of CND.25 In their hydrogel system, CND was concluded to be essential for the tissue-adhering property and was attributed to protein adsorption onto the large negatively charged surface area of CND. In concordance with their results, we observed that P1.5k/ DOX gels did not adhere to tissue, indicating that CND is essential for the tissue-adhering properties of P3k/CND/DOX hybrid gels. Furthermore, P3k3/C0.8/D70 hybrid gel was soft, but tenacious, and did not break even when the hybrid gel was bent, stretched, and twisted (Figure 5D), indicating its mechanical compatibility with soft tissues. 3.5. DOX Release Profiles of PLGA-PEG-PLGA/CND/ DOX Hybrid Gels. Antitumor therapeutic application requires that the loaded DOX be released in a controlled manner from the gel to exert its biological function. The release kinetics of DOX from P3k/CND/DOX hybrid gels prepared using different P3k concentrations and containing different DOX concentrations were studied in PBS at 37 °C using DOXcontaining P1.5k gels as controls. Under physiological conditions (pH 7.4), DOX was released from P1.5k gels (15 wt %) significantly faster than from P3k/CND/DOX hybrid gels (Figure 6A). Burst release from P1.5k gels during the first 6 h resulted in 35% of the DOX being released, regardless of the DOX concentration in the gel. Following this burst, DOX continued to be released at a constant, slower rate, and then stopped after 72 h because the gels collapsed and DOX precipitated together with the polymers. DOX burst release from the P1.5k gel is attributed to uncontrolled diffusion of DOX due to marked swelling of the P1.5k gel shortly after gel forming. In contrast, P3k/CND/DOX hybrid gels provided sustained DOX release for over 20 days without an initial burst release (Figure 6B−D). The DOX release profile was bimodal, with relatively rapid release kinetics in the initial 120 h; early phase followed by slow release kinetics; late phase. Comparison of P3k/CND/DOX hybrid gels with a fixed DOX concentration and various P3k concentrations showed that the release kinetics decreased with an increase in P3k concentration and that this decrease was more pronounced in late phase than early phase. The degradation kinetics followed a similar pattern, as shown in Figure 6B−D. Taken together, these results suggest that the DOX release profile from P3k/CND/DOX hybrid gels in late phase is controlled by the degradation kinetics of P3k in the hybrid gels. In contrast, comparison of the DOX release kinetics of P3k/CND/DOX hybrid gels with a fixed P3k concentration and various DOX concentrations showed that the release kinetics decreased with an increase in DOX G

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Figure 7. Microscopic images merged with fluorescence images and bright field images of in vivo formed DOX-loaded gels and surrounding tissues at 5 h after subcutaneous injection. Scale bars indicate 1 mm.

to surrounding tissues. In contrast, no burst release was observed with P3k/CND/DOX hybrid gel. These results are consistent with the DOX release profiles obtained in vitro, suggesting that the gels act as a “self-controlled drug release system” in vivo. 3.6. Antitumor Activity of PLGA-PEG-PLGA/CND/DOX Hybrid Gels. The anticancer activity of P3k/CND/DOX hybrid gels was investigated by in vitro experiments using a model human cancer cell line and in vivo experiments using human xenograft tumors in nude mice. The noncytotoxicity of the P3k/CND hybrid gels was confirmed by the Live/Dead assay using L929 cells and HeLa cells (Figure S9 in the SI). The anticancer activity of P3k/CND hybrid gels in vitro was examined using a transwell assay and HeLa cells for 22 days as described in the methods section. Initially, 3.0 × 104 cells were plated in each well and 200 μL of gel samples loaded with DOX was added to the upper chamber. Additional medium (400 μL) was also added to the upper chamber. Every 2 days, the upper chamber was transferred to a new well plated with 3.0 × 104 cells and the cytotoxicity toward HeLa cells was evaluated by the WST-assay. These procedures were repeated every 2 days for 22 days (Figure S10 in the SI). Figure 8A shows the cumulative number of HeLa cells killed by treatment with the gel sample. DOX-loaded P3k/CND hybrid gel (P3k3/C0.8/ D70) was cytotoxic toward HeLa cells, and the cytotoxicity was similar to that of the control P1.5k 15 wt % gel during the first 6 days. The control P3k3/C0.8 gels were not cytotoxic, revealing that the cytotoxic effect was due to the DOX released from the P3k/CND/DOX hybrid gels. More importantly, the hybrid gels displayed sustained cytotoxic activity for at least 22 days, whereas the cytotoxic activity of the control P1.5k/DOX gel ended after 6 days because all of the DOX in the gel had been released after 4 days, as shown in Figure 6A. The long-term cytotoxicity of theP3k/CND/DOX hybrid gel is due to the sustained slow release of DOX. The DOX-release profiles suggest that the gel may remain cytotoxic for considerably longer than shown by the data because approximately 80% of the DOX loaded in the hybrid gel (ca. 56 μg) remained in the gel after 22 days. A DOX delivery system must be effectively taken up by cancer cells and deliver DOX to the interior of cancer cells. The internalization of DOX by HeLa cells can be followed by fluorescence microscopy. Figure 8B shows bright field and fluorescence microscope images of HeLa cells after 12 h of treatment with DOX-loaded gels (hybrid and P1.5k gels). Red fluorescence emitted by DOX is clearly observed in both the cytosol and the cell nuclei counterstained blue with DAPI of

Figure 8. (A) In vitro cell viability over time of human cancer cells (HeLa) treated with P3k/CND/DOX hybrid gels. *Indicates that the gel collapsed (gel-to-sol transition). PBS (⧫); P1.5k/D30 gel (■); P3k3/ C0.8 gel (▲); and P3k3/C0.8/D70 gel (●). (B) CLSM images of HeLa cells treated with P3k/CND/DOX hybrid gels for 12 h. Scale bars indicate 50 μm.

HeLa cells treated with either hydrogels, but cells treated with the hybrid gel showed significantly higher red fluorescence in both the cytosol and cell nucleus than cells treated with control P1.5k/DOX gel. It was recently reported that several cancer cells, including HeLa cells, effectively take up CND by endocytosis.31 As described above, DOX is released from hybrid gels as nanostructures assembled with P3k/CND hybrid micelles through DOX−CND interactions; consequently, these nanostructures could facilitate DOX cellular uptake by acting as nanocarriers for DOX. In order to confirm the hypothesis, DOX cellular uptake of freshly prepared P3k/CND/DOX hybrid micelles were examined. As I expected, higher reddish intensity can be observed inside both cytosol and nucleus (especially in nucleus) after the cells were treated with P3k/ CND/DOX hybrid micelles for 12 h, compared to P1.5k/DOX micelles (Figure S11 in the SI). Thus, this facilitated DOX cellular uptake is attributed to the assembled structures acting as nanocarriers for DOX. It should be noticed that DOX is a weak base with a pKa of 8.30 and, thus, can be easily ionized (protonated) under acidic conditions. This increases the risk of DOX being trapped in biological acidic compartments such as the extracellular space in solid tumors32 and is implicated in DOX resistance. The use of nanocarriers can help overcome this. Therefore, DOX carried by P3k/CND hybrid micelles would not be trapped in the extracellular environment of a solid tumor (ca. pH 6.5). The antitumor activity of P3k/CND/DOX hybrid gels was studied by in vivo experiments using human xenograft tumors in nude mice. When the tumor size reached 300 mm3, 300 μL of hybrid gel precursor solutions (containing 210 μg of DOX) was once directly injected into the vicinity of the tumor. DOXloaded P1.5k gel, free DOX solution in PBS at the same dose, and PBS were used as controls. The change in tumor volume H

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that DOX molecules loaded in the hybrid injectable gels act as cross-linkers to organize new gel networks. Moreover, DOX controls its own release profile by modulating both the crosslinking density and mesh size in gel networks, and thus the PLGA-PEG-PLGA/CND/DOX hybrid injectable gel is a selfcontrolled drug release system, which is a novel concept in controlled drug release. It should be noted that a single injection of PLGA-PEG-PLGA/CND/DOX hybrid gel provides long-term sustained antitumor activity in vivo against human xenograft tumors in nude mice, suggesting the potential of hybrid gels as a valuable local DOX-delivery platform for focal cancer therapy.

over time after treatment was measured to assess the antitumor activity of the various treatments. As shown in Figure 9A, the

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ASSOCIATED CONTENT

S Supporting Information *

Characterization of PLGA-PEG-PLGA copolymers (Table S1 and Figure S1), characterization of P3k/CND hybrid micelles (Figure S2), phase diagrams of control polymer solutions and hybrid solutions (Figures S3, S4, and S5), FTIR spectra of P3k/ CND/DOX hybrid gels (Figure S6), in vitro and in vivo thermogelation (Figure S7), release profiles of P3k/CND/DOX hybrid gels at different pH (Figure S8), Live/Dead assay of P3k/ CND/DOX hybrid gels (Figure S9), illustration of the experiment to examine in vitro anticancer activity (Figure S10), and cellular uptake of P3k/CND/DOX hybrid micelles and control P1.5k micelles (Figure S11). This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 9. (A) In vivo antitumor activity of DOX-loaded hybrid gels against human cancer cells implanted in nude mice. PBS as a sham control (⧫); DOX solution (▲); P1.5k/D70 gel (■); and P3k3/C0.8/ D70 gel (●). The inset is a photograph of a mouse 7 days after treatment with P1.5k/D70 gel. (B) Photographs of the excised tumors from animals sacrificed 21 days after injection.

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AUTHOR INFORMATION

Corresponding Author

tumor volume increased almost 3-fold after 21 days in the PBStreated mice. In contrast, effective decrease in tumor size was observed following either free DOX or P1.5k/DOX gel treatments after 7days, but then the tumors started to grow again. The termination of anticancer activity by DOX-loaded P1.5k gel could be attributed to the rapid DOX release profile, as shown in Figure 6A, and trapping of protonated DOX in the extracellular environment of the solid tumor. It should be noted that severe dermatitis caused by DOX was observed at the injection site in mice treated with P1.5k/DOX gel and free DOX solution in PBS (inset in Figure 9A). In contrast, a gradual decrease in tumor volume was observed in mice treated with P3k/CND/DOX hybrid gel. Most importantly, the decrease was sustained for at least 21 days after a single injection of hybrid gel, without any dermatitis. Figure 9B shows tumor tissues isolated from the mice at 21 days after injection. The anticancer activity of P3k/CND/DOX hybrid gel was confirmed. The longterm sustained anticancer activity of P3k/CND/DOX hybrid gel could be attributed to its sustained slow release of DOX and enhanced cellular internalization of released DOX by forming nanostructures composed of DOX with P3k/CND hybrid micelles. Thus, it is concluded that long-term sustained slow release of DOX without “burst release” achieved by using injectable P3k/CND/DOX gel is essential for a DOX-delivery system for focal cancer chemotherapy.

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Laponite was kindly supplied by Wilbur-Ellis Co. This work was partly supported by JSPS KAKENHI Grant Number 24700487.

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REFERENCES

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4. CONCLUSION We have developed a novel biodegradable injectable in situforming gel through the self-assembly of biocompatible PLGAPEG-PLGA micelles, CND, and DOX. PLGA-PEG-PLGA/ CND/DOX hybrid solutions undergo temperature-responsive sol−gel transition between room temperature and body temperature at very low polymer concentration. We discovered I

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