Article pubs.acs.org/Macromolecules
Dual Stimuli-Responsive Redox-Active Injectable Gel by Polyion Complex Based Flower Micelles for Biomedical Applications Shiro Ishii,† Junya Kaneko,† and Yukio Nagasaki*,†,‡,§ †
Department of Materials Science, Graduate School of Pure and Applied Sciences, ‡Master’s School of Medical Sciences, Graduate School of Comprehensive Human Sciences, and §Satellite Laboratory, International Center for Materials Nanoarchitectonics (WPI-MANA), National Institute for Materials Science (NIMS), University of Tsukuba, Tennoudai 1-1-1, Tsukuba 305-8573, Japan S Supporting Information *
ABSTRACT: We developed and evaluated a redox-active injectable gel (RIG) comprising poly[4-(2,2,6,6-tetramethylpiperidine-N-oxyl)aminomethylstyrene]-b-poly(ethylene glycol)-bpoly[4-(2,2,6,6-tetramethylpiperidine-N-oxyl)aminomethylstyrene] (PMNT−PEG−PMNT) triblock copolymer and poly(acrylic acid) (PAAc). Cationic PMNT−PEG−PMNT and anionic PAAc formed polyion complexes (PIC) flower micelles without aggregation, which was confirmed by FRET analysis. We confirmed that the PIC flower micelles exhibited irreversible sol−gel phase transitions with increasing both temperatures and ionic strengths. The PIC flower micelles had a low viscosity at room temperature. The viscosity increased with increasing temperatures and ionic strengths, and the RIG was formed under physiological conditions. The RIG was able to provide sustained release of an anionic model drug for more than 4 weeks without an initial burst. These results indicated that the RIG has potential as an injectable system for pharmaceutical and biomedical applications such as a local delivery carrier for the controlled release of charged drugs such as proteins or siRNA.
1. INTRODUCTION Injectable, stimuli-responsive hydrogels have attracted attention for pharmaceutical and biomedical applications such as drug delivery for low and large molecules and tissue engineering.1−4 Preformed gels need surgical intervention for administration. Therefore, it is costly and inconvenient for the patient. In contrast, injectable hydrogels are flowable, aqueous solutions before administration. After injection into the body, they form a gel immediately in response to environmental changes such as temperature, pH, enzymes, and light.5−8 Therefore, only a minimally invasive treatment is needed for therapies using the injectable stimuli-responsive hydrogels; the patient can avoid surgery for administration.9 ABA-type triblock copolymers have been widely studied for stimuli-responsive hydrogel systems.7,10 For example, poly(DLlactide-co-glycolide)-b-poly(ethylene glycol)-b-poly(DL-lactideco-glycolide) (PLGA−PEG−PLGA), poly(ε-caprolactone)-bPEG-b-poly(ε-caprolactone) (PCL−PEG−PCL), and others can be used for temperature-sensitive hydrogel systems.11−17 The aqueous solutions of these triblock copolymers are in a sol state at room temperature. They form gels under the physiological temperature (37 °C) after administration because of increased hydrophobic interactions. Poly(β-amino ester)-bPEG-b-poly(β-amino ester) (PAE−PEG−PAE), poly(amidoamine)-b-PEG-b-poly(amidoamine) (PAMAM−PEG− PAMAM), and others can be used for pH- and temperaturesensitive hydrogel systems.7,18,19 The aqueous solutions of these triblock copolymers are prepared in an acidic pH. These © XXXX American Chemical Society
solutions do not form gels when only temperature is changed because the B-segments of these triblock copolymers are ionized under an acidic pH. When pH and temperature are changed to the physiological condition, the B-segments are deionized; this results in enhancement of the hydrophobic interactions leading to the formation of a gel. The pH- and temperature-sensitive hydrogel systems have potential as drug delivery carriers due to their ability to form ionic complexes with oppositely charged therapeutic agents.20 Recently we have developed and reported a redox-active injectable gel (RIG) system by using poly[4-(2,2,6,6tetramethylpiperidine-N-oxyl)aminomethylstyrene]-b-poly(ethylene glycol)-b-poly[4-(2,2,6,6-tetramethylpiperidine-Noxyl)aminomethylstyrene] (PMNT−PEG−PMNT) triblock copolymer, which possesses reactive oxygen species (ROS) scavenging nitroxide radicals as side chains on the PMNT segment (Figure 1).21 The cationic PMNT segment in PMNT−PEG−PMNT forms polyion complexes (PIC) with anionic poly(acrylic acid) (PAAc) to form flower-like micelles, which exhibit temperature- and ionic-strength-responsive irreversible gelation under physiological conditions. The PIC flower micelle solutions were injected subcutaneously into the paw of a mouse, and RIG was formed successfully in situ. The RIG showed prolonged retention time at the local injection site Received: February 11, 2015 Revised: April 8, 2015
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DOI: 10.1021/acs.macromol.5b00305 Macromolecules XXXX, XXX, XXX−XXX
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Figure 1. Schematic illustration of temperature- and ionic-strength-responsive redox-active injectable gel (RIG) system. Polyion complex (PIC) flower micelles are formed by self-assembly via electrostatic interaction between cationic poly[4-(2,2,6,6-tetramethylpiperidine-N-oxyl)aminomethylstyrene]-b-poly(ethylene glycol)-b-poly[4-(2,2,6,6-tetramethylpiperidine-N-oxyl)aminomethylstyrene] (PMNT−PEG−PMNT) and anionic poly(acrylic acid) (PAAc). PIC flower micelle solution shows irreversible sol−gel transition with increasing temperature and ionic strength.
compared to low-molecular-weight (LMW) nitroxide radical compounds and LMW nitroxide radical compounds physically entrapped into non-nitroxide-radical-containing hydrogels (TEMPO@nRIG). The RIG inhibited neutrophil infiltration and cytokine production better than the LMW nitroxide radical compounds and TEMPO@nRIG because of the prolonged site-specific retention of the RIG; this led to the suppression of the local inflammation related to oxidative stress. These results indicated that the RIG has potential as an innovative approach to treatment of local inflammations. In addition, since the RIG is a polyion complex gel, by controlling the charge balance of the matrix, it is anticipated to be a better drug delivery carrier not only for hydrophobic drugs but also for also charged molecules than previously reported drug carriers, which has no ROS-scavenge ability. The redox PIC micelle is thus unique, and it was confirmed that it forms a gel in response to the in vivo conditions. However, detailed characteristics of redox flower micelle prepared by polyion complex and its gels have not been studied in detail. The flower micelle with PIC as a driving force of hydrophobic core is unique, and no other publications have been available thus far. In this study, we evaluated the material properties of the RIG in detail. Formation of the flower micelles via polyion complexes was confirmed by fluorescence energy transfer method. The gelation mechanism of the PIC flower micelles, the sol−gel transition properties, and the rheological properties with increasing temperatures and ion strengths were investigated using a rheometer. As basic properties of the developed material, the critical micelle concentration (CMC) of the PIC flower micelles and swelling properties of the gel formed by cross-linking of collapsed polyions in the PIC flower micelles were confirmed. In addition, the in vitro release property of a model drug from the RIG was investigated to evaluate the
potential to apply the RIG as a pharmaceutical drug carrier for a sustained drug release.
2. EXPERIMENTAL SECTION 2.1. Materials. Poly(acrylic acid) (PAAc) (Mn = 5 000) (Wako Pure Chemical Industries, Ltd., Osaka, Japan), 4-amino-2,2,6,6tetramethylpiperidine-N-oxyl (4-amino-TEMPO) (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), 5-aminofluorescein (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), and poly(ethylene glycol) (PEG) possessing sulfanyl groups at both ends (SH-PEG-SH) (Mn = 10 000) (NOF Corporation Co., Ltd., Tokyo, Japan) were used without further purification. Purification of 2,2′-azobis(isobutyronitrile) (AIBN; Kanto Chemical Co., Inc., Tokyo, Japan) from methanol was achieved by recrystallization. Chloromethylstyrene (CMS) was kindly provided by Seimi Chemical Co., Ltd. (Kanagawa, Japan), and purified on a silica gel column to remove nitrophenol and other inhibitors, followed by vacuum distillation in a nitrogen atmosphere. 2.2. Synthesis of PMNT−PEG−PMNT Triblock Copolymer. PMNT−PEG−PMNT was synthesized as previously reported.21 Briefly, poly(chloromethylstyrene)−PEG−poly(chloromethylstyrene) (PCMS−PEG−PCMS) triblock copolymer was synthesized by radical telomerization of CMS using SH-PEG-SH (Mn = 10 000) as a telogen. To obtain PMNT−PEG−PMNT, the chloromethyl groups on the PCMS segment of the triblock copolymer were converted to nitroxide radicals via the amination of PCMS−PEG−PCMS with 4-aminoTEMPO in dimethyl sulfoxide (DMSO) (see Supporting Information). 2.3. Preparation of Nitroxide Radical-Containing PIC Flower Micelles (RIG Precursor). PMNT−PEG−PMNT triblock copolymer and poly(acrylic acid) (PAAc) were dissolved in a phosphate buffer (PB) solution (50 mM, pH 6.2) at a concentration of 5 mg/mL. Nitroxide radical-containing PIC flower micelles were prepared as previously reported.21 Briefly, the PIC flower micelle was prepared by stirring the PMNT−PEG−PMNT solution, a drop at a time, into the PAAc solution at various molar ratios, including an r of 2:1, 1:1, and 1:2 [r = molar unit of the cationic amine groups of PMNT−PEG− B
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micelles.24,25 We confirmed that the PMNT−PEG−PMNT triblock copolymers formed PIC flower-like micelles coupled with PAAc, as shown below. Figure 2 and Table 1 show the
PMNT:molar unit of the anionic carboxyl groups of PAAc, obtained from α−pH curves (Figure S3)]. The particle size of the PIC flower micelles was measured by dynamic light scattering (DLS) using Zetasizer Nano series ZEN3600 (Malvern Instruments Ltd., Worcestershire, UK). PIC flower micelle solutions at different molar ratios (r = 2:1, 1:1, and 1:2) were concentrated using a centrifugal evaporator (EYELA CVE 3100, Tokyo Rikakikai Co., Ltd., Tokyo, Japan). The critical micelle concentration (CMC) of the PIC flower micelles was determined using pyrene as a fluorescence probe. Detailed methods are described in the Supporting Information. 2.4. Preparation of Fluorescein-Labeled PAAc (FL-PAAc) Loaded RIG Precursor. Detailed methods are described in the Supporting Information. 2.5. Swelling Ratio Measurement. The gelation was conducted by heating the PIC flower micelle solution (55 mg/mL, pH 6.2, 550 mM PB) at 37 °C in the water bath. The obtained RIG was weighed (Wgel) after removing the extra water on the surface. The RIG was then freeze-dried and weighed (Wdried gel) again. The degree of swelling (swelling ratio) was calculated as follows:22
degree of swelling (swelling ratio) = (Wgel − Wdried gel)/ Wdried gel Figure 2. Size distribution of polyion complexes (PIC) flower micelles (5 mg/mL, pH 6.2) at various molar ratios measured using DLS.
2.6. Rheological Evaluation. Rheological evaluations of the RIG were conducted using a rheometer (MCR302, Anton Paar). A parallel plate with 20 mm diameter and a gap of 0.2 mm was used. The PIC flower micelle solution was put between the plates using a micropipet. Rheological properties (storage modulus G′, loss modulus G″, and complex viscosity) of temperature dependencies from 15 to 45 °C were measured at a fixed frequency of 1 Hz, a heating/cooling rate of 1 °C/min, and strain amplitudes from 0.5% to 15%, which was within the linear viscoelastic regime. 2.7. Sol−Gel Phase Transition Properties (Phase Diagram Measurement). Storage modulus G′ and loss modulus G″ were measured as described above. The crossover point, G′ = G″, is commonly used as a phase transition point.23 Sol−gel phase transition temperatures were determined, and phase diagrams were prepared using this criterion. 2.8. In Vitro Release of FL-PAAc from the RIG. Using fluorescein-labeled PAAc (FL-PAAc) as a model drug, the in vitro release property was evaluated. The gelation was conducted by heating 300 μL of FL-PAAc loaded RIG precursor solution (55 mg/mL, r = 1:1, pH 6.2, 550 mM PB) in a 1.5 mL tube at 37 °C in a water bath. Then, 150 μL of PBS was added to the top of the RIG in the tube and incubated in a shaker at 100 rpm at 37 °C. At predetermined intervals, 100 μL of the release medium was taken from the tube, and 100 μL of fresh PBS was added. Release medium (50 μL) was added to 50 μL of 100 g/L SDS for the collapse of the released polyion complexes, including the FL-PAAc. The fluorescent intensity (Ex: 485 nm, Em: 535 nm) was measured using a plate reader (PerkinElmer), and the release ratio of FL-PAAc was determined from the fluorescent intensity.
Table 1. Characterization of Polyion Complexes (PIC) Flower Micelles (5 mg/mL, pH 6.2) at Various Molar Ratios Measured Using DLS molar ratio, r
size (nm)
pdl
2:1 1:1 1:2
67.9 43.0 35.1
0.204 0.172 0.170
results of the DLS measurements. The nanoparticles with several tens of nanometers were confirmed regardless of the ratios, r, from 2:1 to 1:2. No aggregation of the micelles was observed at room temperature. An increase in the molar ratio (r) increased the size of the flower micelle. In order to confirm the complex formation between PMNT−PEG−PMNT and PAAc, FL-PAAc was used because an energy transfer is observed when FL is in close proximity to nitroxide radicals.26,27 The fluorescence intensity of FL-PAAc loaded PIC flower micelle reduced, compared to that of FL-PAAc, because of quenching of the fluorescence by TEMPO in the PIC core, which was caused by the fluorescence resonance energy transfer (FRET) (Figure 3).26,27 These results indicated that the PMNT segments were in the PIC core, and the PIC flower micelles were formed. The critical micelle concentration (CMC) of the PIC flower micelles (r = 1:1, pH 6.2, 50 mM PB, 25 °C) was determined using pyrene as a fluorescence probe. Pyrene preferentially localizes in the hydrophobic core of aggregates and the photophysical properties of pyrene change.28,29 Figure S4 shows plots of the I3/I1 intensity ratio as a function of logarithm of the concentration of the PIC flower micelles under various concentrations of NaCl. The I3/I1 intensity ratio showed an abrupt increase above a certain concentration, which indicated the formation of the PIC flower micelles. The CMC value was estimated the intersection of the tangent to the curve at the inflection with the horizontal tangent through the points at low concentration. The CMC values of PIC flower micelles with 0 mM NaCl, 150 mM NaCl, and 500 mM NaCl were 314, 356, and 384 μg/mL, respectively. An increase in the concentration of NaCl increased the CMC value, which indicated that the PIC flower micelles were destabilized because of the increased
3. RESULTS AND DISCUSSION 3.1. Synthesis of PMNT−PEG−PMNT Triblock Copolymer and Preparation of Nitroxide Radical-Containing PIC Flower Micelles (RIG Precursor). PCMS−PEG−PCMS triblock copolymer was successfully synthesized by the radical telomerization of CMS using SH-PEG-SH (Mn = 10 000) as a telogen and confirmed by GPC and 1H NMR analysis (Figure S1). PMNT−PEG−PMNT triblock copolymer was successfully synthesized by amination of the chloromethyl group of PCMS−PEG−PCMS with 4-amino-TEMPO and confirmed by 1H NMR analysis. Confirmation occurred when the signal for the methylene protons on the chloromethyl groups in the PCMS unit at 4.5 ppm completely disappeared by amination (Figure S2). ABA-type triblock copolymers, which possess hydrophobic A segments and a hydrophilic B segment, form flower-like C
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Figure 3. Relative fluorescence intensity of fluorescein-labeled poly(acrylic) acid (FL-PAAc) loaded polyion complexes (PIC) flower micelles to FL-PAAc.
electrostatic shielding effect.30 Although we were not able to make an easy comparison of the CMC values between our PIC flower micelles and previous reported PIC micelles because of different media conditions, the CMC values of the PIC flower micelles were the same order as previous reported PIC micelles (140−620 μg/mL).30,31 The CMC values of poly(oxyethylene tert-octylphenol) (Triton X-100) and sodium dodecyl sulfate (SDS), typical surfactant micelles, at 25 °C, are 5.8 and 2.3 mg/ mL, respectively.31 It is noticed that the CMC values of our PIC flower micelles are much lower than those of the surfactant micelles, which indicates our PIC flower micelles are more stable than the typical surfactants micelles. 3.2. Swelling Ratio. Figure 4 shows the swelling ratios of the RIG at different molar ratios (r = 2:1, 1:1, and 1:2) just
Figure 5. Change in storage modulus G′ and loss modulus G″ of polyion complexes (PIC) flower micelles (55 mg/mL, r = 1:1, 150 mM NaCl, pH 6.2, 550 mM PB) with increasing temperatures (from 15 to 45 °C), followed by decreasing temperatures (from 45 to 15 °C).
27.6 °C. This indicated that the PIC flower micelles were destabilized as the molecular motion of the PEG segment increased because of increased thermal motion energy.21 When the temperature decreased from 45 to 15 °C, the RIG remained in the gel state (G′ > G″). These results indicated that the RIG is a temperature-responsive irreversible gel. In contrast, conventional ABA-type triblock copolymers, such as PLGA− PEG−PLGA, form temperature-responsive reversible gel, which is caused by the aggregation of flower micelles induced by increased hydrophobic interaction.11 It is assumed that the gel formed by our PIC flower micelles occurs through the formation of ionic cross-linkages between the cationic PMNT− PEG−PMNT and anionic PAAc; this is caused by a part of destruction of the flower-like structure with a PEG outer shell due to changes in temperature and ionic strength and not by aggregation of the flower micelles. It is interesting to note that storage modulus of RIG was more than 1000 Pa, which was much higher than conventional flower-type micelle gel such as PLGA−PEG−PLGA (most of them are less than 100 Pa).32−35 The electrostatic cross-linking might improve the mechanical strength of RIG. 3.4. Sol−Gel Phase Transition Properties. Using the crossover point G′ = G″ as a sol−gel phase transition point, sol−gel phase transition temperatures were determined. Figures 6a and 6b show the sol−gel phase transition diagrams of the PIC flower micelles with different concentrations of PIC flower micelles (r = 1:1, 0 mM NaCl, pH 6.2, 550 mM PB) and different concentrations of NaCl (55 mg/mL of PIC flower micelles, r = 1:1, pH 6.2, 330 mM PB, 550 mM PB), respectively. At room temperature, the PIC flower micelles exhibited a sol state, even at a high concentration of micelles and high ionic strength of NaCl. From these results and the results of the DLS measurements, it was suggested that the PEG outer shell prevented the aggregation of the micelles and the destruction of the polyion cores under low temperature. By increasing the temperature, the sol-to-gel phase transitions were
Figure 4. Swelling ratios of redox-active injectable gel (RIG, 55 mg/ mL) at various molar ratios.
after the preparation at 37 °C in vitro. They had swelling ratios of approximately 7−8, regardless of the molar ratio. Both poly(ethylene glycol) and poly(acrylic) acid are highly hydrophilic. The difference of the molar ratio did not affect the swelling properties in the range of the molar ratio from 2:1 to 1:2. 3.3. Gelation Mechanism. To understand the gelation mechanism of the RIG, storage modulus G′ and loss modulus G″ of the PIC flower micelle solution (55 mg/mL, r = 1:1, 150 mM NaCl, pH 6.2, 550 mM PB) were measured while increasing the temperature (from 15 to 45 °C), followed by decreasing the temperature (from 45 to 15 °C). The result is shown in Figure 5. When the temperature was increased from 15 to 45 °C, sol−gel phase transition (G′ = G″) occurred at D
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micelle solution also affected the sol−gel phase transition temperature. Our RIG system thus responds to ionic strength in addition to temperature. 3.5. Rheological Properties. Figure 7a shows the temperature dependencies of the complex viscosity of the PIC flower micelles with different concentrations of the PIC flower micelles (r = 1:1, 0 mM NaCl, pH 6.2, 550 mM PB). The viscosity of the PIC flower micelles at 22, 33, 55, and 66 mg/mL increased abruptly at approximately 34, 33, 30, and 28 °C, respectively. Figure 7b shows the temperature dependencies of the complex viscosity of the PIC flower micelles (55 mg/mL, r = 1:1, pH 6.2, 550 mM PB) with different concentrations of NaCl. The viscosity of the PIC flower micelles with 0 mM NaCl, 150 mM NaCl, 300 mM NaCl, and 420 mM NaCl increased abruptly at approximately 30, 28, 26, and 24 °C, respectively. From the sol−gel phase transition behavior, it was confirmed that the increase in the complex viscosity of the PIC flower micelles was induced by temperature- and ionic-strength-responsive gelation. Figure 7c shows the temperature dependencies of the complex viscosity of the PIC flower micelles (55 mg/mL) with various molar ratios, r, of 2:1, 1:1, and 1:2. The viscosity of the PIC flower micelles with r = 2:1, 1:1, and 1:2 increased abruptly at approximately 32, 30, and 30 °C, respectively, which indicated the formation of a gel regardless of the ratio from 2:1 to 1:2. As reported previously, the PIC flower micelles with an r of 2:1 form temperature-responsive reversible gels caused by the aggregation of micelles, while the PIC flower micelles with an r of 1:1 and 1:2 form irreversible gels caused by the ionic cross-linkage between cationic PMNT−PEG−PMNT and anionic PAAc.21 It is assumed that this difference in the gelation mechanisms led to the difference in the viscosity properties in the same concentrations of PIC flower micelles; the PIC flower micelles with an r of 2:1 have a lower complex viscosity and higher sol−gel phase transition temperature than the PIC micelles with an r of 1:1 and 1:2. Because the PIC flower micelles have a low viscosity at room temperature, the micelles can be injected easily into the body. After injection into the body, the viscosity increases, along with an increase in temperature and ionic strength, causing the formation of a gel in the physiological condition. Therefore, the RIG is ideal as an injectable system for biomedical applications. Our PIC flower micelles form gel in response to temperature and ionic strength. The gelation condition can be controlled by the concentration of the PIC flower micelles and by the ionic strength of the PIC flower micelle solution. In case an injectable gel system is applied to catheter-based therapy, an injectable gel that responds only to temperature cannot be used because of gelation in the catheter caused by body temperature. On the other hand, our temperature- and ionic-strengthresponsive PIC flower micelles can be applied to catheter-based therapy. By controlling gelation conditions, it is possible for the PIC flower micelles to keep the sol state in the catheter at body temperature and to form the gel at the target site along with increasing the ionic strength when they are released from the catheter and exposed to the physiological ionic condition. Thus, our RIG system can be applied to local therapy for deeply placed diseased sites using the catheter. 3.6. In Vitro Release of FL-PAAc from RIG. Using FLPAAc as a model drug, the in vitro release property was investigated to evaluate the possibility of applying the RIG to pharmaceutical drug carriers for sustained drug release. Figure 8 shows the in vitro release profile of FL-PAAc from the RIG. The
Figure 6. Phase diagrams of polyion complexes (PIC) flower micelles. (a) Concentration dependency (r = 1:1, 0 mM NaCl, pH 6.2, 550 mM PB). (b) Ionic strength dependency (r = 1:1, 55 mg/mL, pH 6.2), concentration of phosphate buffer (PB) in PIC flower micelle solution: 330 mM (closed square) and 550 mM (closed circle).
shown, as described in section 3.3. An increase in the concentration of PIC flower micelles lowered the temperature of the sol−gel phase transition. Increasing the concentration of micelles accelerated gelation. It is important to note that most ABA-type triblock copolymers reported so far need a concentration of more than approximately 5 wt % to form a gel.11,12,15 Our PIC flower micelles for the RIG system exhibited gelation at a concentration as low as approximately 2 wt %. It was assumed that the difference of the gelation mechanism between PIC flower micelles and conventional ABA-type triblock copolymers led to the difference of the concentration of the polymers for the gelation. Gelation at a lower concentration of polymer is desirable for in vivo use because of the low viscosity before gelation, which is easier for injections. An increase in the concentration of NaCl lowered the temperature of the sol−gel phase transition, which indicated that the PIC flower micelles were destabilized because of the increased electrostatic shielding effect of the ion pairs.21,30 The concentration of PB in the PIC flower E
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Figure 8. In vitro release profile of fluorescein-labeled poly(acrylic acid) (FL-PAAc) from redox-active injectable gel (RIG).
release was maintained for more than 4 weeks without an initial burst. In the case of polymeric drug carriers comprising hydrophobic polymers such as PLGA microspheres, the initial burst of the drug is caused by the immediate release from the pores and the surface of the microspheres.36−38 In contrast, it was assumed that the RIG had no pore owing to the hydrogel with hydration of hydrophilic PEG. In addition, it was considered that the cationic PMNT−PEG−PMNT of the RIG had an ionic interaction with anionic FL-PAAc throughout the gel and that the gel was gradually degraded from the surface, which induced the release of the drug. It is assumed that these properties of the RIG contributed to the suppression of the initial burst and controlled release. This result indicated that the RIG could be applied to the local delivery carrier for the controlled release of charged drugs such as proteins or siRNA.
4. CONCLUSIONS The material properties of RIG/PIC flower micelles comprising PMNT−PEG−PMNT and PAAc were evaluated. The PIC flower micelles exhibited irreversible sol−gel phase transitions by increasing temperature and ionic strength. At room temperature, the PIC flower micelles had a low viscosity. The viscosity increased with the increase of temperature and ionic strength, and they formed gels under the physiological condition. The RIG was able to provide a sustained release of an anionic model drug for more than 4 weeks without an initial burst. Our study indicated that RIG has potential as an injectable system for pharmaceutical and biomedical applications such as a local delivery carrier for the controlled release of charged drugs. In addition, it is anticipated that the RIG can provide a combination therapy for inflammation-related diseases by an incorporated drug in the gel and ROS scavenging nitroxide radicals conjugated in the PMNT−PEG−PMNT.
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ASSOCIATED CONTENT
S Supporting Information *
All details in synthesis of PMNT−PEG−PMNT triblock copolymer, titration of PMNT−PEG−PMNT and PAAc, preparation of fluorescein labeled PAAc (FL-PAAc) loaded RIG precursor, and critical micelle concentration (CMC). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.5b00305.
Figure 7. Temperature dependencies of complex viscosity of polyion complexes (PIC) flower micelles (a) with different concentrations of PIC flower micelles (r = 1:1, 0 mM NaCl, pH 6.2, 550 mM PB), (b) with different concentrations of NaCl (55 mg/mL, r = 1:1, pH 6.2, 550 mM PB), and (c) with various molar ratios (55 mg/mL, 0 mM NaCl, pH 6.2, 550 mM PB). F
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(27) Scaiano, J. C.; Laferrière, M.; Galian, R. E.; Maurel, V.; Billone, P. Phys. Status Solidi A 2006, 203 (6), 1337−1343. (28) Goon, P.; Manohar, C.; Kumar, V. V. J. Colloid Interface Sci. 1997, 189 (1), 177−180. (29) Ohya, Y.; Takeda, S.; Shibata, Y.; Ouchi, T.; Maruyama, A. Macromol. Chem. Phys. 2010, 211 (16), 1750−1756. (30) Yuan, X.; Harada, A.; Yamasaki, Y.; Kataoka, K. Langmuir 2005, 21 (7), 2668−2674. (31) Harada, A.; Togawa, H.; Kataoka, K. Eur. J. Pharm. Sci. 2001, 13 (1), 35−42. (32) Nikouei, N. S.; Vakili, M. R.; Bahniuk, M. S.; Unsworth, L.; Akbari, A.; Wu, J.; Lavasanifar, A. Acta Biomater. 2015, 12, 81−92. (33) Park, S. H.; Choi, B. G.; Joo, M. K.; Han, D. K.; Sohn, Y. S.; Jeong, B. Macromolecules 2008, 41 (17), 6486−6492. (34) Xu, Y.; Shen, Y.; Xiong, Y.; Li, C.; Sun, C.; Ouahab, A.; Tu, J. Drug Dev. Ind. Pharm. 2014, 40 (9), 1264−1275. (35) Yu, L.; Zhang, H.; Ding, J. Angew. Chem. 2006, 45 (14), 2232− 5. (36) Cohen, S.; Yoshioka, T.; Lucarelli, M.; Hwang, L.; Langer, R. Pharm. Res. 1991, 8 (6), 713−720. (37) Park, T. G.; Cohen, S.; Langer, R. Macromolecules 1992, 25 (1), 116−122. (38) Shieh, L.; Tamada, J.; Tabata, Y.; Domb, A.; Langer, R. J. Controlled Release 1994, 29 (1−2), 73−82.
AUTHOR INFORMATION
Corresponding Author
*Tel/Fax +81 29 853 5749; e-mail
[email protected] (Y.N.). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS A part of this work was supported by Grant-in-Aid for Scientific Research S (25220203) and the World Premier International Research Center Initiative (WPI Initiative) on Materials Nanoarchitronics of the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.
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DOI: 10.1021/acs.macromol.5b00305 Macromolecules XXXX, XXX, XXX−XXX
