Facile Synthesis of a H2O2-Responsive Alternating Copolymer Bearing Thioether Side Groups for Drug Delivery and Controlled Release.

A novel amphiphilic alternating copolymer with thioether side groups (P(MSPA-a-EG)) was synthesized through an amine-epoxy click reaction of 3-(methylthio)propylamine (MSPA) and ethylene glycol diglycidyl ether. P(MSPA-a-EG) was characterized in detail by nuclear magnetic resonance (NMR), gel permeation chromatography, Fourier transformed infrared, differential scanning calorimeter, and thermogravimetric analysis to confirm the successful synthesis. Due to its amphiphilic structure, P(MSPA-a-EG) could self-assemble into spherical micelles with an average diameter of about 151 nm. As triggered by H2O2, theses micelles could disassemble because hydrophobic thioether groups are transformed to hydrophilic sulfoxide groups in MSPA units. The oxidant disassemble process of micelles was systemically studied by dynamic light scattering, transmission electron microscopy, and 1H NMR measurements. The MTT assay against NIH/3T3 cells indicated that P(MSPA-a-EG) micelles exhibited good biocompatibility. Furthermore, they could be used as smart drug carriers to encapsulate hydrophobic anticancer drug doxorubicin (DOX) with 4.90% drug loading content and 9.81% drug loading efficiency. In vitro evaluation results indicated that the loaded DOX could be released rapidly, triggered by H2O2. Therefore, such a novel alternating copolymer was expected to be promising candidates for controlled drug delivery and release.

Introduction

In the past decades, various stimuli-responsive polymeric nanocarriers have been developed to overcome the disadvantages of traditional chemotherapy agents, including poor bioavailability, nonspecific selectivity, low accumulation in tumor tissue, adverse side effects, etc.[1−3] Up to now, a wide range of stimuli, such as redox,[4,5] pH,[6−10] oxidation,[11−14] overexpressed enzyme,[15] ultrasound,[16] magnetic field,[17] and light,[18,19] have been introduced into polymeric nanocarriers to achieve the controlled drug delivery and release in targeted cancer cells while minimizing the toxicity to normal cells. Among these stimuli, reactive oxygen species (ROS) have been widely used to develop various ROS-responsive polymers for target-specific drug delivery on the basis of the different ROS content between the pathological sites and their surroundings.[20−24] Usually, there are four ROS including hydrogen peroxides (H2O2), hydroxyl radicals (OH·), superoxides (O2–), and peroxynitrites (ONOO–). Among them, H2O2 is the most major ROS factor in biological microenvironment. Thus, a lot of H2O2-responsive polymeric materials have been designed and widely applied in drug delivery systems for cancer therapy in recent years.[25−27] However, most H2O2-responsive polymeric materials were still prepared through the complicated synthetic process.[28−30] Therefore, it is still a challenge to develop a facile synthetic method of H2O2-responsive polymers.

Alternating copolymers (ACPs) are an important kind of linear copolymers with two different structure units arranged alternately in their main chains and widely used as plasticizing agents,[31] chemical sensors,[32] photoelectricity,[33,34] and so on. Recently, Khan et. al. reported a facile method to synthesize ACPs by the robust, efficient, and orthogonal click chemistry under mild reaction conditions.[35] Our group also synthesized a serial of amphiphilic ACPs through the click reactions of amine-epoxy/amine-thiol and the obtained ACPs could self-assemble into various architectures, such as nanotubes,[36] vesicles,[37] and sea urchin like assemblies.[38] In addition, some of them were used as electrode materials.[39,40] Thus, we want to use this facile method of thiol-epoxy/amine-epoxy click reactions to further design and synthesize ACPs with excellent biocompatibility and stimulus-responsive properties for biomedical applications.

Herein, we reported a novel H2O2-responsive amphiphilic ACP P(MSPA-a-EG) with thioether side groups that was conveniently synthesized by the amine-epoxy click reaction from 3-(methylthio)propylamine (MSPA) and ethylene glycol diglycidyl ether (EGDE) at room temperature. The self-assembly of P(MSPA-a-EG) in water and the corresponding oxidant disassemble process were studied in detail by dynamic light scattering (DLS), transmission electron microscopy (TEM), and 1H nuclear magnetic resonance (NMR) measurements. In addition, we also investigated the potential of this H2O2-responsive alternating copolymer as a smart carrier for controlled drug release (Scheme ).

Scheme 1

Schematic Illustration of Doxorubicin (DOX)-Loaded and H2O2-Responsive Drug Release Behavior of P(PSMA-a-EG) Micelles

Results and Discussion

Synthesis and Characterization of P(MSPA-a-EG)

As shown in Scheme , P(MSPA-a-EG) was synthesized from MSPA and EGDE by one-step amine-epoxy click polymerization at room temperature without any catalyst. The resulting ACP was characterized by NMR, Fourier-transform infrared spectroscopy (FTIR), and gel permeation chromatography (GPC) techniques. The 1H NMR spectra of EGDE, MSPA, and P(MSPA-a-EG) are displayed in Figure A. Compared with their 1H NMR spectra in CDCl3, the peaks at 2.6 ppm (a), 2.79 ppm (a′), and 3.16 ppm (b) belonging to the epoxy protons of EGDE disappeared completely, and two new peaks at 2.5 ppm (a) and 3.83 ppm (b) belonging to methylene protons (−N–CH–CH(OH)−) and methyne protons (−N–CH2–CH(OH)−), respectively, appeared in the 1H NMR spectrum of P(MSPA-a-EG). The peak at 1.47 ppm (5) belonging to the amine protons of MSPA also disappeared completely in the 1H NMR spectrum of P(MSPA-a-EG). Furthermore, the peak area integral ratio (Sb/Sd/Sc/S2+4+a/S1/S3) of all protons in P(MSPA-a-EG) was approximately equal to its theoretical value of 2:4:4:8:3:2, which also confirmed the chemical structure of the resulting alternating copolymer. In addition, the 13C NMR spectrum of P(MSPA-a-EG) in Figure B further verified its chemical structure. The GPC curve, FTIR spectrum, thermogravimetric analysis (TGA) curve, and differential scanning calorimetry (DSC) curve of P(MSPA-a-EG) are shown in Figure . As shown in Figure A, the number-averaged molecular weight (Mn) and polydispersity index (PDI) of P(MSPA-a-EG) were 2110 and 2.66, respectively, which indicated that the amine-epoxy click polymerization was successful. The FTIR spectrum of P(MSPA-a-EG) is exhibited in Figure B. The strong broad peak at 3382 cm–1 was attributed to the stretching vibration of −OH, which indicated that strong multiple hydrogen bonds would form among these hydroxyl groups.[41] The peaks at 2903 and 2846 cm–1 could be ascribed to the asymmetric and symmetric stretching vibrations of −CH2– respectively. The peak at 1447 cm–1 belonged to the bending vibration of −CH2–. The strong peak at 1110 cm–1 was assigned to the stretching vibration of C–O. The thermal properties of P(MSPA-a-EG) were studied by TGA and DSC measurements, and the results are shown in Figure C,D. The thermal stability of P(MSPA-a-EG) was relatively high, with the initial decomposition temperature of around 304 °C (Figure C). The glass transition temperature of P(MSPA-a-EG) was about −36.8 °C (Figure D). All the above experimental results confirmed that P(MSPA-a-EG) was synthesized successfully.

Scheme 2

Synthesis of P(MSPA-a-EG) through the Amine-Epoxy Click Copolymerization

Figure 1

(A) 1H NMR spectra of EGDE, MSPA, and P(MSPA-a-EG). (B) 13C NMR spectrum of P(MSPA-a-EG).

Figure 2

(A) GPC curve, (B) FTIR spectrum, (C) TGA curve, and (D) DSC curve of P(MSPA-a-EG).

Self-Assembly Behavior of P(MSPA-a-EG)

Based on the amphiphilic structure of P(MSPA-a-EG) with alternating hydrophobic MSPA units and hydrophilic EG units in the main chain, it could self-assemble spontaneously in water. The formation of P(MSPA-a-EG) micelles was confirmed by fluorescence technique with Nile red as a fluorescent probe. The fluorescence intensity of Nile red increased dramatically when the concentration of P(MSPA-a-EG) increased to a certain value, which verified the formation of micelles and the encapsulation of Nile red into the hydrophobic core of micelles (Figure A). In addition, the maximum emission wavelength of Nile red in the P(MSPA-a-EG) solution exhibited about 26 nm blue shift from 659 to 633 nm when the concentration of P(MSPA-a-EG) was increased from 3.9 × 10–3 to 0.5 mg/mL (Figure B). This further confirmed that the Nile red was transferred from the water into the hydrophobic environment as a result of the formation of micelles, which was consistent with previous report.[42] Accordingly, the critical micelle concentration (CMC) of P(MSPA-a-EG) was calculated to be about 63.4 μg mL–1 (Figure C). The size and morphology of P(MSPA-a-EG) micelles were measured by DLS and TEM measurements, respectively. As shown in Figure A, the DLS curve indicated that P(MSPA-a-EG) was able to assemble into nanoparticles with an average size of about 151 nm and the particle-size distribution index (PDI) of 0.249. The TEM image in Figure B exhibits that P(MSPA-a-EG) micelles were spherical with an average diameter of approximately 130 nm, which was slightly smaller than that measured by DLS. This was attributed to the dry state of micelles in the TEM measurement but the wet state of micelles in DLS measurement.

Figure 3

(A) Fluorescence emission spectra of Nile red in different concentrations of P(MSPA-a-EG) solution. (B) Blue shift of maximum emission wavelength of Nile red in different concentrations of P(MSPA-a-EG) solution. (C) Emission intensity of Nile red versus the different concentrations of P(MSPA-a-EG).

Figure 4

(A) DLS curve and (B) TEM image of P(MSPA-a-EG) micelles.

H2O2 Responsiveness of P(MSPA-a-EG) Micelles

Generally, hydrophobic thioether groups are easy to transform into hydrophilic sulfoxide groups in an oxidative environment. Here, H2O2 was employed as the oxidant to study the oxidation responsiveness of P(MSPA-a-EG) micelles. The aqueous dispersions of P(MSPA-a-EG) micelles were incubated with H2O2 at various concentrations from 0 to 166.6 mM for 12 h, and the final photographs are shown in Figure A. With the increasing concentration of H2O2, the turbid aqueous dispersions of P(MSPA-a-EG) micelles were gradually changed into transparent solutions, which indicated the disassembly of P(MSPA-a-EG) micelles in the oxidative process. The UV–vis spectrophotometer was used to detect the transmittance of aqueous dispersions of P(MSPA-a-EG) micelles, and the results are shown in Figure B. When the concentration of H2O2 was improved to 22.3 mM, the transmittance was increased to 98.6%, which confirmed that almost all the micelles dissociated at this concentration of H2O2. Meanwhile, the diameter changes of P(MSPA-a-EG) micelles with different amounts of H2O2 were measured by DLS and TEM. Both results exhibited the disassembly of P(MSPA-a-EG) micelles after oxidation. In detail, the diameter of P(MSPA-a-EG) micelles decreased from 151 nm to 6 nm by DLS with the increase in concentration of H2O2 from 0 to 166.6 mM (Figure C).

Figure 5

(A) Photographs of (B) transmittance and (C) diameter changes of P(MSPA-a-EG) micelles solution after treatment with different concentrations of H2O2. (D) TEM images of P(MSPA-a-EG) micelles after treatment with different concentrations of H2O2.

Meanwhile, the TEM images in Figure D indicate that the size of P(MSPA-a-EG) micelles decreased continuously with the increase in concentration of H2O2. When the concentration of H2O2 was increased to 166.6 mM, lots of tiny fragments of several nanometers were observed. The disassembly of P(MSPA-a-EG) micelles was ascribed to the oxidation of hydrophobic thioether groups into hydrophilic sulfoxide groups or even sulfone groups, which endowed the change of P(MSPA-a-EG) from amphiphilic to hydrophilic.

To further verify the oxidation of thioether groups into sulfoxide groups or even sulfone groups, all the above samples were freeze-dried and then characterized by 1H NMR measurement. As shown in Figure A, the proton signals at 2.06 and 2.42 ppm belonging to methyl (−SCH) and methylene (−CHSCH3) adjacent to the sulfur atom in the thioether group gradually disappeared with the increase in concentration of H2O2. Meanwhile, some new proton signals appeared at 2.59, 2.62, and 1.91 ppm, which could be attributed to methyl (−SOCH), methylene (−CHSOCH3), and methylene (−CHCH2SOCH3). When the concentration of H2O2 increased to 22.3 mM, the proton signals adjacent to the sulfur atom in the thioether groups disappeared completely, which indicated that all thioether groups were oxidized into sulfoxide groups. The oxidation extent of P(MSPA-a-EG) micelles was estimated by the following equation, and the results are shown in Figure B.The oxidation extent of P(MSPA-a-EG) micelles was calculated as 22 and 85.3% after treatment with H2O2 at concentrations of 2.3 and 8.9 mM, respectively. Furthermore, the oxidation extent of P(MSPA-a-EG) micelles reached 100% when the concentration of H2O2 was 22.3 mM or higher. This H2O2-concentration-dependent behavior was consistent with the results of UV–vis and DLS measurements.

Figure 6

(A) 1H NMR spectra of P(MSPA-a-EG) micelles after treatment with different concentrations of H2O2. (B) Oxidation extent of P(MSPA-a-EG) micelles at various concentrations of H2O2.

Cell Cytotoxicity Assay

As a nanocarrier for drug delivery, the cytotoxicity to normal cells or biocompatibility is a key parameter for the biomedical applications of P(MSPA-a-EG). Thus, the in vitro cytotoxicity of P(MSPA-a-EG) was evaluated by MTT assay against the NIH/3T3 cell line. The NIH/3T3 cells were incubated with aqueous solutions of P(MSPA-a-EG) at different concentrations from 0.005 to 1 mg mL–1 for 24 h and the results are displayed in Figure . Obviously, the cell viability even remained 80.0% when the concentration of P(MSPA-a-EG) increased to 1 mg mL–1. Therefore, P(MSPA-a-EG) had good biocompatibility and could be used as materials for drug delivery.

Figure 7

Cell cytotoxicity of P(MSPA-a-EG) against NIH/3T3 cells after 24 h incubation. The data are presented as average ± standard deviation (n = 6).

DOX-Loaded and in Vitro H2O2-Responsive Drug Release

Here, anticancer drug DOX was selected as a model drug to evaluate the potential of H2O2-responsive P(MSPA-a-EG) micelles as a smart drug delivery system for drug control release. According to the standard curve of DOX, drug-loading content (DLC) and drug-loading efficiency (DLE) of P(MSPA-a-EG) micelles loaded with DOX were calculated as 4.9 and 9.81%, respectively. Then, the in vitro drug release of DOX-loaded P(MSPA-a-EG) micelles was investigated in pure phosphate-buffered saline (PBS) and PBS with 5 or 20 mM H2O2. As shown in Figure , the cumulative release of drug from DOX-loaded P(MSPA-a-EG) micelles in PBS was only about 23.4% for 24 h. When the concentration of H2O2 was 5 mM in PBS, the cumulative release of drug from DOX-loaded P(MSPA-a-EG) micelles was just increased to 31.4% within the same time. This indicated that a small part of hydrophobic thioether in P(MSPA-a-EG) was oxidized to hydrophilic sulfoxide at 5 mM H2O2 and resulted in the partial dissociation of the micelles. When the concentration of H2O2 in PBS was further improved to 20 mM, the cumulative release of drug from DOX-loaded P(MSPA-a-EG) micelles was significantly increased to 55.6%, which was ascribed to the rapid dissociation of P(MSPA-a-EG) micelles under the higher concentration of H2O2. Thus, H2O2-responsive drug release can be realized through the transition from amphiphilic to hydrophilic of P(MSPA-a-EG) on the basis of thioether groups oxidized to sulfoxide groups. Overall, P(MSPA-a-EG) micelles could be used as a potential biomaterial for drug delivery and controlled release.

Figure 8

In vitro drug release behavior of DOX-loaded P(MSPA-a-EG) micelles with different concentrations of H2O2 at 37 °C.

Conclusions

Novel amphiphilic ACP P(MSPA-a-EG) was synthesized successfully by amine-epoxy click copolymerization of MSPA and EGDE. H2O2-responsive micelles with an average diameter of about 151 nm were constructed by the self-assembly of P(MSPA-a-EG). Triggered by H2O2, hydrophobic thioether groups in the micelle cores were oxidized into hydrophilic sulfoxide groups and resulted in the rapid dissociation of P(MSPA-a-EG) micelles. In vitro cell cytotoxicity assay indicated that P(MSPA-a-EG) micelles exhibited good biocompatibility against normal cells. Anticancer drug DOX could be loaded into P(MSPA-a-EG) micelles effectively and realize H2O2-triggered rapid drug release. Such H2O2-responsive ACP P(MSPA-a-EG) micelles based on an amphiphilic-to-hydrophilic transition would be promising candidates as smart drug carriers for drug delivery and controlled release.

Experimental Section

Materials

3-(Methylthio)propylamine (MSPA, 98%, TCI), Nile red (99%, Acros), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, Sigma), triethylamine (TEA, 99%, Adamas), acetone (99.5%, Adamas), and ethanol (99.5%, Adamas) were used as received. Ethylene glycol diglycidyl ether (EGDE) was purchased from Adamas and purified by distillation under reduced pressure.

Measurements

GPC measurement was performed on an HLC-8320GPC (TOSOH, EcoSEC GPC System) system at 40 °C with dimethylformamide as the mobile phase at a flow rate of 0.6 mL min–1. 1H NMR and 13C NMR spectra were obtained on a Varian Mercury Plus 400 MHz spectrometer with deuterium chloroform (CDCl3) as a solvent at 20 °C. Tetramethylsilane was used as an internal standard. The DSC measurement was carried out on a Thermal Advantage DSC Q2000 autosampler (TA Instruments) equipped with a refrigerator cooling system. The polymer (9.1 mg) was placed in aluminum pans (nonhermetic) (30 μL) scanned at a heating rate of 10 °C min–1 from −80 to 80 °C under dry nitrogen atmosphere. The data were treated by using Universal Analysis 2000 V 4.3 A software from TA Instruments. FTIR spectrum was recorded on a PerkinElmer Spectrum 100 FTIR spectrometer by KBr sample holder method. TGA was measured on a PerkinElmer Q5000IR thermobalance by using nitrogen as the purging gas at a heating rate of 20 °C min–1. Dynamic light scattering (DLS) measurement was performed under a 3,000 HS (Malvern Instruments, Ltd.) equipped with 125 mW laser light operating at λ = 633 nm with a scattering angle of 90°. Transmission electron microscopy (TEM) studies was observed under a JEOL 2010 instrument operated at 200 kV. One little drop of the micelle solution (0.2 mg mL–1) was dropped onto a carbon-coated copper grid. Then, the grid was immersed into liquid nitrogen and freeze-dried in vacuum at −50 °C before measurement. Fluorescent spectra were measured on QC-4-CW spectrometer, made by Photon Technology International, Int. USA/CAN. The excitation wavelength was set at 550 nm, and the emission was monitored from 570 to 750 nm. Ultraviolet–visible (UV–vis) absorption of the sample solutions was measured at room temperature by using a Thermo Electron-EV300 UV–vis spectrophotometer. The slit-width was set as 1 nm with a scan speed of 480 nm min–1.

Synthesis of P(MSPA-a-EG)

P(MSPA-a-EG) was synthesized from MSPA and EGDE as monomers by the amine-epoxy click reaction. Typically, MSPA (2.613 g, 15 mmol) and EGDE (1.578 g, 15 mmol) were added into a 25 mL round flask. After stirring for 48 h at room temperature, the polymer was purified by dialysis against ethanol for 48 h (MWCO = 3500 g mol–1). By rotary evaporation to remove ethanol, the yellowish viscous liquid P(MSPA-a-EG) was obtained.

Preparation of P(MSPA-a-EG) Micelles

Briefly, 10.0 mg P(MSPA-a-EG) was dissolved in 0.5 mL ethanol completely. Then, the solution was added dropwise into 5 mL deionized water under slight stirring for 10 min. Subsequently, the solution was dialyzed in deionized water for 12 h (MWCO = 1000 g mol–1), during which the deionized water was renewed every 4 h. Finally, P(MSPA-a-EG) micelle aqueous solution was obtained.

Critical Micellization Concentration (CMC) of P(MSPA-a-EG)

To determine the CMC value of P(MSPA-a-EG), Nile red was used as a fluorescent probe. Twenty-five microliters of Nile red acetone solution (1.6 × 10–4 mol L–1) was added into 4 mL of aqueous solution of P(MSPA-a-EG) with different concentrations (from 0.00753 to 1 mg mL–1), while the final concentration of Nile red in each solution was kept at 1.6 × 10–6 mol L–1. Then, the samples were exposed to air overnight to remove acetone completely. The fluorescence emission spectra of all samples were recorded on a fluorescence spectrometer at the excitation wavelength of 550 nm.

H2O2-Responsiveness of P(MSPA-a-EG) Micelles

The blank P(MSPA-a-EG) micelles (1.5 mL, 10 mg mL–1) were mixed with 0.5 mL H2O2 solutions at different concentrations. After treatment for 12 h at room temperature, the appearance and transmittance of these samples were recorded by using a digital camera and a UV–vis spectrophotometer at the wavelength of 500 nm. In addition, the diameter changes of P(MSPA-a-EG) micelles was monitored by DLS. Finally, all the samples were lyophilized and characterized by 1H NMR in CDCl3.

Preparation of DOX-Loaded P(MSPA-a-EG) Micelles

The typical preparation of DOX-loaded P(MSPA-a-EG) micelles was as follows: a predetermined amount of DOX·HCl and one molar equivalent of trimethylamine (TEA) were dissolved in 1 mL of DMSO solution containing 15 mg of P(MSPA-a-EG) completely. Then, the mixture was slowly added into 8 mL of deionized water under slight stirring at room temperature for 20 min. Subsequently, the mixture was transferred to a dialysis bag (MWCO = 1000 g mol–1) and dialyzed against deionized water for 24 h. To determine the loading amount of DOX, 1 mL of DOX-loaded micelle solution was lyophilized and then redissolved in DMSO. The total loading amount of DOX was determined by the UV absorbance of the solution at 500 nm. The drug-loading content (DLC) and drug-loading efficiency (DLE) were calculated according to the following equationsHere, Wloaded, Wtotal, and Wpolymer representing the weight of the loaded DOX, total DOX, and P(MSPA-a-EG), respectively.

In Vitro H2O2-Triggered Drug Release

Two milliliters of DOX-loaded P(MSPA-a-EG) micelles solution (1 mg mL–1) was transferred into a dialysis bag (MWCO = 1000 g mol–1). Then, the dialysis bag was immersed in 30 mL PBS, or PBS with 5 mM, or 20 mM H2O2 in a shaking water bath at 37 °C. At predetermined time intervals, 3 mL of the external buffer solution was withdrawn and replaced with 3 mL of fresh PBS, or PBS with 5 mM or 20 mM H2O2. The cumulative released amount of DOX was determined by using the fluorescence measurement by QC-4-CW spectrometer at the excitation wavelength of 485 nm. All DOX-released experiments were carried out in triplicate, and the results are shown as the average data with standard deviations.

Cell Culture

NIH/3T3 normal cells (a mouse embryonic fibroblast cell line) were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplied with 10% FBS and antibiotics (50 U mL–1 penicillin and 50 U mL–1 streptomycin) at 37 °C in a humidified atmosphere containing 5% CO2.

MTT Assay

The biocompatibility of P(MSPA-a-EG) was evaluated by MTT assay. NIH/3T3 cells were seeded in 96-well plates at 8 × 103 cells per well in 200 μL of DMEM. After incubation overnight, the DMEM was removed and fresh DMEM with P(MSPA-a-EG) at different concentrations (0.005 to 1 mg mL–1) added. The cells without the treatment were used as control. The cells were incubated for another 24 h. Then, 20 μL of 5 mg mL–1 MTT solution in PBS was added to each well. After the cells were incubated for 4 h, the DMEM containing unreacted MTT was carefully removed. Then, 200 μL of DMSO was added into each well to dissolve the blue formazan crystal, and the absorbance at a wavelength of 490 nm was measured by a BioTek Synergy H4 hybrid reader. The blank was subtracted to the measured optical density values, and the cell viability was expressed as percentage of the values obtained for the untreated control cells.