Facile Preparation of Reduction-Responsive Micelles Based on Biodegradable Amphiphilic Polyurethane with Disulfide Bonds in the Backbone

In this paper, we synthesized a biodegradable amphiphilic polymer of polyurethane-polyethylene glycol with disulfide bonds in the main chain (PEG-PU(SS)-PEG). DLS and SEM showed that the polymer could self-assemble into micelles in aqueous solution and could be used to load the hydrophobic anticancer drug DOX. Intriguingly, drug release in vitro indicated that DOX-loaded PEG-PU(SS)-PEG micelles had good stability under the extracellular physiological environment, but the disulfide bonds broke rapidly and DOX was released quickly under the intracellular reducing conditions. CCK-8 assays showed that DOX-loaded PEG-PU(SS)-PEG micelles had a high in vitro antitumor activity in C6 cells, whereas blank PEG-PU(SS)-PEG micelles were nontoxic to C6 cells. It was also found that there was strong and persistent accumulation of DOX-loaded PEG-PU(SS)-PEG as compared with PEG-PU-PEG both by the cell internalization tests and the flow cytometry measurements. Hence, PEG-PU(SS)-PEG micelles will have a potential use for clinical treatment of cancer in the future.


Introduction
In recent years, the early detection and effective treatment of cancer still represent a huge challenge due to the disease's complexity, diversity, and dynamics [1,2]. So far, there is no complete effective solution to the treatment of cancer. Traditional methods of treating cancer include surgical resection, radiotherapy, and chemotherapy, among which chemotherapy is the most common method [3]. However, intravenous administration of many highly effective chemotherapeutic agents is often hampered by their low solubility in aqueous environments [4]. Conventional anticancer chemotherapeutic drugs currently on the market face severe systemic side effects, multidrug resistance (MDR), and deadly metastasis of cancer cells. To address these limitations of chemotherapeutic agents, many drug delivery systems (DDSs) have been studied in order to deliver drugs to tumor sites without harming healthy tissues and organs [5][6][7]. In this case, polymeric micelles have shown great advantages and potential as delivery vehicles for chemotherapeutic drugs, overcoming the problems caused by conventional free drugs, such as poor solubility, poor stability, fast clearance, poor selectivity, and adverse effects on healthy cells, among others [8]. Nanomicelles have good advantages, including improving the solubility of insoluble drugs, being more stable in the body, aiming at the

Characterization
1 H NMR spectra were recorded on a Bruker 400 MHz (Bruker, Billerica, MA, USA) resonance spectrometer. The molecular weight and molecular weight distribution of the polymer were determined by PL GPC50 gel permeation chromatography (GPC). The GPC was equipped with 10E4, 2M Jordi (Agilent, Santa Barbara, CA, USA) gel column (DMF was used as eluent and the flow rate was 1.0 mL/min). The particle size and zeta potential of micelles were measured with a Zetasizer Nano-ZS dynamic light-scattering (DLS) instrument (Malvern, Malvern, UK). The morphology of micelles was observed with scanning electron microscope (SEM) SU-8010 (Hitachi, Tokyo, Japan). The fluorescence intensity of DOX was detected by an F-4600 (Hitachi,Tokyo, Japan) fluorescence spectrophotometer at 25 °C. CCK-8 experimental data were measured by a Synergy H4 mixed multifunction enzyme scale instrument (BioTek, Winooski, VT, USA). Scheme 1. The illustration of the preparation of DOX-loaded PEG-PU(SS)-PEG micelles for reduction-responsive intracellular drug release.

Characterization
1 H NMR spectra were recorded on a Bruker 400 MHz (Bruker, Billerica, MA, USA) resonance spectrometer. The molecular weight and molecular weight distribution of the polymer were determined by PL GPC50 gel permeation chromatography (GPC). The GPC was equipped with 10E4, 2M Jordi (Agilent, Santa Barbara, CA, USA) gel column (DMF was used as eluent and the flow rate was 1.0 mL/min). The particle size and zeta potential of micelles were measured with a Zetasizer Nano-ZS dynamic light-scattering (DLS) instrument (Malvern, Malvern, UK). The morphology of micelles was observed with scanning electron microscope (SEM) SU-8010 (Hitachi, Tokyo, Japan). The fluorescence intensity of DOX was detected by an F-4600 (Hitachi, Tokyo, Japan) fluorescence spectrophotometer at 25 • C. CCK-8 experimental data were measured by a Synergy H4 mixed multifunction enzyme scale instrument (BioTek, Winooski, VT, USA).

Synthesis of PEG-PU(SS)-PEG and PEG-PU-PEG
Under the nitrogen protection, LDI (0.119 g, 0.525 mmol), PLA-SS-PLA (0.50 g, 0.5 mmol), 7.6 mL DMF, and 6 mg Sn(Oct) 2 were added sequentially to the reactor and reacted at 65 • C for 48 h. After cooling to room temperature, PEG-OH (0.25 g, 0.025 mmol) was added dropwise to the above solution and the reaction was continued at 25 • C for 48 h. Then the reaction solution was dripped into ice-ethyl ether for sedimentation. The polymer was centrifuged and dried for 48 h (PEG-OH was treated with toluene to remove water; toluene and DMF were re-distilled before the experiment). Yield: 82%. The specific synthetic route is shown in Scheme 2.
The synthesis procedure of PEG-PU-PEG is basically the same as PEG-PU(SS)-PEG, except that PLA-SS-PLA diol is replaced by PLA diol.

Synthesis of PEG-PU(SS)-PEG and PEG-PU-PEG
Under the nitrogen protection, LDI (0.119 g, 0.525 mmol), PLA-SS-PLA (0.50 g, 0.5 mmol), 7.6 mL DMF, and 6 mg Sn(Oct)2 were added sequentially to the reactor and reacted at 65 °C for 48 h. After cooling to room temperature, PEG-OH (0.25 g, 0.025 mmol) was added dropwise to the above solution and the reaction was continued at 25 °C for 48 h. Then the reaction solution was dripped into ice-ethyl ether for sedimentation. The polymer was centrifuged and dried for 48 h (PEG-OH was treated with toluene to remove water; toluene and DMF were re-distilled before the experiment). Yield: 82%. The specific synthetic route is shown in Scheme 2.
The synthesis procedure of PEG-PU-PEG is basically the same as PEG-PU(SS)-PEG, except that PLA-SS-PLA diol is replaced by PLA diol.

Preparation and Characterization of PEG-PU(SS)-PEG Micelles
PEG-PU(SS)-PEG micelles were prepared via a dialysis method. The specific procedure was as follows: 2 mg of PEG-PU(SS)-PEG was dissolved in 1 mL of dimethyl sulfoxide (DMSO) under stirring at room temperature for 12 h. Deionized water (1.5 mL) was slowly added dropwise to the DMSO solution of PEG-PU(SS)-PEG. After the addition was completed, the prepared micellar solution was loaded into a pre-prepared dialysis bag (SPECTRA/POR, MWCO: 3500, (Spectrum Labs, Rancho Dominguez, CA, USA) and dialyzed against PB (10 mM, pH 7.4) for 24 h.
The particle size and distribution of the PEG-PU(SS)-PEG micelles were determined by DLS. Micellar solutions were filtered using a 0.45 μm filter before testing. The morphology of the PEG-PU(SS)-PEG micelles was observed by scanning electron microscope (SEM). Sample Preparation: The micellar solution was diluted to 0.1 mg/mL. A 10 μL portion of micellar solution was dropped on the silicon pellet and dried at room temperature.
In this study, the critical micelle concentration (CMC) of PEG-PU(SS)-PEG micelles was determined by using pyrene as a fluorescence probe. Thirteen kinds of micellar solution with a different concentration gradient were prepared. Then, a 30 μL portion of pyrene-acetone solution (1.622 × 10 −5 g/mL) was added under dark conditions. The excitation wavelength of the

Preparation and Characterization of PEG-PU(SS)-PEG Micelles
PEG-PU(SS)-PEG micelles were prepared via a dialysis method. The specific procedure was as follows: 2 mg of PEG-PU(SS)-PEG was dissolved in 1 mL of dimethyl sulfoxide (DMSO) under stirring at room temperature for 12 h. Deionized water (1.5 mL) was slowly added dropwise to the DMSO solution of PEG-PU(SS)-PEG. After the addition was completed, the prepared micellar solution was loaded into a pre-prepared dialysis bag (SPECTRA/POR, MWCO: 3500, (Spectrum Labs, Rancho Dominguez, CA, USA) and dialyzed against PB (10 mM, pH 7.4) for 24 h.
The particle size and distribution of the PEG-PU(SS)-PEG micelles were determined by DLS. Micellar solutions were filtered using a 0.45 µm filter before testing. The morphology of the PEG-PU(SS)-PEG micelles was observed by scanning electron microscope (SEM). Sample Preparation: The micellar solution was diluted to 0.1 mg/mL. A 10 µL portion of micellar solution was dropped on the silicon pellet and dried at room temperature.
In this study, the critical micelle concentration (CMC) of PEG-PU(SS)-PEG micelles was determined by using pyrene as a fluorescence probe. Thirteen kinds of micellar solution with a different concentration gradient were prepared. Then, a 30 µL portion of pyrene-acetone solution (1.622 × 10 −5 g/mL) was added under dark conditions. The excitation wavelength of the spectrofluorometer was set to 330 nm and the fluorescence intensity of the micellar solution was measured in the scan range from 350 to 500 nm.

Degradation of PEG-PU(SS)-PEG Micelles under Reducing Conditions
The degradation of the PEG-PU(SS)-PEG micelles under reducing conditions (10 mM DTT) was followed by using DLS to measure the particle size of the micelles. In brief, two micellar solutions (2 mL) were prepared, one of which was bubbled under nitrogen to remove the oxygen. The well-dosed DTT was added to a glass cell of PEG-PU(SS)-PEG micelles (0.1 mg/mL) under nitrogen protection. PEG-PU(SS)-PEG micelles without DTT were used as a control. The two glass sample cells were then sealed with rubber stoppers, shaken evenly, and placed in a 37 • C shaker (200 rpm). The DLS was measured to track the change in particle size of the micelles at selected time points.

Preparation of Encapsulated-DOX PEG-PU(SS)-PEG Micelles and Determination of Drug Loading
In this experiment, the common anticancer drug doxorubicin (DOX) was chosen as a model drug.
Since it was a fluorescent-sensitive substance, it was necessary to protect it from light during operation.
According to the theoretical drug loading (10% or 20%), the required DOX was dissolved in the DMSO solution of PEG-PU(SS)-PEG and sonicated for 30 min. Then, 1.5 mL of water was added dropwise into the above solution under vigorous stirring and the solution was sonicated again for 1 h. The mixed solution was then transferred into a dialysis bag (SPECTRA/POR, MWCO: 3500) and dialyzed against PB (10 mM, pH 7.4) for 24 h. The entire experiment was processed in dark conditions.
In order to determine the content of DOX, a certain amount of lyophilized drug-loaded micelles was dissolved in DMSO and sonicated for 1 h. The fluorescence intensity was measured with an F-4600 fluorescence spectrophotometer, wherein the corresponding drug loading content (DLC) and drug loading efficiency (DLE) of DOX were calculated according to the fluorescence standard curve. DLC and DLE were calculated based on Equations (1) and (2) DOX-loaded micelles were prepared in three dialysis bags (MWCO = 12,000-14,000) and immersed into 20 mL of the appropriate media. At the scheduled time, 4 mL of release media was removed and replenished with an equal volume of fresh release media. After 24 h, the sampling ended and the fluorescence of DOX was determined by an F-4600 fluorescence spectrophotometer to determine the content of drug released at each time point. Each release experiment was run in parallel three times.

CCK-8 Assays
The cytotoxicity of PEG-PU(SS)-PEG micelles was evaluated using C6 cells. C6 cells were cultivated in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), 1% L-glutamic acid, antibiotics penicillin (100 IU/mL), and streptomycin (100 µg/mL) at 37 • C in a humidified atmosphere of 5% CO 2 . C6 cells were digested when they were cultured to about 80% of the culture flask and then seeded in the 96-well plates (1 × 10 4 cells/well). This experiment had five groups of test groups (micellar concentrations of 0.2, 0.4, 0.6, 0.8, and 1 mg/mL), a control group, and a blank group. After incubation for 24 h, the medium was sucked away and replaced by 100 µL of fresh medium containing various concentrations of micelle for another 24 h. Afterward, a 10 µL portion of CCK-8 solution was added to each well for 0.5-4 h of incubation. The absorbance (A) of each well was measured using a Synergy Model H4 hybrid multi-plate reader, and the blank medium well was used as a zero group for zero adjustment. The experiment is performed in multiples. According to the plate reader results, cell viability was calculated based on Equation (3): In the formula: A T : Absorbance of test group A C : Absorbance of control group A B : Absorbance of blank group As described above, the antitumor activity of DOX-loaded PEG-PU(SS)-PEG micelles and free DOX·HCl was also studied by CCK-8 assays. C6 cells were cultivated in RPMI 1640 media supplemented with 10% FBS, 1% L-glutamic acid, antibiotics penicillin (100 IU/mL), and streptomycin (100 µg/mL) at 37 • C in a humidified atmosphere of 5% CO 2 . C6 cells were digested when they were cultured to about 80% of the culture flask and then seeded in 96-well plates (5 × 10 3 cells/well). This experiment had seven groups of test groups (DOX concentrations of 0.625, 1.25, 2.5, 5, 10, 20, and 40 µg/mL), a control group, and a blank group. After incubation for 24 h, the medium was sucked away and replaced by 100 µL of fresh medium containing various concentrations of micelle for 48 h. Afterward, a 10 µL portion of CCK-8 solution was added to each well for 0.5-4 h of incubation. The absorbance (A) of each well was measured using a Synergy H4 hybrid multi-plate reader, and the blank medium well was used as a zero group for zero adjustment. The experiment is performed in multiples. According to the plate reader results, the cell viabilities were calculated as described above.

Cell Internalization
The cellular uptake and intracellular drug release behaviors of DOX-loaded PEG-PU(SS)-PEG micelles were examined in C6 cells using a fluorescence microscope. C6 cells were cultivated in RPMI 1640 media supplemented with 10% FBS, 1% L-glutamic acid, antibiotics penicillin (100 IU/mL), and streptomycin (100 µg/mL) at 37 • C in a humidified atmosphere of 5% CO 2 . C6 cells were digested when they were cultured to about 80% of the culture flask and then seeded in 24-well plates (6 × 10 4 cells/well). After incubation for 24 h, the medium was sucked away and replaced by fresh medium containing DOX-loaded PEG-PU(SS)-PEG micelles, DOX-loaded PEG-PU-PEG micelles, or DOX·HCl (final DOX concentration of 40 µg/mL) for 0.5 h, 2 h, and 4 h. The medium in each well was removed and washed three times with phosphate buffered saline (PBS, 10 mM, pH 7.4). Thereafter, the cells were fixed with 1 mL of 4% paraformaldehyde for 30 min. After removal of the paraformaldehyde, the cells were rinsed with PBS another three times, and stained with 4,6-diamidino-2-phenylindole (DAPI) for 15 min. Finally, the excess staining solution was washed three times with PBS and fluorescence images of cells were obtained with a fluorescence microscope.

Synthesis of Reduction-Sensitive Degradable PEG-PU(SS)-PEG Triblock Copolymer
PEG-PU(SS)-PEG was synthesized via condensation reaction of polylactide diol (PLA-SS-PLA) and diisocyanate (LDI), then reacted with PEG-OH. The polymer was characterized by 1 H NMR and GPC. The 1 H NMR spectrum of PEG-PU(SS)-PEG polymers showed that the peaks at chemical shift δ 1.67 and δ 5.20 corresponded to the hydrogen of methyl and methyne of PLA-SS-PLA, respectively ( Figure 1). The peak at δ 2.95 corresponded to methylene hydrogen of PLA-SS-PLA. The chemical shift of 3.66 is the signal of methylene hydrogen in the PEG backbone, and δ 1.24 and δ 1.97 are signals for methylene hydrogen in LDI. GPC data (Table 1) showed that PEG-PU(SS)-PEG had a single peak and a narrower molecular weight distribution (the Polymer dispersity index (PDI) value was 1.03). All the results demonstrated the successful synthesis of PEG-PU(SS)-PEG in this experiment. In addition, PEG-PU-PEG without disulfide bonds in the backbone was synthesized as a control.

Formation and Characterization of PEG-PU(SS)-PEG Micelles
In this study, PEG-PU(SS)-PEG micelles were prepared via a dialysis method. The size of the PEG-PU(SS)-PEG micelles was 116.7 nm measured by DLS at 25 °C, which indicated that the PEG-PU(SS)-PEG micelles had a small size and uniform distribution and reached a monodisperse state ( Figure 2a). As shown in Figure 2b, SEM revealed that the micelles were individual particles with great dispersion and regular spherical shape, which was in line with those obtained from DLS. As

Formation and Characterization of PEG-PU(SS)-PEG Micelles
In this study, PEG-PU(SS)-PEG micelles were prepared via a dialysis method. The size of the PEG-PU(SS)-PEG micelles was 116.7 nm measured by DLS at 25 • C, which indicated that the PEG-PU(SS)-PEG micelles had a small size and uniform distribution and reached a monodisperse state (Figure 2a). As shown in Figure 2b, SEM revealed that the micelles were individual particles with great dispersion and regular spherical shape, which was in line with those obtained from DLS. As the previous work demonstrated [29], such small size and a homogeneous distribution are helpful to the cell internalization of PU micelles. The CMC of PEG-PU(SS)-PEG micelles was determined by a fluorescence method using pyrene as a probe. As shown in Table 2, PEG-PU(SS)-PEG micelles had a lower CMC value of 1.6 mg/L than that of PEG-PU-PEG, which might be related to the difference in molecular weight of the hydrophobic block, as demonstrated in some references [30,31]. Due to the low CMC, the micelles are more stable in water and capable of long circulation in vivo, which is advantageous for the drug carrier to accumulate at the tumor site through enhanced permeability and retention (EPR) effects. The stability of the micelle can also be demonstrated by the good redissolving status of the freeze-drying PEG-PU(SS)-PEG micelle. the previous work demonstrated [29], such small size and a homogeneous distribution are helpful to the cell internalization of PU micelles. The CMC of PEG-PU(SS)-PEG micelles was determined by a fluorescence method using pyrene as a probe. As shown in Table 2, PEG-PU(SS)-PEG micelles had a lower CMC value of 1.6 mg/L than that of PEG-PU-PEG, which might be related to the difference in molecular weight of the hydrophobic block, as demonstrated in some references [30,31]. Due to the low CMC, the micelles are more stable in water and capable of long circulation in vivo, which is advantageous for the drug carrier to accumulate at the tumor site through enhanced permeability and retention (EPR) effects. The stability of the micelle can also be demonstrated by the good redissolving status of the freeze-drying PEG-PU(SS)-PEG micelle.

Reduction-Responsive Size Change of PEG-PU(SS)-PEG Micelles
It is well-known that disulfide bonds are easily broken down into thiol under reducing conditions. In this work, in order to investigate the potential intracellular degradation of PEG-PU(SS)-PEG micelles, the change of particle size and size distribution in reducing environment were monitored by DLS. It is worth noting that in the presence of 10 mM DTT, the micelles underwent rapid and significant swelling and the average particle size increased. As shown in Figure 2c, the size distribution of PEG-PU(SS)-PEG micelles after incubation for 6 h with 10 mM DTT increased significantly, with the average particle size increasing from 101.3 to 254.8 nm, while the PDI also

Reduction-Responsive Size Change of PEG-PU(SS)-PEG Micelles
It is well-known that disulfide bonds are easily broken down into thiol under reducing conditions. In this work, in order to investigate the potential intracellular degradation of PEG-PU(SS)-PEG micelles, the change of particle size and size distribution in reducing environment were monitored by DLS. It is worth noting that in the presence of 10 mM DTT, the micelles underwent rapid and significant swelling and the average particle size increased. As shown in Figure 2c, the size distribution of PEG-PU(SS)-PEG micelles after incubation for 6 h with 10 mM DTT increased significantly, with the average particle size increasing from 101.3 to 254.8 nm, while the PDI also increased from 0.15 to 0.463. After 24 h, the micelles clearly broke, DLS showed that it divided into many peaks, and some aggregates appeared. In contrast, the size of the micelles did not change much in the non-reducing environment. It was likely that the disulfide bonds between the hydrophilic shell and the hydrophobic core of the PEG-PU(SS)-PEG micelles were reductively degraded. The hydrophilic shells fell off, and the micelles swelled, resulting in instability or aggregation of the PEG-PU(SS)-PEG micelles. Degradation experiments showed that PEG-PU(SS)-PEG micelles were responsive to the reducing environment and could break rapidly under reducing conditions that simulate the nucleus and cytoplasm.

Loading and In Vitro Release of DOX
To evaluate the loading capacity and the in vitro release capacity of PEG-PU(SS)-PEG micelles as a drug carrier, DOX was used as a model drug. DOX is one of the most potent chemotherapeutic agents, and its principle of action is to insert itself into DNA and thus inhibit the synthesis of nucleic acids [32,33].
DOX-loaded PEG-PU(SS)-PEG micelles were prepared via a dialysis method, and DOX-loaded PEG-PU-PEG micelles was used as a control. It was found that PEG-PU(SS)-PEG micelles could encapsulate DOX efficiently. Table 3 indicates that the encapsulation efficiency of PEG-PU(SS)-PEG micelles was slightly higher than that of PEG-PU-PEG micelles, which was approximately 65%. The size of DOX-loaded PEG-PU-PEG micelles had no appreciable change, which was approximately 116.2-144.6 nm. These data demonstrated that drug-loaded micelles were easily phagocytosed by the cells, thereby being beneficial for transporting DOX into the cells. The fluorescence of drug-loaded micelles was tested by a fluorescent spectrophotometer, and then the DLC and DLE were calculated according to the standard curve of DOX.
To further prove the stability of PEG-PU(SS)-PEG micelles and explore the probability for future clinical treatment, reconstitution experiments were performed on freeze-dried samples. First, a sample of 10% DOX-loaded PEG-PU-PEG micelles was freeze-dried for 24 h, then an equal volume of water was added to redissolve the sample, and the size of the micelles was measured again using DLS. The size of the redissolved micelles was 159.72 ± 0.54 nm, and the PDI was 0.142 ± 0.06 (Figure 2d). Compared with the micelles before freeze-drying, the size of the redissolved micelles changed slightly, further indicating that DOX-loaded PEG-PU-PEG micelles had a good physical stability, which was conducive to its use as a drug carrier. In vitro drug release experiments of drug-loaded micelles were carried out in a constant temperature shaker (200 rpm, 37 • C). As shown in Figure 3, owing to the reduction-degradable character of disulfide bonds in the backbone, approximately 75.25% of DOX was released within 2 h from drug-loaded PEG-PU(SS)-PEG micelles, and approximately 97.54% of DOX was released within 24 h from drug-loaded PEG-PU(SS)-PEG micelles. In contrast, only 25.2% of DOX was released within 24 h from reduction insensitive PEG-PU-PEG micelles under the same conditions. The results of the release experiment fully demonstrated that DOX-loaded PEG-PU(SS)-PEG micelles had reduction sensitivity and could release DOX rapidly under reducing conditions. The release results were consistent with the previous results that the micelles swell under reducing conditions of 10 mM DTT in the degradation reaction of PEG-PU(SS)-PEG micelles.

Cellular Uptake
Fluorescence microscopes were widely used in the study of intracellular drug delivery and release behavior. Taking DOX-loaded PEG-PU(SS)-PEG micelles as an example, the cellular uptake and intracellular release of DOX-loaded PEG-PU(SS)-PEG micelles in C6 cells were observed by fluorescence microscope. The experiment was divided into three groups: DOX-loaded PEG-PU(SS)-PEG micelles, DOX-loaded PEG-PU-PEG micelles, and free DOX·HCl, with three replicates in each group. As shown in Figure 4, C6 cells following 2 h incubation with DOX-loaded PEG-PU(SS)-PEG micelles showed strong red fluorescence of DOX in the cytoplasm. In contrast, DOX-loaded PEG-PU-PEG micelles showed only weak red fluorescence of DOX. Furthermore, the fluorescence intensity of DOX-loaded PEG-PU(SS)-PEG micelles incubated with C6 cells for 4 h significantly increased in the nucleus and cytoplasm. However, DOX-loaded PEG-PU-PEG micelles incubated with C6 cells for 4 h only showed some DOX fluorescence in the cytoplasm, and almost no red fluorescence appeared in the nucleus. In line with CCK-8 assays, free DOX was delivered more quickly into the nuclei of the C6 cells. In addition, the average fluorescence intensity of the reduction-sensitive PEG-PU(SS)-PEG micelles was obviously higher than that of the non-sensitive PEG-PU-PEG micelles at all intervals. The above experimental results showed that PEG-PU(SS)-PEG micelles could respond to the intracellular reducing environment and the disulfide bonds cleavage would lead to the disintegration of PEG-PU(SS)-PEG micelles and accelerate the release of DOX in tumor cells.

Cellular Uptake
Fluorescence microscopes were widely used in the study of intracellular drug delivery and release behavior. Taking DOX-loaded PEG-PU(SS)-PEG micelles as an example, the cellular uptake and intracellular release of DOX-loaded PEG-PU(SS)-PEG micelles in C6 cells were observed by fluorescence microscope. The experiment was divided into three groups: DOX-loaded PEG-PU(SS)-PEG micelles, DOX-loaded PEG-PU-PEG micelles, and free DOX·HCl, with three replicates in each group. As shown in Figure 4, C6 cells following 2 h incubation with DOX-loaded PEG-PU(SS)-PEG micelles showed strong red fluorescence of DOX in the cytoplasm. In contrast, DOX-loaded PEG-PU-PEG micelles showed only weak red fluorescence of DOX. Furthermore, the fluorescence intensity of DOX-loaded PEG-PU(SS)-PEG micelles incubated with C6 cells for 4 h significantly increased in the nucleus and cytoplasm. However, DOX-loaded PEG-PU-PEG micelles incubated with C6 cells for 4 h only showed some DOX fluorescence in the cytoplasm, and almost no red fluorescence appeared in the nucleus. In line with CCK-8 assays, free DOX was delivered more quickly into the nuclei of the C6 cells. In addition, the average fluorescence intensity of the reduction-sensitive PEG-PU(SS)-PEG micelles was obviously higher than that of the non-sensitive PEG-PU-PEG micelles at all intervals. The above experimental results showed that PEG-PU(SS)-PEG micelles could respond to the intracellular reducing environment and the disulfide bonds cleavage would lead to the disintegration of PEG-PU(SS)-PEG micelles and accelerate the release of DOX in tumor cells.  Flow cytometry assays are widely used to quantitatively determine the cell endocytosis and uptake of DOX-loaded micelles and FITC (fluoresceine isothiocyanate)-labeled micelles [34][35][36]. It has been reported that only the fluorescence of DOX released by the self-disassembling of DOXloaded nanocarriers could be observed and measured by flow cytometry [34]. Therefore, the intensity of intracellular fluorescence was directly related to the amount of DOX released from the cells. As Flow cytometry assays are widely used to quantitatively determine the cell endocytosis and uptake of DOX-loaded micelles and FITC (fluoresceine isothiocyanate)-labeled micelles [34][35][36]. It has been reported that only the fluorescence of DOX released by the self-disassembling of DOX-loaded nanocarriers could be observed and measured by flow cytometry [34]. Therefore, the intensity of intracellular fluorescence was directly related to the amount of DOX released from the cells. As shown in Figure 5, the fluorescence intensity of C6 cells following 4 h incubation with DOX-loaded PEG-PU(SS)-PEG micelles was far greater than that of C6 cells following 4 h incubation with DOX-loaded PEG-PU-PEG micelles. In other words, DOX-loaded PEG-PU(SS)-PEG micelles were responsive to the reducing environment in tumor cells and released doxorubicin more quickly and more completely. The results of the flow cytometry assay were also consistent with the results measured by fluorescence microscope previously.

Cell Viability Analysis of DOX-Loaded PEG-PU(SS)-PEG Micelles
The cytotoxicity and antitumor activity of reduction-responsive PEG-PU(SS)-PEG micelles were studied by CCK-8 assays. C6 cells were incubated with DOX-loaded PEG-PU(SS)-PEG micelles for 24 h at different DOX concentrations (0.625-40 μg/ml), and DOX-loaded PEG-PU-PEG micelles and free DOX·HCl were used as controls. DOX-loaded PEG-PU(SS)-PEG micelles showed significant dose-dependent antitumor activity compared to their counterparts, and led to more efficient delivery and release of DOX to the cytoplasm and nucleus. The IC50 was 7.02 μg /mL (DOX equivalents), lower than DOX-loaded PEG-PU-PEG micelles (25.68 μg/mL) ( Figure 6). The reason is that the cellular entry enhancement and intracellular drug release can rapidly increase the concentration of drugs in the tumor cells, effectively killing cancer cells [37]. Under the same conditions, the antitumor activity of free DOX·HCl to C6 cells was slightly higher than that of other two formulations. This is because free DOX·HCl is a small molecule that directly enters the nucleus by passive diffusion, whereas DOXloaded PEG-PU(SS)-PEG micelles and DOX-loaded PEG-PU-PEG micelles enter the cells to release DOX through endocytosis. In the biocompatibility experiment, the blank PEG-PU(SS)-PEG micelles did not show any cytotoxicity at a concentration up to 1 mg/mL, and the cells still had a viability of over 90% (Figure 7). These results demonstrated that reduction-responsive PEG-PU(SS)-PEG micelles had good biocompatibility and are likely to be used as pharmaceutical nanovehicles. In fact, the PU micelles based on the hydrophilic segment PEG and the biodegradable hydrophobic segments with excellent biocompatibility have been widely used in drug carriers, tissue engineering, and other biomedical fields [17][18][19][20]. The cytotoxicity and antitumor activity of reduction-responsive PEG-PU(SS)-PEG micelles were studied by CCK-8 assays. C6 cells were incubated with DOX-loaded PEG-PU(SS)-PEG micelles for 24 h at different DOX concentrations (0.625-40 µg/mL), and DOX-loaded PEG-PU-PEG micelles and free DOX·HCl were used as controls. DOX-loaded PEG-PU(SS)-PEG micelles showed significant dose-dependent antitumor activity compared to their counterparts, and led to more efficient delivery and release of DOX to the cytoplasm and nucleus. The IC 50 was 7.02 µg /mL (DOX equivalents), lower than DOX-loaded PEG-PU-PEG micelles (25.68 µg/mL) ( Figure 6). The reason is that the cellular entry enhancement and intracellular drug release can rapidly increase the concentration of drugs in the tumor cells, effectively killing cancer cells [37]. Under the same conditions, the antitumor activity of free DOX·HCl to C6 cells was slightly higher than that of other two formulations. This is because free DOX·HCl is a small molecule that directly enters the nucleus by passive diffusion, whereas DOX-loaded PEG-PU(SS)-PEG micelles and DOX-loaded PEG-PU-PEG micelles enter the cells to release DOX through endocytosis. In the biocompatibility experiment, the blank PEG-PU(SS)-PEG micelles did not show any cytotoxicity at a concentration up to 1 mg/mL, and the cells still had a viability of over 90% (Figure 7). These results demonstrated that reduction-responsive PEG-PU(SS)-PEG micelles had good biocompatibility and are likely to be used as pharmaceutical nanovehicles. In fact, the PU micelles based on the hydrophilic segment PEG and the biodegradable hydrophobic segments with excellent biocompatibility have been widely used in drug carriers, tissue engineering, and other biomedical fields [17][18][19][20].

Conclusions
In this work, biodegradable amphiphilic polymers PEG-PU(SS)-PEG with disulfide bonds in the main chain were successfully synthesized. In aqueous solution, the resulting PEG-PU(SS)-PEG polymers could self-assemble into micelles and load DOX to kill glioma cells with high expression.

Conclusions
In this work, biodegradable amphiphilic polymers PEG-PU(SS)-PEG with disulfide bonds in the main chain were successfully synthesized. In aqueous solution, the resulting PEG-PU(SS)-PEG polymers could self-assemble into micelles and load DOX to kill glioma cells with high expression.

Conclusions
In this work, biodegradable amphiphilic polymers PEG-PU(SS)-PEG with disulfide bonds in the main chain were successfully synthesized. In aqueous solution, the resulting PEG-PU(SS)-PEG polymers could self-assemble into micelles and load DOX to kill glioma cells with high expression. The new polymer micelles have the following advantages and highlights: (1) The micelles have good biocompatibility and hydrophobic segment biodegradability. (2) The micelles have small particle sizes and are favorable for drug delivery. (3) The micelles are stable under normal physiological conditions and can be circulated in human body fluid for a long time. (4) The micelles have a reduction responsiveness and can rapidly release the loaded drug DOX. On one hand, in vitro drug release experiments showed that the micelles exhibited excellent colloidal stability in the extracellular environment, which avoided the early release of drugs due to the dissociation of micelles. On the other hand, the micelles showed obvious reduction responsiveness after they had been endocytosed into cancer cells. Under the influence of the reducing substance GSH, the disulfide bond in the main chain broke, triggered the release of the loaded drug, and achieved the high release of the DOX. Cell experiments confirmed that treatment with DOX-loaded PEG-PU(SS)-PEG micelles significantly inhibited C6 cell growth compared to other groups. The above results indicate that PEG-PU(SS)-PEG micelles have good biocompatibility, stability, and reduction responsibility, and it is expected they will be used as a promising anticancer drug carrier for cancer treatment in the future.