Stable, Bioresponsive, and Macrophage-Evading Polyurethane Micelles Containing an Anionic Tripeptide Chain Extender.

Polymeric nanocarriers have been extensively used in medicinal applications for drug delivery. However, intravenous nanocarriers circulating in the blood will be rapidly cleared from the mononuclear macrophage system. The surface physicochemical characterizations of nanocarriers are the primary factors to determine their fate in vivo, such as evading the reticuloendothelial system, exhibiting long blood circulation times, and accumulating in the targeted site. In this work, we develop a series of polyurethane micelles containing segments of an anionic tripeptide, hydrophilic mPEG, and disulfide bonds. It is found that the long hydrophilic mPEG can shield the micellar surface and have a synergistic effect with the negatively charged tripeptide to minimize macrophage phagocytosis. Meanwhile, the disulfide bond can rapidly respond to the intracellular reduction environment, leading to the acceleration of drug release and improvement of the therapeutic effect. Our results verify that these anionic polyurethane micelles hold great potential in the development of the stealth immune system and controllable intracellular drug transporters.

Introduction

In recent years, drug delivery nanocarriers, including polymeric micelles, polymeric nanoparticles, liposomes, and inorganic or other solid particles, have been widely used for the diagnosis and therapy of malignant tumors.[1−4] The physicochemical properties of nanocarriers such as size, surface hydrophobicity, and surface charge may affect their interaction with blood components, leading to rapid recognition and elimination by the mononuclear macrophage system (MPS), and hence influence the endurance of blood circulation, biodistribution, and the accumulation at the target sites.[5−8] The hydrophilicity of the surface of nanocarriers is an important factor to affect their fate in vivo. The grafting of hydrophilic poly(ethylene glycol) (PEG) onto the surface of nanocarriers has been widely utilized to increase the hydrophilicity of nanocarriers[9] and create a thermodynamic shield,[10,11] which can counteract the hydrophobic and electrostatic interactions between nanocarriers and opsonins or MPS, leading to minimization of the rapid opsonization, suppression and elimination of the reticuloendothelial system (RES), and a prolonged blood circulation time.[12−16]

The surface charge of nanocarriers is the primary factor that determines their stability and longevity in the blood circulation, the mechanism and efficiency of cellular uptake, the bioavailability, and the therapeutic effect in vivo. The negatively charged surface of nanocarriers can help prolong the time of blood circulation and minimize the clearance by MPS.[17,18] For example, Yamamoto et al.[19] verified that the negatively charged PEG–PDLLA micelles can significantly minimize nonspecific uptake by the spleen and liver due to the electrostatic repulsion between micellar and cellular surfaces. Elci et al.[20] demonstrated that positively charged AuNPs were rapidly cleared from the blood circulation, whereas the negatively charged and the neutral nanoparticles circulated longer. Moreover, the negative surface charge of nanocarriers could significantly minimize the bovine serum albumin adsorption compared with positively charged samples.[21,22]

Desired nanocarriers for drug delivery systems should be able to overcome not only extracellular barriers, but also the intracellular barriers.[23] The design of stimuli-responsive nanocarriers that release drugs in response to intracellular signals has received great interest. In recent years, redox-responsive drug delivery systems involving disulfide bonds have been developed for cellular imaging[24−26] or cell-specific drug release.[27−30] The redox potentials in the cytoplasm are distinctly discrepant between tumor and normal tissues due to the different concentrations of glutathione (GSH) tripeptide, which is the most abundant redox agent in the cytoplasm.[31,32] It has been demonstrated that the intracellular GSH concentration reaches up to 10 mM,[33] while the concentration drops to about 2–20 μM in the extracellular fluids.[34] Furthermore, the GSH amount in tumor tissues is at least 4-fold higher than that in the normal tissue in mice.[35] The different reduction potentials in different cell regions have been exploited for triggered disulfide bond cleavage, thereby causing disintegration of micelles and drug unloading.

In our previous study, we have reported the construction of several multifunctional polyurethane micelles as drug delivery systems.[36−38] We have demonstrated that the cationic polyurethane micelles carried out an effective internalization.[39,40] However, the positive surface charge of polyurethane micelles could accelerate the elimination by macrophages and increase the accumulation in the liver, spleen, and kidney.[38,41−43] In this work, we aim to develop anionic polyurethane micelles containing the hydrophilic PEG chain and disulfide bond, which can minimize the recognition and elimination by RES owing to the electrostatic repulsion and hydrophilic shell protection and respond to the redox potentials in tumor cells, to improve the biodistribution and maintain the therapeutic effect of polymeric micelles.

To provide the negative charge of polyurethane, we designed and synthesized a tripeptide chain extender (TCE), the sequence of which is lysine–lysine–phenylalanine (Lys–Lys–Phe), containing an anionic carboxyl group. l-Cystine diethyl ester (CYSD) containing a disulfide is incorporated into the polyurethane to provide redox stimuli sensitivity. Meanwhile, to better understand the relationship between the anionic groups and mPEG chains of different molecular weights, and clarify their influence on the physical, chemical, and biological properties of micelles, polyurethanes with different contents of TCE, mPEG, and disulfide bond were synthesized and systematically characterized. These polyurethanes form micelles with a negatively charged surface by self-assembly (Scheme ). The self-assembly ability, stability in the physiological environment, redox-stimulated response, drug loading, and drug-release profile of these anionic micelles were investigated. Furthermore, confocal laser scanning microscopy (CLSM) and flow cytometry were performed to evaluate the cellular uptake and phagocytosis of the micelles.

Scheme 1

Design and Construction of Four Types of Polyurethane Micelles as a Drug Delivery System for Anticancer Therapy

Results and Discussion

Synthesis of the Tripeptide Chain Extender

To introduce the negatively charged groups into the polyurethane micelles, the tripeptide chain extender (TCE) was designed and synthesized. The structure and synthetic route of the tripeptide are shown in Scheme . The sequence of the tripeptide is Lys–Lys–Phe. Diamino groups of the first lysine (Lys) in the sequence can ensure that the tripeptide incorporated into the polyurethane skeleton as a chain extender, and the carboxyl group from phenylalanine (Phe) provides a negative charge.

Scheme 2

Synthesis Route of the Tripeptide Chain Extender

Mass spectrometry was carried out to prove the successful synthesis of TCE as listed in the Materials and Methods section. The structure of the TCE was further confirmed by 1H NMR and FTIR spectra. As shown in Figure A, peaks at 7.16 ppm are assigned to the benzene ring from Phe (−C6H5). The peaks at 4.13, 4.03, and 3.15 ppm are attributed to the hydrogen protons linked by chiral carbon atoms. The signals near 7.9 and 7.5 ppm are assigned to the amide group (−CONH−). Signal peaks of other hydrogen protons can be found as shown in Figure A.

Figure 1

(A) 1H NMR spectrum and (B) FTIR spectrum of the tripeptide chain extender. The solvent of 1H NMR is DMSO-d6.

In the FTIR spectrum (Figure B), the absorption bands around 3448 and 3298 cm–1 are attributed to vibrational modes associated with the primary amine and secondary amine. The peak at 1631 cm–1 belongs to the C=O stretching, and the characteristic peaks of the carboxyl group appear at 1560 and 1381 cm–1. The band near 700 cm–1 is assigned to the out-of-plane bending mode of =C–H in benzene. These results indicate that the tripeptide chain extender has been successfully synthesized.

Synthesis of Anionic Polyurethanes

To explore the effects of different contents of anionic carboxyl groups and various molecular weights of the hydrophilic shell in micelles on the phagocytosis by macrophages, a series of polyurethanes were synthesized by changing the feed ratio of TCE and the molecular weight of mPEG (Table ). The polyurethanes are named as TxC50mEy, in which T, C, and mE represent TCE, CYSD, and mPEG, respectively; x represents the molar ratio of TCE and y represents the molecular weight of mPEG. Polyurethanes without TCE or mPEG were also prepared as control samples (T0C50mE1900 and T50C50mE0). As shown in Table , the molecular weights (Mn) of the polyurethanes range from 23 000 to 60 000 g/mol with relatively narrow distributions (PDI 1.28–1.67).

Table 1

Theoretical Composition and Molecular Weights of Anionic Polyurethanes

a	feed ratio						molecular weightsb
				chain extender
samplesa	LDI	PCL	mPEG	PDO	tripeptide	CYSD	Mn	Mw	Mw/Mn
T50C50mE0	4	2	0	0	1	1	60 536	88 504	1.46
T50C50mE500	4	1.6	0.8	0	1	1	23 652	39 560	1.67
T50C50mE1900	4	1.6	0.8	0	1	1	49 054	67 321	1.37
T50C50mE5000	4	1.6	0.8	0	1	1	39 762	54 182	1.36
T25C50mE1900	4	1.6	0.8	0.5	0.5	1	48 620	68 273	1.4
T0C50mE1900	4	1.6	0.8	1	0	1	31 736	40 735	1.28

Anionic polyurethanes are denoted as TxC50mEy, where T is for tripeptide chain extender, C is for CYSD, mE is for mPEG, x is for the molar fraction of the tripeptide chain extender, and y is the molecular weight of mPEG, respectively.

Molecular weights and molecular weight distributions were determined by GPC.

To clarify the structure, Figure shows the 1H NMR spectra of anionic polyurethanes. The characteristic proton shifts near 7.2 ppm result from the benzene ring protons of the TCE (−C6H5). There is no peak near 7.2 ppm in the spectrum of T0C50mE1900 without TCE. The shift at 3.1 ppm is attributed to the methylene protons attached to the disulfide bond (−CH2–S–S−). The peaks of different methylene protons of the PCL segments are observed at 1.32 ppm (−CH2CH2CH2−), 1.55 ppm (−CH2CH2CH2−), 2.26 ppm (−CH2COO−), and 3.96 ppm (−CH2O−), respectively. The chemical shifts of methylene (−CH2–OCO−) and methyl protons (−CH3) in the ethoxyl group of LDI are at 4.44 and 1.18 ppm, respectively. The methylene protons of the mPEG are found at 3.51 ppm. No mPEG peaks are seen for the control sample T50C50mE0. The 1H NMR spectra indicate that the polyurethanes with the different carboxyl groups and various molecular weights of mPEG were successfully synthesized.

Figure 2

1H NMR spectra of anionic polyurethanes: (a) T50C50mE0, (b) T50C50mE500, (c) T50C50mE1900, (d) T50C50mE5000, (e) T25C50mE1900, and (f) T0C50mE1900 in DMSO-d6. The chemical structures of the main components for anionic polyurethanes are also presented in this figure.

The FTIR spectra were used to characterize the specific chemical groups in anionic polyurethanes. As shown in Figure , a broad stretching band around 3395 cm–1 is mainly attributed the N–H stretching vibration associated with the amino groups in urethane. There are no peaks at about 2200 cm–1 (stretching vibration of −N=C=O), indicating that LDI has been completely reacted into urea or urethane groups. In the carbonyl region of 1650–1750 cm–1, the strong peaks at approximately 1732 cm–1 are assigned to the carbonyl (C=O) in LDI and PCL. The peak at 1650 cm–1 is assigned to the hydrogen-bonded carbonyl of TCE and CYSD, while the control sample T0C50mE1900 without TCE has a correspondingly lower single at 1650 cm–1. Meanwhile, the absorption bands in the infrared fingerprint region near 705 cm–1 caused by the benzene ring have obviously enhanced with the incorporation of TCE. These results further demonstrated that the polyurethanes had been successfully synthesized.

Figure 3

FTIR spectra of anionic polyurethanes.

Preparation and Characterization of Anionic Polyurethane Micelles

To investigate the effects of the anion carboxyl groups on the self-assembly behavior of the micelles, the size, size distribution, ζ-potential, and morphology of the micelles in the physiological environment were measured by DLS and TEM. The results obtained from DLS are listed in Table . The size ranges from 84 to 137 nm with PDI 0.13–0.37. As the molecular weight of mPEG increases, the micellar size shows an obviously downward trend owing to the hydrophilicity improvement, which is validated in our previous reports.[44] All of the micelles show a surface negative potential from −5 to −33 mV. The absolute values of the ζ-potential decrease as the mPEG segment chain grows as well as the TCE content decreases. In particular, the longest chain of mPEG (Mn = 5000) completely shields the carboxyl anion on the surface of micelles, the ζ-potential of which is −5 mV, similar to the T0C50mE1900 micelles without carboxyl groups. The shortest mPEG (Mn ≤ 500) has little ability to cover anionic groups. Simultaneously, with the content of the carboxyl anion decreased, the ζ-potentials of micelles grafted with mPEG1900 increased from −18.77 to −6.20 mV. As observed by TEM (Figure ), the morphologies of representative anionic polyurethane micelles (T0C50mE1900 and T50C50mE1900) display an irregular sphere and individual dispersion, and the diameters are calculated to be about 100 nm according to the scale, which is quite consistent with DLS results (Table ). In addition, the incorporation of an anionic tripeptide has slight influence on the morphology of micelles.

Table 2

Size, Size Distribution, and ζ-Potential of Anionic Polyurethane Micelles

sample	size (d.nm)	PDI	ζ-potential (mV)
T50C50mE0	109.7 ± 13.9	0.37	–33.53 ± 2.63
T50C50mE500	105.6 ± 1.1	0.13	–32.97 ± 0.81
T50C50mE1900	84.6 ± 0.6	0.23	–18.77 ± 0.50
T50C50mE5000	96.5 ± 1.3	0.29	–5.82 ± 0.02
T0C50mE1900	137.4 ± 7.5	0.16	–6.20 ± 0.30
T25C50mE1900	111.7 ± 1.2	0.14	–16.73 ± 0.91

Figure 4

TEM images of anionic polyurethane micelles.

To further understand the distribution of the tripeptide in the micelle structure, dissipative particle dynamics (DPD) simulation was carried out by a computer. Blue, red, green, and yellow colors in the simulated structures of anionic polyurethane micelles represent PCL, tripeptide, mPEG, and CYSD, respectively (Figure ). It is found that mPEG-free T50C50mE0 can still self-assemble into micelles but the particle size is not uniform. As shown in Figure , the size and size distribution become smaller and narrower with the increasing chain length of mPEG. In addition, TCE is found to be mainly distributed between the PCL core and the mPEG shell, and a small amount of TCE is buried into the PCL core, which might be due to the structure of the tripeptide. In detail, the phenylalanine can provide strong hydrophobicity, which improves the interaction of TCE and the PCL core, while the two hydrophilic lysines prompt TCE migration to the micellar surface. The distribution of the tripeptide can explain why longer mPEG chains can completely shield the negative surface charge of micelles.

Figure 5

Computer-simulated structures of anionic polyurethane micelles. Blue, red, green, and yellow represent PCL, tripeptide, mPEG, and CYSD, respectively.

To evaluate the effects of anionic carboxyl groups on the stability of micelles under physiological conditions, the size change of micelles was monitored in PBS solution at 37 °C for 24 h (Figure ). Apparently, it can be found that the size of micelles without mPEG (T50C50mE0) changed significantly from 60 to 80 nm. Other micelles display extreme stability in PBS solution within 24 h. These results indicate that the mPEG segments could provide micelles good steric stabilization, and the anionic TCE with the carboxyl group cannot affect the stabilization of micelles in the buffer solution.

Figure 6

Change of size of anionic polyurethane micelles for 24 h of incubation at 37 °C in PBS solution (pH 7.4). Significance levels were set at *P < 0.05. All data presented as mean ± SD (n = 3).

The redox-stimulated response of the disulfide bond under a reductive environment was evaluated by monitoring the size change of micelles in PBS with 10 mM GSH. As shown in Figure , the size of the micelles containing disulfide bonds exhibits a dramatic increase in 1 h, indicating that the disulfide bond can respond to the reductive stimulus and rapidly cleave under the 10 mM GSH environment.

Figure 7

Changes of size distributions for polyurethane micelles incubated in a reductive environment (10 mM GSH) for 24 h.

Drug Loading and Release

The chemotherapeutic drug PTX was used to evaluate the drug-loading capacity of anionic polyurethane micelles. Two drug-loading parameters of anionic polyurethane micelles, LC and EE, are plotted in Figure A. All of the micelles have a good drug-loading capacity with the same PTX feeding amount (10%); the maximum LC and EE are ∼6 and ∼50%, respectively. The incorporation of anionic TCE cannot affect the drug-loading ability of anionic polyurethane micelles. In addition, the size of micelles has basically no change after the encapsulation of PTX except the size of T0C50mE1900 (Figure B).

Figure 8

(A) Drug-loading content (%) and encapsulation efficiency (%) of PTX in anionic polyurethane micelles. (B) Size of anionic polyurethane micelles containing PTX. Significance levels were set at *P < 0.05, **P < 0.01. All data are presented as mean ± SD (n = 3).

To further study the release profiles of PTX-loaded anionic polyurethane micelles, we examined the PTX release under an imitated physiological environment and reducing environment in vitro. As depicted in Figure A, there is a strong correlation between the release rates of PTX and the length of mPEG segments. The PTX accumulative release rates of T0C5mE1900 and T50C50mE1900 were about 60% within 120 h, while a serious initial burst release in the profile of the T50C50mE0 occurred due to the lack of mPEG segments, and the accumulative PTX release was near 100% after 120 h. This phenomenon may confirm that the longer hydrophilic mPEG shell would inhibit PTX release.[44] In addition, T50C50mE0 without mPEG is relatively unstable in PBS solution, which would be a reason for the rapid drug release. As shown in Figure B, the release profiles of T50C50mE1900 and T0C50mE1900 are almost the same under the physiological environment, which indicates that anionic TCE has a slight impact on the release profiles of polyurethane micelles. However, the release rates of T0C50mE1900 and T50C50mE1900 micelles were sharply increased in PBS with 10 mM GSH, and the cumulative release amounts increased from ∼60 to ∼95%. It could be anticipated that a higher concentration of GSH in tumor cells could trigger intracellular PTX release and subsequently increase the cytotoxicity of micelles.

Figure 9

(A) Release profiles of PTX-loaded anionic polyurethane micelles with different molecular weights of mPEG. (B) Release profiles of polyurethane micelles in PBS (pH 7.4) with or without 10 mM GSH solution. Significance levels were set at *P < 0.05. All data are presented as mean ± SD (n = 3).

In Vitro Cellular Uptake

The analyses on flow cytometry and CLSM were carried out to evaluate the internalization efficiency of anionic polyurethane micelles with a varying content of TCE and mPEG with different molecular weights in tumor cells (HeLa). T50C50mE0 displayed the strongest fluorescence signal due to the lack of a hydrophilic mPEG chain as a sheltered enclosure (Figure ). Moreover, as shown in Figure , the fluorescence signal decreased along with the increase of the length of the mPEG chain or/and content of TCE. Evidently, the fluorescence intensity of FITC-labeled anionic polyurethane micelles is observed at the cell membrane. Simultaneously, the fluorescence signal is difficult to be observed in the cytoplasm within 3 h of incubation (Figure B). Most of the anionic polyurethane micelles attach to the cell membrane, which due to the longer hydrophilic mPEG chain would provide the superior stabilization by steric repulsion. In addition, TCE would supply the negative surface charge, which can inhibit the internalization of anionic polyurethane micelles. Therefore, the polyurethane micelles with mPEG segments and the anionic TCE can hardly enter HeLa cells within 3 h. Future studies on incorporating the monoclonal antibody through carboxylic acid reactions will be carried out to improve the internalization by tumor cells.

Figure 10

(A) Flow cytometry and (B) CLSM images on incubation with polyurethane micelles for 3 h.

In Vitro Cytocompatibility and Antitumor Activity

To assess the biocompatibility of anionic polyurethane micelles, L929 fibroblast cells were employed to evaluate the cytotoxicity by a CCK-8 cell viability assay. As shown in Figure A,B, the cell viability of all micelles is higher than 85% even at a high concentration (0.1 mg/mL) within 72 h of incubation. It indicates that these anionic polyurethane micelles have good biocompatibility. Moreover, these blank micelles also do not inhibit the growth of tumor cells (HeLa cells) (Figure C,D).

Figure 11

Viability of L929 mouse fibroblasts (A, B) and HeLa cells (C, D) treated by drug-free anionic polyurethane micelles for 24 h (A, C) and 72 h (B, D).

The antineoplastic potential of the PTX-loaded anionic micelles was investigated against HeLa cells in vitro by a CCK-8 cell viability assay. The commercialized anticancer drug Taxol was set as a positive control. The cytotoxicity toward HeLa cells is dependent on the length of mPEG segments and the content of the anionic groups in the anionic polyurethane micelles. As shown in Figure , the half maximal inhibitory concentrations (IC50’s) of T50C50mE0, T50C50mE500, T50C50mE1900, T50C50mE5000, and Taxol were calculated to be 0.447, 1.377, 4.074, 8.054, and 0.088 μg/mL, respectively. Particularly, the IC50 of micelles with a long mPEG chain (Mn ≥ 1900) is 10–20 times higher than that of the micelles without an mPEG chain, which is because a longer hydrophilic mPEG chain can effectively hamper cell uptake of micelles as well as decrease the PTX release from polyurethane micelles. On the other hand, micelles incorporated with the anionic carboxyl groups have slightly increased the IC50 value from 3.664 (T0C50mE1900) to 4.074 (T50C50mE1900) μg/mL. The negatively charged carboxyl groups could prevent the micelles from internalizing into the tumor cells due to the electric repulsion between the micellar surface and the cytomembrane. The lowest cytotoxicity of PTX-loaded T50C50mE5000 can be associated with the synergistic effects of a longer mPEG chain and the anionic carboxyl groups of the micelles.

Figure 12

Cytotoxicity of anionic polyurethane micelles against HeLa cells after 24 h (A, B) and 72 h (C,D) of incubation. Insets show the IC50 values of various PTX formulations toward HeLa cells for 72 h of incubation.

Phagocytosis of Macrophages on Anionic Polyurethane Micelles

Phagocytosis is one of the main mechanisms of the innate immune defense to remove and clear extraneous materials in the blood or tissue, which would greatly reduce the bioavailability of drug delivery systems. To evaluate the effect of anionic TCE and the hydrophilic mPEG segments on the clearance of macrophage phagocytosis, the murine raw264.7 cells were employed to study the phagocytosis (Figure ). It was found that the fluorescent signals of T50C50mE1900 and T50C50mE5000 were much weaker than those of T50C50mE500 and T50C50mE0 (Figure A,C). Moreover, the fluorescent intensity gradually darkened with the increase of anionic TCE (Figure B,D). Importantly, the T50C50mE5000 micelles have not been swallowed by macrophages within 3 h. These results indicate that the hydrophilic mPEG chain and anionic TCE have a synergistic effect, owing to the dual role of the electrostatic repulsion and hydrophilic protection, against the elimination of micelles by macrophages. Therefore, the negative surface charge as well as the mPEG segments have a great ability to help the drug delivery system to escape the interception by macrophages in the immune system. Such micelles contain disulfide bonds, which would quickly respond to the GSH stimulation in tumor cells, and thus can help accelerate the drug release at tumor cells to improve the biodistribution and enhance the therapeutic effect.

Figure 13

CLSM images of Raw264.7 cells incubated with FITC-labeled anionic polyurethane micelles for 1 h (A, B) and 3 h (C, D).

Conclusions

A series of anionic polyurethanes containing anionic tripeptide chain extenders and disulfide bonds have been designed and prepared, and these polyurethanes can be self-assembled in an aqueous solution. It was found that the longer mPEG chains can completely screen the negative surface charge of micelles due to the TCE distributed between the hydrophobic PCL core and the hydrophilic mPEG shell. The anionic polyurethane micelles have good stability under the imitated physiological environment. In addition, it is verified that the disulfide bond provided by CYSD quickly responded to the GSH stimulation, leading to the disintegration of micelles and acceleration of drug release at tumor cells. Moreover, longer hydrophilic mPEG chains and a negatively charged micellar surface have a synergistic effect on weakening the macrophage phagocytosis as well as on the internalization by tumor cells, due to the electrostatic repulsion and hydrophilic protection. Therefore, the anionic TCE and hydrophilic PEG shell have great potential to help micelles evade the MPS uptake, minimize the clearance of RES, prolong the blood circulation time, and increase the accumulation at the tumor site. Future studies on incorporating the monoclonal antibody through carboxylic acid reactions will be carried out to simultaneously improve the immune evasion and therapy efficacy of the anionic polyurethane micelles in vivo.

Materials and Methods

Materials

mPEG (Mn = 500, 1900, and 5000) was purchased from Alfa Aesar (U.K.), and PCL diols (Mn = 2000) was purchased from Dow Chemical (USA). Both of them were dehydrated under vacuum before use. Paclitaxel and Taxol were obtained from Jinhe Biotechnology Co., Ltd. (Shanghai, China) and West China Hospital, Sichuan University, respectively. Fluorescein isothiocyanate isomer I (FITC, 90%) was purchased from Acros Organics (USA). 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) was obtained from Roche Diagnostics (Germany). l-Cystine diethyl ester was purchased from Shanghai Bajiu Industry Co., Ltd. (China), and H-Lys(Boc)-OH and Phe-OMe were purchased from Sichuan Anpaibo Biotechnology Co., Ltd. (China). Dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (HOSU) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (China). CCK-8 was purchased from Tongren Institute of Chemistry (Japan).

Synthesis of the Tripeptide Chain Extender

H-Lys(Boc)-OH was dissolved in a mixture of methanol and saturated NaHCO3 aqueous solution, and the solution was cooled to ∼0 °C in an ice-water bath. Acetic anhydride was added dropwise into the solution. The mixture solution was stirred for 1 h. Then, the methanol was removed by vacuum rotary evaporation at 45 °C, after which the pH of the mixture solution was adjusted to 3 using 1 M HCl. The mixture solution was extracted by ethyl acetate three times, and the organic layer was washed sequentially with saturated NaHCO3 solution, saturated NaCl solution, and deionized water, dried with anhydrous Na2SO4 for 24 h, and filtered to obtain Boc-Lys-acy (yield: 82%). Boc-Lys-acy and Phe-OMe were added into the CH2Cl2. The pH value of the mixture solution was adjusted to 7–8 using N-methylmorpholine, and the temperature was decreased to ∼0 °C using the ice-water bath. DCC and HOSU were added and the mixture was stirred at room temperature for 48 h. After complete reaction, dicyclohexylurea (DCU) was removed by filtration and the solution was washed sequentially with 0.6 M citric acid, saturated NaHCO3 solution, saturated NaCl solution, and deionized water, dried with anhydrous Na2SO4 for 24 h, and then filtered. The solution was concentrated with a rotary evaporator. Then, the solution of diethyl ether/petroleum ether (v/v, 1:1) was added to recrystallize and obtain the product of Boc-lys(acy)-Phe-OMe (yield: 81%). Boc-lys(acy)-Phe-OMe was dissolved in ethyl acetate, and AcOEt/HCl was added dropwise into the mixture solution to deprotect the BOC group. Then, the residual solvent in the product was removed using vacuum rotary evaporation. A small amount of menthol was added to dissolve the product, after which a large amount of diethyl ether was added to precipitate and obtain the purified product lys(acy)-Phe-OMe (yield: 88%). DCC and HOSU were added to the CH2Cl2 solution of lys(acy)-Phe-OMe and Boc-lysine, which was cooled in the ice-water bath to 0 °C. Next, the mixture solution was stirred for 48h at room temperature. Subsequently, DCU was removed by filtration and the collected solution was concentrated with a rotary evaporator. To the mixture, 0.5 M HCl was added, and it was extracted with ethyl acetate three times. The organic layer was washed sequentially with saturated aqueous solution of NaHCO3, saturated NaCl solution, and deionized water, dried with anhydrous Na2SO4 for 24 h, and filtered to obtain the product Boc-lys-lys(acy)-Phe-OMe (yield: 83%). Boc-lys-lys(acy)-Phe-OMe was converted to the final tripeptide with HCl and saturated ethyl acetate, and then dilute NaOH solution was used to adjust the pH to 8. The solvents were removed by vacuum rotary evaporation at 50 °C, and the residue was dissolved in anhydrous methanol. Finally, the methanol solution was dried with a rotary evaporator to gain the crude product. It was further purified with silica gel column chromatography (yield: 77%).

Mass spectrum (positive, m/z): theoretical: 485.56; observed: 486.09.

Synthesis and Characterization of Polyurethanes

A series of anionic polyurethanes were synthesized from l-lysine diisocyanate (LDI), mPEG, cystine, CYSD, and PCL according to our previous report.[45] Briefly, pre-polymerization of LDI and PCL was carried out in the presence of 0.1% stannous octoate catalyst and under the protection of nitrogen at 60 °C for 1 h. The resulting solution was cooled down to room temperature, after which the chain extender CYSD and tripeptide were added to react at room temperature for 1 h (for T0C50mE1900, the addition of 1,3-propanediol (PDO) is needed to react at 80 °C for an extra 2 h). Subsequently, mPEG as the terminated chain was added and reacted at 90 °C for 6 h. The final products were precipitated by diethyl ether and washed at least three times, after which the products were dried under vacuum at 50 °C for 2 days.

The polyurethanes were characterized by proton nuclear magnetic resonance (1H NMR), Fourier transform infrared spectroscopy (FTIR), and gel permeation chromatography (GPC). 1H NMR spectra were obtained on a Bruker AV II-400 MHz spectrometer, using dimethyl sulfoxide (DMSO-d6) as a solvent. FTIR spectroscopy was performed by a Nicolet 6700 FTIR spectrometer using the KBr tablet method. GPC was examined on Waters-1515; the mobile phase was N,N-dimethylformamide (DMF)/50 mM LiBr with a flow rate of 1 mL/min at 40 °C.

Preparation and Characterization of Polyurethane Micelles

The anionic polyurethane (50 mg) was first dissolved in 10 mL of N,N-dimethylacetamide (DMAc), and then the resulting polymer solution was added dropwise into 30 mL of deionized water. Subsequently, the obtained solution was dialyzed by a dialysis bag (MWCO = 3500 g/mol) in a large amount of deionized water for at least six cycles. Finally, the solution was passed through a syringe filter with a pore size of 0.45 μm to obtain the micelles.

Dynamic light scattering (DLS, Malvern zetasizer Nano ZS) was used to measure the size, size distribution, and ζ-potential of anionic micelles at 25 °C with an angle of 90°. A transmission electron microscope (TEM, Hitachi model H-600-4) was used to observe the morphology of polyurethane micelles. The micelles were negatively stained by 1% phosphotungstic acid and dropped onto the copper grid, and then observed at 75 kV.

Stability and Redox Stimuli-Responsiveness of Polyurethane Micelles

To study the stability of the anionic micelles, the micelles were incubated at 37 °C in a shaker under PBS (pH 7.4) with or without 10 mM GSH for 24 h. The change of size was determined at the scheduled time by DLS.

Drug Loading and Release in Vitro

Paclitaxel (PTX) was first dissolved in acetone, and the acetone was evaporated by nitrogen in a glass vial to form a transparent film. The micelles were added into the vial under ultrasonication for 2 h at room temperature. The unloaded PTX drug was filtered by a syringe filter with a pore size of 0.45 μm. The concentration of PTX was calculated by a high-performance liquid chromatography (HPLC) system equipped with a reverse-phase C18 column (Agilent 1260 series). The eluent was a mixture of acetonitrile and water (60:40 v/v) with 1 mL/min flow rate. The UV detector was set at 227 nm and the temperature was set at 30 °C.The PTX release profile was investigated using the dialysis method as previously described.[36] Briefly, the PTX-loaded micelle solution (15 mL) was added to the dialysis bag. The bag was immersed into 100 mL of PBS (0.01 M) solution containing 1 M sodium salicylate. The experiment was performed in an incubator shaker, which was maintained at 37 °C and 100 rpm. At desired time intervals, 1 mL solution of the media was taken out, and an equal amount of fresh buffer solution was replenished. The concentration of PTX in the release media was measured by HPLC.

Cell Culture

The murine fibrosarcoma cell line (L929), human cervical carcinoma cell line (HeLa), and murine macrophage cell line (Raw264.7), which were purchased from West China Medical Center of Sichuan University (Sichuan, China), were cultivated in RPMI 1640 media containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The cells were cultivated in a humidified atmosphere of 5% CO2 at 37 °C.

Cellular Uptake in Vitro

Confocal laser scanning microscopy (CLSM) and flow cytometry microscopy were used to observe the internalization behavior and intracellular distribution of the anionic micelles. In brief, Raw264.7 and HeLa cells were plated in a six-well plate with a density of 1 × 105 cells/well and cultivated in 1640 media containing 10% FBS and 1% penicillin. FITC-labeled anionic micelles were prepared by physical entrapment of the fluorescent probe FITC. Next, the FITC-labeled micelles with cultured cells were transferred into wells and incubated for 1 and 3 h (HeLa cells only incubated for 3 h). Then, each well was washed three times using 0.1 M PBS. For the CLSM test, the cells were fixed with 4% paraformaldehyde for 30 min and stained with DAPI for 10 min. For flow cytometry, the cells were collected by trypsin treatment and centrifugation. After removing the supernatant, 0.1 M PBS (0.5 mL) was used to resuspend the cells. Finally, the cells were analyzed by Cytomics FC 500 (Beckman Coulter).

Cytotoxicity Assay

The cytotoxicity of anionic micelles was evaluated using L929 and HeLa cells with proliferation in vitro. The cells were cultured in a 96-well plate with a density of 5000 cells/well. PTX-loaded or drug-free anionic micelles with different concentrations were transferred into the wells with cultured cells and incubated for 24 and 72 h. The cell viability was evaluated using a CCK-8 assay kit.

Statistics

The Statistical Package for the Social Sciences (version 17.0) software was used for statistical analyses. The reported data are expressed as means ± standard deviations (SD). Student’s t-test or one-way analysis of variance at a 95% confidence level (P < 0.05) was conducted to assess the statistical significance within the data.