Bioconjugate Chem. 2009, 20, 1095–1099
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COMMUNICATIONS Shell-Detachable Micelles Based on Disulfide-Linked Block Copolymer As Potential Carrier for Intracellular Drug Delivery Ling-Yan Tang,† Yu-Cai Wang,*,‡ Yang Li,† Jin-Zhi Du,‡ and Jun Wang*,† Hefei National Laboratory for Physical Sciences at Microscale and School of Life Science, Hefei, Anhui 230027, People’s Republic of China, and Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 230026, People’s Republic of China. Received April 2, 2009; Revised Manuscript Received April 28, 2009
Aiming at development of a micellar nanoparticle system for intracellular drug release triggered by glutathione in tumor cells, a disulfide-linked biodegradable diblock copolymer of poly(ε-caprolactone) and poly(ethyl ethylene phosphate) was synthesized. It formed biocompatible micelles loaded with doxorubicin in aqueous solution but detached the shell material under glutathione stimulus, resulting in rapid drug release with destruction of micellar structure. These glutathione-sensitive micelles also rapidly released the drug molecules intracellularly and led to enhanced growth inhibition to A549 tumor cells, suggesting that this nanoparticle system may have potential for improving drug delivery efficacy.
Despite the discovery of many anticancer drugs for cancer therapy, clinical outcomes have been disappointing due to the severe side effects. Various nanovehicles such as polymeric micelles and vesicles (1-3), liposomes (4), and nanogels (5, 6) have been developed to overcome the problems. Such nanoparticle-based vehicles can passively accumulated in solid tumor tissues owning to the enhanced permeation and retention effect and improve the therapeutic efficiency (7). However, the concentration of active anticancer drug within cancer cells is often insufficient due to the inefficient release of drug from the vehicle into the cytoplasm, resulting in a requirement for higher drug dosage. The promising approach to improving the efficacy is to develop carrier systems that can release the drug triggered by intracellular stimuli, such as pH (8-11), glutathione (12-15), and enzyme (16). Upon reaching the targeted tumor, such carriers can be rapidly localized intracellularly and subsequently provoked by the stimuli to release the drug, hence inducing aggressive activity within tumor cells and leading to maximal therapeutic efficacy with reduced side effects. Chemical reaction involving the reductive degradation of disulfide bonds of polymers by intracellular glutathione (GSH) has been widely investigated for responsive drug and gene delivery (17-19). GSH is a thiol-containing tripeptide and reduces disulfide bonds in the cytoplasm. It has been demonstrated that the intracellular concentration of GSH (ca. 10 mM) is significantly higher than the level in the cell exterior (ca. 2 * To whom correspondence should be addressed. Jun Wang, School of Life Science, University of Science and Technology of China; 443 Huangshan Road, Hefei, Anhui 230027, P.R. China. Fax: 86-5513600402. E-mail:
[email protected]; Or Yu-Cai Wang, Department of Polymer Science and Engineering, University of Science and Technology of China; 96 Jinzhai Road, Hefei, Anhui 230026, P.R. China. E-mail:
[email protected]. † Hefei National Laboratory for Physical Sciences at Microscale and School of Life Science. ‡ Department of Polymer Science and Engineering.
µM) (17). We have previously demonstrated that micellar nanoparticles assembled from diblock copolymer of poly(εcaprolactone) (PCL) and hydrophilic poly(ethyl ethylene phosphate) (PEEP) is potential as a drug carrier (20, 21). In the present work, aiming at development of micellar nanoparticle system for intracellular drug release triggered by GSH in tumor cells, we synthesized a disulfide-linked diblock copolymer of PCL and PEEP (PCL-SS-PEEP), which formed biocompatible micelles in aqueous solution, and detached the shell material under GSH stimulus, resulting in rapid drug release with destruction of micellar structure (Scheme 1). PCL-SS-PEEP was synthesized by a coupling reaction between the thiol end group functionalized poly(ε-caprolactone) (PCL-SH) with the pyridyldisulfide groups at the end of poly(ethyl ethylene phosphate). PCL-SH was obtained through polymerization of ε-caprolactone initiated by 2-(2,4-dinitrophenylthio)ethanol in the presence of stannous octoate (Sn(Oct)2) to give a precursor polymer PCL-DNP, followed by deprotection of dinitrophylenyl groups with mercaptoethanol. As shown in Figure 1, the 1H NMR spectrum of PCL-SH indicates that proton signals at δ 7.76, 8.37, and 9.05 from 2-(2,4-dinitrophenylthio) groups of PCL-DNP are no longer present, demonstrating the successful deprotection. The degree of polymerization (DP) of PCL-SH is 32, which is calculated from the integration ratio of resonance at δ 4.02 of PCL-DNP against those at δ 7.76, 8.37, and 9.05. In another aspect, the pyridyldisulfide end group functionalized poly(ethyl ethylene phosphate) (PEEP-Py) was synthesized through ring-opening polymerization of ethyl ethylene phosphate, initiated by 2-(2-pyridyldithio)-ethanol and Sn(Oct)2. The 1H NMR spectrum of PEEP-Py given in Figure 1 shows resonances at δ 7.13, 7.56, 7.70 and 8.50, assigned to protons of pyridyl end groups. The DP of PEEP is 22, calculated based on the integration ratio of resonances at δ 4.1-4.3 against those at δ 7.13, 7.56, 7.70 and 8.50. The molecular weights determined by gel permeation chromatography (GPC) analyses are summarized in Table 1.
10.1021/bc900144m CCC: $40.75 2009 American Chemical Society Published on Web 05/13/2009
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Scheme 1. Synthesis Pathway of the Disulfide-Linked PCL-SS-PEEP and Schematic Illustration of Intracellular Drug Release
The coupling reaction of PCL-SH and PEEP-Py was carried out at room temperature under N2 atmosphere carefully to minimize the air oxidation and homodimerization. The feed ratio of PEEP-Py to PCL-SH was set at 1.2:1. The excessive PEEP-
Py was removed by precipitating the product in methanol, followed by thorough dialysis in water. GPC profile of the disulfide-linked diblock copolymer PCL-SS-PEEP compared with the precursor polymers PCL-SH and PEEP-Py (Supporting
Figure 1. 1H NMR spectra of diblock copolymer PCL-SS-PEEP and the precursors.
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Table 1. Characterization of the Disulfide-Linked Block Copolymer and the Precursors polymer
Mna
Mwa
PDIa
Mnb
PCL-SH PEEP-Py PCL-SS-PEEP
4780 2900 6420
5500 3950 8670
1.15 1.36 1.35
3650 3480 6950
a
Determined from GPC. b Determined from 1H NMR.
Information Figure S1) shows a significant shift toward higher molecular weights, suggesting successful coupling. 1H NMR of PCL-SS-PEEP in Figure 1 also shows the complete disappearance of pyridyl group resonances at δ 7.13, 7.56, 7.70 and 8.50, indicating successful conversion of PEEP-Py to the targeted copolymer. Like previously reported diblock copolymer PCL-b-PEEP (21), PCL-SS-PEEP is amphiphilic and self-assembles into micelles in aqueous solution. The critical micellization concentration of PCL-SS-PEEP is 3.1 × 10-3 mg mL-1, which was determined by fluorescent measurements with pyrene as the probe (20). Using the dialysis method, anticancer drug doxorubicin (DOX) was encapsulated into the core of the micelles with 2.8% drug loading content. Transmission electron microscopy image shown in Figure 2A exhibits the spherical morphology of DOX-loaded PCL-SS-PEEP micelles, with a diameter around 70 nm, which is consistent with that observed by dynamic light scattering (DLS) measurements. The disulfide bridge linkage between PCL and PEEP block makes PCL-SS-PEEP micelles reductively breakable with response to GSH. To demonstrate the responsiveness, micelles were treated with 10 mM of GSH, and at different time intervals, the particle sizes were measured by DLS. As shown in Figure 2B, the average diameter of GSH-treated micelles immediately raised, reaching about 1000 nm within 10 min. Further incubation resulted in microsized particles and severe aggregations. However, without GSH treatment, PCL-SS-PEEP micelles showed negligible size variation within the same period of time. The variation of particle size in the presence of GSH was caused by the cleavage of disulfide linkages, which led to detachment
of the PEEP shells from the micellar nanoparticles and resulted in enhanced hydrophobic PCL core material interaction. The effect of PEEP shell detachment to the DOX release from the drug-loaded PCL-SS-PEEP micelles was directly observed by measuring DOX fluorescence of the micelle solution with treatment of 10 mM of DL-dithiothreitol (DTT). The relative fluorescence intensities of treated micelles to the nontreated micelles were plotted in Figure 2C. It has been reported that incorporation of DOX in the hydrophobic core of micelles decreases the fluorescent intensity of DOX when compared with free DOX at the same concentration (22, 23). The enhanced DOX fluorescence of DTT-treated DOX-loaded micelles therefore should be due to the rapid release of drug from the micelles. The drug release was also quantitatively studied by incubation of nanoparticles in GSH-containing medium. The DOX release profiles are displayed in Figure 2D. The DOX release from micelles was around 40% in 4 days in the absence of GSH, indicating lack of stability of micelles; however, in the presence of 10 mM of GSH corresponding to an intracellular level, the DOX release from the micelles was much faster, reaching almost complete drug release. These results demonstrate that cleavage of disulfide bridge linkages accelerated the drug release. It can be assumed that, under the stimulus of GSH, the disulfide bridge linkages of PCL-SS-PEEP are reduced and broken, which destabilizes the micellar nanoparticles and results in accelerated DOX release. The intracellular behavior of DOX-loaded PCL-SS-PEEP micelles was evaluated in A549 cells to assess whether these GSH-responsive micelles function in the intracellular environment. Most tumor cells have an elevated GSH level compared with normal cells (24-26). As a model system, A549 cells were incubated with glutathione monoester (GSH-OEt) for 2 h to manipulate the intracellular GSH concentration. GSH-OEt can penetrate cellular membranes and rapidly generates high intracellular GSH concentration with ethyl ester hydrolyzation in cytoplasm (13, 24). After pretreatment with 10 mM of GSHOEt, the cells were further incubated with DOX-loaded PCLSS-PEEP micelles for 2 h. The DOX fluorescence of the cells
Figure 2. Transmission electron microscopy image of doxorubicin-loaded PCL-SS-PEEP micelles (A); dynamic light scattering measured PCLSS-PEEP micelle size changes with glutathione (GSH) treatment (B); relative fluorescence intensity of doxorubicin-loaded PCL-SS-PEEP micelles with treatment of DL-dithiothreitol (DTT) (C); in Vitro release of doxorubicin from PCL-SS-PEEP micelles in PBS (pH 7.4) at 37 °C with or without GSH treatment (D).
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Figure 3. (A,B) Confocal laser scanning microscopy observation of nonpretreated (A) and GSH-OEt pretreated A549 cells (B) after 2 h incubation with DOX-loaded PCL-SS-PEEP micelles. (C) Flow cytometric analyses of nonpretreated and GSH-OEt pretreated A549 cells incubated with DOX-loaded PCL-SS-PEEP micelles for 2 h. Nonpretreated A549 cells without DOX-loaded micelle treatment was used as the control. The relative geometrical mean fluorescence intensities (GMFI) of cells are shown as an inset. (D) Viability of non-pretreated and pretreated A549 cells with either 10 or 20 mM of GSH-OEt incubated with DOX-loaded PCL-SS-PEEP micelles for 24 h. * p < 0.05, ** p < 0.01 by Student’s t-test.
was observed under confocal laser scanning microscopy and analyzed by flow cytometric analyses. The results were compared with the A549 cells not pretreated with GSH-OEt but incubated for 2 h with the same dose of DOX-loaded PCL-SSPEEP micelles. As shown in Figure 3A,B, stronger DOX fluorescence was observed in the cytoplasm of GSH-OEt pretreated cells. Flow cytometric analyses shown in Figure 3C also exhibit similar results, and relative geometrical mean fluorescence intensities (GMFI) of GSH-OEt pretreated cells are about 2-fold of nonpretreated cells. It is noteworthy that incubating DOX-loaded PCL-b-PEEP micelles without reducible disulfide bridges did not result in significant DOX fluorescence difference between non-pretreated and pretreated A549 cells, indicating that GSH-OEt pretreatment did not affect the endocytosis ability of A549 cells. On the other hand, broken disulfide linkages of PCL-SS-PEEP micelles enhanced the DOX fluorescence (Figure 2C). Therefore, the enhanced intracellular DOX fluorescence in GSH-OEt pretreated cells is due to the rapid intracellular DOX release from the DOX-loaded PCLSS-PEEP micelles. Pretreatment of A549 cells with GSH-OEt up-regulated the intracellular GSH concentration, which subsequently accelerated the DOX release from the micelles owning to GSH-responsive disulfide bridge linkage degradation. Such an enhanced intracellular release of DOX from PCLSS-PEEP micelles led to improved cytotoxicity. DOX-loaded PCL-SS-PEEP micelles at the same dose were incubated with A549 cells, which were either non-pretreated or pretreated with GSH-OEt at 10 or 20 mM for 2 h. Cell viability measured by MTT assay shows significant differences between GSH-OEt pretreated and nonpretreated cells, as shown in Figure 3D. Incubation of DOX-loaded PCL-SS-PEEP micelles at a DOX dose of 3.4 µg mL-1 with non-pretreated A549 cells for 24 h did not exhibit significant inhibition to the cell proliferation, while the viability of GSH-OEt pretreated cells significantly decreased with increased GSH-OEt concentration for the pretreatments. However, proliferation of A549 cells incubated with free DOX was not affected by the treatment of GSH-OEt
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(Supporting Information Figure S2). On the other hand, treatments of both GSH-OEt and DOX free PCL-SS-PEEP micelles did not result in significant cytotoxicity to A549 cells under the same conditions (Supporting Information Figure S3). These results demonstrated that the destabilization of PCL-SS-PEEP micelles by intracellular GSH accelerated DOX release, and the cell proliferation inhibition was thus enhanced due to the faster intracellular DOX release. To further confirm such a response to intracellular GSH concentration, viability of incubation of A549 cells with DOX-loaded PCL-SS-PEEP micelles was examined after buthionine sulfoximine (BSO) pretreatments. BSO is an inhibitor of intracellular GSH synthesis (27) and does not affect the proliferation of A549 cells at the tested concentrations up to 0.5 mM (Supporting Information Figure S4). With the pretreatment of BSO at higher concentration, DOX-loaded PCL-SS-PEEP micelles exhibited lower cytotoxicity (Supporting Information Figure S4). This is due to that incubation of A549 cells with BSO lowered down the intracellular GSH concentration; therefore, destabilization of DOX-loaded PCL-SS-PEEP micelles was suppressed, resulting in less DOX release from the micelles. In conclusion, micelle nanoparticles self-assembled from a disulfide-linked diblock copolymer PCL-SS-PEEP have been prepared for enhanced intracellular drug delivery. The micelles exhibit GSH-responsive structure change, and the intracellular DOX release is accelerated at higher GSH concentration. The enhanced intracellular DOX release leads to more significant growth inhibition to A549 cells. This GSH-triggered drug release system has the potential to maximize the delivery efficiency for cancer chemotherapy.
ACKNOWLEDGMENT This work was supported by grants from the National Natural Science Foundation of China (50733003, 20774089), the Ministry of Sciences and Technology of the People’s Republic of China (2006CB933300, 2009CB930300), and “Bairen” Program of Chinese Academy of Sciences. Supporting Information Available: Experimental procedures, gel permeation chromatography analyses, and MTT assayed cytotoxicity. This material is available free of charge via the Internet at http://pubs.acs.org.
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BC900144M
