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Light and pH Dual-Degradable Triblock Copolymer Micelles for Controlled Intracellular Drug Release Qiao Jin,* Tongjiang Cai, Haijie Han, Haibo Wang, Yin Wang, Jian Ji* A novel amphiphilic ABA-type triblock copolymer poly(ethylene glycol)-b-poly(ethanedithiol-altnitrobenzyl)-b-poly(ethylene glycol) (PEG-b-PEDNB-b-PEG) is successfully prepared by sequential thiol-acrylate Michael addition polymerization in one pot. PEG-b-PEDNB-b-PEG is designed to have light-cleavable o-nitrobenzyl linkages and acid-labile β-thiopropionate linkages positioned repeatedly in the main chain of the hydrophobic block. The light and pH dual degradation of PEGb-PEDNB-b-PEG is traced by gel permeation chromatography (GPC). Such triblock copolymer can self-assemble into micelles, which can be used to encapsulate anticancer drug doxorubicin (DOX). Because of the different degradation chemistry of o-nitrobenzyl linkages and β-thiopropionate linkages, DOX can be released from the micelles by two different manners, i.e., light-induced rapid burst release and pH-induced slow sustained release. Confocal laser scanning microscopy (CLSM) results indicated that DOX-loaded micelles exhibited faster drug release in A549 cells after UV irradiation. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) results show that the DOX-loaded micelles under UV light degradation exhibit better anticancer activity against A549 cells than that of the nonirradiated ones.
1. Introduction Owing to the impressive progress in materials science and pharmaceutics, polymeric micelles have emerged as a most promising and valuable technology platform for targeted and controlled drug delivery.[1–9] Generally, micelles have many distinct advantages as potential drug carriers, including improved drug solubility, decreased side effects, prolonged circulation time, passive targeting of tumor tissues via enhanced permeability and retention (EPR) effect,[10] and improved drug bioavailability. Dr. Q. Jin, T. Cai, H. Han, H. Wang, Y. Wang, Prof. J. Ji MOE Key Laboratory of Macromolecule Synthesis and Functionalization of Ministry of Education, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China E-mail: [email protected]; [email protected]
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In order to maximize the therapeutic effect, overstable micelles are not desirable since the release of the drug might be inhibited when the micelles arrive at the site of action. Therefore, various stimuli-responsive drug nanocarriers were developed to overcome the problems and achieve smart drug release.[11,12] In general, fast drug release can be achieved if the core of the micelles becomes hydrophilic under different stimulus since the micelles will be disassembled.[13,14] On the other hand, fast drug release can also be realized if the core of the micelles can be degraded in response to external stimuli.[15,16] Such degradation-induced drug release is even more promising for polymer-based drug delivery applications. Stimuliresponsive degradation enables fast and controlled release of encapsulated therapeutic drugs from micelles in targeted cells. Furthermore, it also ensures the clearance of the polymer carriers after drugs are delivered to the body, which is very important since the nondegradable polymers
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DOI: 10.1002/marc.201400171
Light and pH Dual-degradable Triblock Copolymer Micelles for Controlled Intracellular Drug Release
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preparation of a new amphiphilic ABAtype triblock copolymer by sequential thiol-acrylate Michael addition polymerization in one pot (Scheme 1). Such triblock copolymer was designed to possess two stimuli-responsive functionalities positioned repeatedly in the main chain of the hydrophobic middle block, namely, light-cleavable o-nitrobenzyl linkages and acidlabile β-thiopropionate linkages. The o-nitrobenzyl linkages can be degraded very fast upon light irradiation. In addiScheme 1. Schematic illustration of the synthesis of ABA-type triblock copolymer PEG-b-PEDNB-b-PEG. tion, β-thiopropionate linkages can be selectively degraded under mild acidic conditions in a very slow rate. Therefore, we anticipate cannot be eliminated through metabolism and will result that drugs can be released from the micelles of such triin prolonged inflammation. Therefore, various stimuliblock copolymers by two different manners, i.e., lightresponsive degradable micelles were developed as drug induced rapid burst release and pH-induced slow susnanocarriers by incorporating cleavable linkages into the tained release, which have a great potential as efficient main chain of the polymers, such as disulfide, acid-labile intracellular smart drug delivery platforms. bonds, light-cleavable linkages, and so on.[17–22] Lightdegradable polymeric micelles are especially attractive since light provides a possibility for realizing remote and spatiotemporal drug release by facile tuning the wave2. Experimental Section length, energy, and site of irradiation.[23–28] Combination of All experimental details are provided in the Supporting light and interventional therapy, site-specific drug release Information. can be realized by taking advantage of optical fiber. On the other hand, pH-degradable micelles attract more and more attention because of the existence of pH gradient 3. Results and Discussion between the extra- and intracellular space.[29] Such pHdegradable micelles, while being stable at a physiological 3.1. Synthesis and Characterization pH, can be degraded in acidic cancer cells, facilitating the of PEG-b-PEDNB-b-PEG intracellular release of anticancer drugs.[30,31] Acid-labile Click chemistry has made tremendous impact in linkages, such as acetal, orthoester, imine, hydrazine, and polymer synthesis during the last decade. The ABA-type β-thiopropionate linkages are widely used to construct amphiphilic triblock copolymer poly(ethylene glycol)-bpH-degradable drug nanocariers since they can be cleaved poly(ethanedithiol-alt-nitrobenzyl)-b-poly(ethylene glycol) under mild acidic conditions (pH ≈ 5.0).[32–35] (PEG-b-PEDNB-b-PEG) with two stimuli-responsive funcVery recently, ABC-type triblock copolymers with tionalities in the main chain was synthesized by sequena redox-cleavable disulfide and a photocleavable tial thiol-acrylate Michael addition polymerization in one o-nitrobenzyl group at the two junctions of the three pot as illustrated in Scheme 1. PEDNB, which was periodiblocks were reported.[28] They can self-assemble to lightcally inserted with light-cleavable o-nitrobenzyl linkages and redox-responsive micelles. However, the release and acid-labile β-thiopropionate linkages, was obtained of Nile Red was limited since the removal of one type by mixing dithiol monomer EDOL (M1) and diacrylate of hydrophilic polymer chains from the micelle corona monomer NPDA (M2) with stoichiometric imbalance (M1/ by a reducing agent or light irradiation cannot result in M2 = 1.15:1.0 in mole ratio) in the presence of triethylamine. the disruption of the micelles. In order to design stimThe 1H NMR spectrum of PEDNB was exhibited in Figure 1A. uli-responsive degradable micelles with a more versatile and complex level of control, it is of great interest to All peaks could be well assigned with the proposed cheminsert multi-stimuli-cleavable units in the main chain of ical structures. In order to make sure PEDNB polymer chains the hydrophobic block in a controlled way. The resulting contained thiol groups at both ends, the dithiol monomer micelles might exhibit much better controlled drug EDOL was used in excess. As expected, the characteristic resrelease behaviors because of the main chain degradaonance signals of acrylate groups around 5.5–6.5 ppm was bility in response to two or more stimuli. In this research, not observed, which further confirmed that the end groups we reported an extremely simple synthetic route for the of PEDNB were thiol groups, not acrylate groups.
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Subsequently, triblock copolymer PEG-b-PEDNB-b-PEG was successfully prepared by adding excessive acrylate monomer poly(ethylene glycol) methyl ether acrylate (PEGMEA) into PEDNB solution. The molecular weight of PEGb-PEDNB-b-PEG was found to be 10 200 by GPC (Mw/Mn = 1.19), which was also in perfect agreement with the theoretically calculated value (≈9600). The 1H NMR spectrum of PEG-b-PEDNB-b-PEG was shown in Figure 1B with all the peaks labeled. Since the DP of PEGMEA is known, the DP of PEDNB block can therefore be calculated to be about 11 by comparing the well-defined peak integrals of OCH3 (3.37 ppm) of PEG block and C6H3 (7.51 ppm) of PEDNB block. It is also very close to the theoretically calculated DP (14.3), which is another proof of quantitative chain-end functionalization. According to the DP of PEDNB block calculated by 1H NMR, the molecular weight of PEG-b-PEDNBb-PEG was calculated to be 8700. 3.2. Self-Assembly and Degradation of Triblock Copolymer PEG-b-PEDNB-b-PEG The triblock copolymer micelles were prepared by dialysis. Dynamic light scattering (DLS) measurements showed that the PEG-b-PEDNB-b-PEG copolymers formed nano-sized aggregates. The intensity-average hydrodynamic Figure 1. 1H NMR spectra of A) PEDNB and B) PEG-b-PEDNB-b-PEG in CDCl3. * indicates diameter (Dh) of the aggregates was residual NMR solvent. 78 nm with low polydispersity index (PDI = 0.19). The transmission electron microscopy (TEM) image displayed a spherical morphology At 100% conversion with a stoichiometric ratio of with an average micelle diameter of about 64 nm (Figure 1.15:1.0, the degree of polymerization (DP) of PEDNB S5, Supporting Information). The smaller size observed by could be theoretically calculated to be 14.3 using the folTEM as compared to that measured by DLS is most likely lowing equation.[36] due to shrinkage of hydrophilic shells upon drying the DP = (1 + r )/ (1 − r ), samples. The critical micelle concentration (CMC) of the copolymers was determined using Nile Red as the hydrowhere phobic fluorescent probe. The estimated CMC value of the r = [ M2]/[ M 1] micelles was 29.46 mg L−1. It was important to note that the CMC value was much smaller than those of low-molecTherefore, the molecular weight of PEDNB could be ular-weight surfactants and was comparable with those of theoretically calculated to be 5505. The number-average other micelle-like polymeric aggregates.[37] molecular weight (Mn ) of PEDNB was 5800 as determined by GPC in Figure S4 (Supporting Information), which comAs is known, the triblock copolymer PEG-b-PEDNBpared perfectly well to the theoretically calculated molecb-PEG possesses light-cleavable o-nitrobenzyl linkages ular weight. and acid-labile β-thiopropionate linkages positioned
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Light and pH Dual-degradable Triblock Copolymer Micelles for Controlled Intracellular Drug Release
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repeatedly in the main chain of PEDNB block. The light and pH degradation of PEG-b-PEDNB-b-PEG was further investigated. The aqueous solution of PEG-b-PEDNB-b-PEG was irradiated under UV light (365 nm) for different time intervals (from 5 min to 5 h). Figure 2A showed the GPC chromatograms recorded for PEG-b-PEDNB-b-PEG with light irradiation. With increasing irradiation time, the peak was shifting towards higher elution time. At the same time, bimodal GPC chromatograms were observed with concomitant appearance of a shoulder at about 18 min, which indicated the generation of low-molecularweight products, owing to the cleavage of o-nitrobenzyl linkages (Scheme S1, Supporting Information). It is evident that after irradiation for 30 min, the degradation is not complete, which might be attributed to the relatively low intensity of the light source (8 W). However, after irradiation for 5 h, the copolymer can be completely degraded (Figure 2A). In order to investigate the pH degradation of PEG-b-PEDNB-b-PEG, the aqueous solution was stirred at pH 5.0 at regular time intervals and analyzed by GPC. As shown in Figure 2B, the number-average molecular weight of PEG-b-PEDNB-b-PEG decreased as time went on because of the cleavage of β-thiopropionate linkages (Scheme S2, Supporting Information). However, the rate of pH degradation was much slower than that of light degradation, which might be attributed to the relatively slow degradation kinetics of β-thiopropionate linkages. Furthermore, the degradation of the copolymer under UV irradiation at different pH was also investigated. The copolymer was irradiated with UV light for 1 h at pH 7.4 or 5.0. The degradation of the copolymer at pH 5.0 was a little faster than that at pH 7.4, although the distinction was not very obvious (Figure S6, Supporting Information). After light irradiation or acid treatment, the core of the triblock copolymer micelles will be degraded, which might result in the disassembly of micelles. DLS results of the micelles before and after degradation were shown in Figure 3. It should be noted that the degraded products can
be proposed to be hydrophilic because of the generation of carboxyl groups as shown in Schemes S1 and S2 (Supporting Information). As a result, no precipitations or large particles were observed after degradation. The intensity-average Dh of the micelles decreased dramatically after light irradiation with 30 min or acid treatment with 1 week, which confirmed the disassembly of the triblock copolymer micelles. Furthermore, since more low-molecular-weight products were generated after acid treatment with 1 week than that after light irradiation with 30 min as shown in Figure 2, the particle size of acid-degraded micelles is smaller than lightdegraded micelles (Figure 3). Therefore, the micelles will be disassembled after degradation, which is of great importance for drug delivery applications. 3.3. In Vitro Drug Release Study In order to evaluate the potential of the dual-degradable micelles as smart drug nanocarriers, loading and release of DOX (a potent hydrophobic anticancer drug) from PEG-bPEDNB-b-PEG micelles were investigated. The drug-loading content (DLC) was 13.1 wt% measured by fluorescent spectra. Light and pH degradation-triggered drug release behaviors of DOX-loaded micelles were presented in Figure 4. At pH 7.4 without light irradiation, only less than 20% of DOX was released within 48 h. The very slow drug release might come from the overstable micelles, which is not beneficial for cancer therapy. However, more than 50% of DOX can be released within 8 h under light irradiation at pH 7.4. It might be attributed to the very fast light degradation of the core of micelles, which could result in rapid burst release of loaded DOX. Similarly, pH degradationtriggered drug release was further investigated. Compared to the drug release behavior at pH 7.4 without light irradiation, the release of DOX was accelerated at pH 5.0 without light irradiation. We can find the relatively slow sustained release and about 40% of DOX was released within 48 h in this situation. Overall, these results show that the light and
Figure 2. GPC traces of triblock copolymer A) PEG-b-PEDNB-b-PEG after light irradiation; B) after acid treatment (pH 5.0).
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Figure 3. DLS results of the triblock copolymer micelles before degradation and after light irradiation with 30 min or acid treatment with 1 week.
pH dual-degradable triblock copolymer micelles are able to realize rapid burst release or slow sustained release of the encapsulated drugs when it is triggered by UV light or acid environment. By contrast, the drug release would be dramatically suppressed at pH 7.4 without light irradiation. 3.4. Cell Internalization and Intracellular Drug Release Studies The cellular uptake of DOX-loaded PEG-b-PEDNB-b-PEG micelles was studied in A549 cancer cells by flow cytometric analysis. This method was demonstrated by
Figure 4. In vitro drug release behavior of DOX-loaded micelles under different stimulus conditions.
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quantitative fluorometry of DOX from cell internalization. The fluorescence intensity of A549 cells increased greatly with prolonged incubation time (Figure S7, Supporting Information). The rapid enhancement of fluorescence intensity indicated that the DOX-loaded PEG-b-PEDNBb-PEG micelles can be effectively internalized by A549 cells. Furthermore, the light-triggered intracellular DOX release was visualized by CLSM. The A549 cells incubated with DOX-loaded micelles were irradiated under UV light for 30 min before measurement. The A549 cells without UV irradiation were used as a control. As shown in Figure 5, stronger intracellular DOX fluorescence was observed in the cells with UV irradiation, compared to the cells without UV irradiation. It is documented that free DOX shows stronger fluorescence compared with the DOX-loaded nanoparticles at the same DOX concentration due to the self-quenching effect of DOX.[38] Thus, the enhanced fluorescence intensity in the cells with UV irradiation suggested that the core of the micelles was degraded under UV irradiation, which accelerated the intracellular DOX release from PEG-b-PEDNB-b-PEG micelles and resulted in the rapid localization of DOX in cell nucleus. 3.5. Cytotoxicity of DOX-Loaded PEG-b-PEDNB-b-PEG Micelles In order to examine the biocompatibility of PEG-b-PEDNBb-PEG micelles, the cytotoxicity was determined for the PEG-b-PEDNB-b-PEG micelles at different concentrations in human endothelial HUVEC cells using the MTT cell proliferation assay. The viability of HUVEC cells was higher than 90% in a wide polymer concentration range from 0.1 to 1 mg mL−1 (Figure S8, Supporting Information), demonstrating that the polymeric micelles do not have acute or intrinsic cytotoxicity against normal cells. The cytotoxicity and antitumor activity of DOX-loaded PEG-b-PEDNB-b-PEG micelles with and without UV irradiation were also investigated in A549 cells using MTT assay. The free DOX was used as a control. As shown in Figure 6, the DOX-loaded micelles without UV irradiation exhibited low cellular proliferation inhibiting ability even in high DOX concentration (10 μg mL−1), probably because of the overstable micellar structure. However, the DOX-loaded micelles treated with UV irradiation exhibited much higher cytotoxicity with relatively low IC50 value (≈3.15 μg mL−1), which was much lower than that without UV irradiation (>10 μg mL−1). This was due to the rapid DOX release under UV irradiation. Additionally, in order to eliminate the possibility that the increased cytotoxicity of DOX-loaded PEG-b-PEDNB-b-PEG micelles after UV irradiation comes from the cell death under UV light or the cytotoxicity of the byproducts from the degraded PEG-b-PEDNB-b-PEG, the in vitro cytotoxicity of blank
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Light and pH Dual-degradable Triblock Copolymer Micelles for Controlled Intracellular Drug Release
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Information), which showed that the excellent anticancer activity of DOX-loaded PEG-b-PEDNB-b-PEG micelles under UV irradiation was not caused by the UV light or the byproducts from the degraded PEG-b-PEDNB-b-PEG. The low intensity UV irradiation (365 nm, 8 W) showed no cytotoxicity in Figure S9 (Supporting Information), which was similar to previously reported.[40]
4. Conclusions
Figure 5. Representative CLSM images of A549 cells incubated with DOX-loaded PEG-b-PEDNB-b-PEG micelles. a) 2 h, without light irradiation; b) 2 h, with light irradiation. From top to bottom: DAPI (blue), DOX (red), and a merge of the two images.
PEG-b-PEDNB-b-PEG micelles with or without UV irradiation was also investigated by the MTT assay using A549 cells.[39] The blank PEG-b-PEDNB-b-PEG micelles with or without UV irradiation did not exhibit obvious cytotoxicity to A549 cells as shown in Figure S9 (Supporting
A novel amphiphilic ABA-type triblock copolymer PEG-bPEDNB-b-PEG was successfully synthesized by sequential thiol-acrylate Michael addition polymerization in one pot. PEG-b-PEDNB-b-PEG was designed to possess two stimuliresponsive functionalities positioned repeatedly in the main chain of the hydrophobic PEDNB block, namely, light-cleavable o-nitrobenzyl linkages and acid-labile β-thiopropionate linkages. This triblock copolymer can be degraded under UV irradiation or in mild-acid environment as indicated by GPC. DLS and TEM results verified the copolymer can self-assemble into micelles, which can be used to load anticancer drug DOX. In vitro drug release showed that DOX can be released from the micelles by two different manners, i.e., light-induced rapid burst release and pH-induced slow sustained release. The flow cytometry results indicated that DOX-loaded PEG-b-PEDNB-b-PEG micelles can be effectively internalized by A549 cells. The intracellular DOX release behaviors of DOX-loaded micelles towards A549 cells were measured by CLSM, which demonstrated that DOX-loaded micelles exhibited faster drug release in A549 cells treated with UV irradiation, compared to that without UV irradiation. Furthermore, DOX-loaded micelles with UV irradiation showed stronger inhibition of the proliferation of A549 cells than that of the nonirradiated ones. These results indicated that the novel light and pH dual-degradable triblock copolymer micelles could be served as promising drug nanocarriers for controlled intracellular drug release.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Figure 6. Cytotoxicity of free DOX and DOX-loaded PEG-bPEDNB-b-PEG micelles with and without UV irradiation against A549 cells after 24 h incubation.
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Acknowledgements: Financial support from the National Natural Science Foundation of China (Nos. 51333005, 21174126, 51303154, and 51025312), the National Basic Research Program of China (2011CB606203) and Research Fund for the Doctoral Program of Higher Education of China (20120101130013, 20130101120177), the Fundamental Research Funds for the Central Universities (No. 2013QNA4047) and a Project Supported by Scientific Research Fund of Zhejiang Provincial Education Department (Y201326564) are gratefully acknowledged.
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Received: March 21, 2014; Revised: April 15, 2014; Published online: May 22, 2014; DOI: 10.1002/marc.201400171 Keywords: drug release; light-degradable; self-assembly; triblock copolymer
pH-degradable;
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Communication
Light and pH Dual-Degradable Triblock Copolymer Micelles for Controlled Intracellular Drug Release Qiao Jin,* Tongjiang Cai, Haijie Han, Haibo Wang, Yin Wang, Jian Ji* A novel amphiphilic ABA-type triblock copolymer poly(ethylene glycol)-b-poly(ethanedithiol-altnitrobenzyl)-b-poly(ethylene glycol) (PEG-b-PEDNB-b-PEG) is successfully prepared by sequential thiol-acrylate Michael addition polymerization in one pot. PEG-b-PEDNB-b-PEG is designed to have light-cleavable o-nitrobenzyl linkages and acid-labile β-thiopropionate linkages positioned repeatedly in the main chain of the hydrophobic block. The light and pH dual degradation of PEGb-PEDNB-b-PEG is traced by gel permeation chromatography (GPC). Such triblock copolymer can self-assemble into micelles, which can be used to encapsulate anticancer drug doxorubicin (DOX). Because of the different degradation chemistry of o-nitrobenzyl linkages and β-thiopropionate linkages, DOX can be released from the micelles by two different manners, i.e., light-induced rapid burst release and pH-induced slow sustained release. Confocal laser scanning microscopy (CLSM) results indicated that DOX-loaded micelles exhibited faster drug release in A549 cells after UV irradiation. Furthermore, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) results show that the DOX-loaded micelles under UV light degradation exhibit better anticancer activity against A549 cells than that of the nonirradiated ones.
1. Introduction Owing to the impressive progress in materials science and pharmaceutics, polymeric micelles have emerged as a most promising and valuable technology platform for targeted and controlled drug delivery.[1–9] Generally, micelles have many distinct advantages as potential drug carriers, including improved drug solubility, decreased side effects, prolonged circulation time, passive targeting of tumor tissues via enhanced permeability and retention (EPR) effect,[10] and improved drug bioavailability. Dr. Q. Jin, T. Cai, H. Han, H. Wang, Y. Wang, Prof. J. Ji MOE Key Laboratory of Macromolecule Synthesis and Functionalization of Ministry of Education, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China E-mail: [email protected]; [email protected]
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In order to maximize the therapeutic effect, overstable micelles are not desirable since the release of the drug might be inhibited when the micelles arrive at the site of action. Therefore, various stimuli-responsive drug nanocarriers were developed to overcome the problems and achieve smart drug release.[11,12] In general, fast drug release can be achieved if the core of the micelles becomes hydrophilic under different stimulus since the micelles will be disassembled.[13,14] On the other hand, fast drug release can also be realized if the core of the micelles can be degraded in response to external stimuli.[15,16] Such degradation-induced drug release is even more promising for polymer-based drug delivery applications. Stimuliresponsive degradation enables fast and controlled release of encapsulated therapeutic drugs from micelles in targeted cells. Furthermore, it also ensures the clearance of the polymer carriers after drugs are delivered to the body, which is very important since the nondegradable polymers
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DOI: 10.1002/marc.201400171
Light and pH Dual-degradable Triblock Copolymer Micelles for Controlled Intracellular Drug Release
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preparation of a new amphiphilic ABAtype triblock copolymer by sequential thiol-acrylate Michael addition polymerization in one pot (Scheme 1). Such triblock copolymer was designed to possess two stimuli-responsive functionalities positioned repeatedly in the main chain of the hydrophobic middle block, namely, light-cleavable o-nitrobenzyl linkages and acidlabile β-thiopropionate linkages. The o-nitrobenzyl linkages can be degraded very fast upon light irradiation. In addiScheme 1. Schematic illustration of the synthesis of ABA-type triblock copolymer PEG-b-PEDNB-b-PEG. tion, β-thiopropionate linkages can be selectively degraded under mild acidic conditions in a very slow rate. Therefore, we anticipate cannot be eliminated through metabolism and will result that drugs can be released from the micelles of such triin prolonged inflammation. Therefore, various stimuliblock copolymers by two different manners, i.e., lightresponsive degradable micelles were developed as drug induced rapid burst release and pH-induced slow susnanocarriers by incorporating cleavable linkages into the tained release, which have a great potential as efficient main chain of the polymers, such as disulfide, acid-labile intracellular smart drug delivery platforms. bonds, light-cleavable linkages, and so on.[17–22] Lightdegradable polymeric micelles are especially attractive since light provides a possibility for realizing remote and spatiotemporal drug release by facile tuning the wave2. Experimental Section length, energy, and site of irradiation.[23–28] Combination of All experimental details are provided in the Supporting light and interventional therapy, site-specific drug release Information. can be realized by taking advantage of optical fiber. On the other hand, pH-degradable micelles attract more and more attention because of the existence of pH gradient 3. Results and Discussion between the extra- and intracellular space.[29] Such pHdegradable micelles, while being stable at a physiological 3.1. Synthesis and Characterization pH, can be degraded in acidic cancer cells, facilitating the of PEG-b-PEDNB-b-PEG intracellular release of anticancer drugs.[30,31] Acid-labile Click chemistry has made tremendous impact in linkages, such as acetal, orthoester, imine, hydrazine, and polymer synthesis during the last decade. The ABA-type β-thiopropionate linkages are widely used to construct amphiphilic triblock copolymer poly(ethylene glycol)-bpH-degradable drug nanocariers since they can be cleaved poly(ethanedithiol-alt-nitrobenzyl)-b-poly(ethylene glycol) under mild acidic conditions (pH ≈ 5.0).[32–35] (PEG-b-PEDNB-b-PEG) with two stimuli-responsive funcVery recently, ABC-type triblock copolymers with tionalities in the main chain was synthesized by sequena redox-cleavable disulfide and a photocleavable tial thiol-acrylate Michael addition polymerization in one o-nitrobenzyl group at the two junctions of the three pot as illustrated in Scheme 1. PEDNB, which was periodiblocks were reported.[28] They can self-assemble to lightcally inserted with light-cleavable o-nitrobenzyl linkages and redox-responsive micelles. However, the release and acid-labile β-thiopropionate linkages, was obtained of Nile Red was limited since the removal of one type by mixing dithiol monomer EDOL (M1) and diacrylate of hydrophilic polymer chains from the micelle corona monomer NPDA (M2) with stoichiometric imbalance (M1/ by a reducing agent or light irradiation cannot result in M2 = 1.15:1.0 in mole ratio) in the presence of triethylamine. the disruption of the micelles. In order to design stimThe 1H NMR spectrum of PEDNB was exhibited in Figure 1A. uli-responsive degradable micelles with a more versatile and complex level of control, it is of great interest to All peaks could be well assigned with the proposed cheminsert multi-stimuli-cleavable units in the main chain of ical structures. In order to make sure PEDNB polymer chains the hydrophobic block in a controlled way. The resulting contained thiol groups at both ends, the dithiol monomer micelles might exhibit much better controlled drug EDOL was used in excess. As expected, the characteristic resrelease behaviors because of the main chain degradaonance signals of acrylate groups around 5.5–6.5 ppm was bility in response to two or more stimuli. In this research, not observed, which further confirmed that the end groups we reported an extremely simple synthetic route for the of PEDNB were thiol groups, not acrylate groups.
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Subsequently, triblock copolymer PEG-b-PEDNB-b-PEG was successfully prepared by adding excessive acrylate monomer poly(ethylene glycol) methyl ether acrylate (PEGMEA) into PEDNB solution. The molecular weight of PEGb-PEDNB-b-PEG was found to be 10 200 by GPC (Mw/Mn = 1.19), which was also in perfect agreement with the theoretically calculated value (≈9600). The 1H NMR spectrum of PEG-b-PEDNB-b-PEG was shown in Figure 1B with all the peaks labeled. Since the DP of PEGMEA is known, the DP of PEDNB block can therefore be calculated to be about 11 by comparing the well-defined peak integrals of OCH3 (3.37 ppm) of PEG block and C6H3 (7.51 ppm) of PEDNB block. It is also very close to the theoretically calculated DP (14.3), which is another proof of quantitative chain-end functionalization. According to the DP of PEDNB block calculated by 1H NMR, the molecular weight of PEG-b-PEDNBb-PEG was calculated to be 8700. 3.2. Self-Assembly and Degradation of Triblock Copolymer PEG-b-PEDNB-b-PEG The triblock copolymer micelles were prepared by dialysis. Dynamic light scattering (DLS) measurements showed that the PEG-b-PEDNB-b-PEG copolymers formed nano-sized aggregates. The intensity-average hydrodynamic Figure 1. 1H NMR spectra of A) PEDNB and B) PEG-b-PEDNB-b-PEG in CDCl3. * indicates diameter (Dh) of the aggregates was residual NMR solvent. 78 nm with low polydispersity index (PDI = 0.19). The transmission electron microscopy (TEM) image displayed a spherical morphology At 100% conversion with a stoichiometric ratio of with an average micelle diameter of about 64 nm (Figure 1.15:1.0, the degree of polymerization (DP) of PEDNB S5, Supporting Information). The smaller size observed by could be theoretically calculated to be 14.3 using the folTEM as compared to that measured by DLS is most likely lowing equation.[36] due to shrinkage of hydrophilic shells upon drying the DP = (1 + r )/ (1 − r ), samples. The critical micelle concentration (CMC) of the copolymers was determined using Nile Red as the hydrowhere phobic fluorescent probe. The estimated CMC value of the r = [ M2]/[ M 1] micelles was 29.46 mg L−1. It was important to note that the CMC value was much smaller than those of low-molecTherefore, the molecular weight of PEDNB could be ular-weight surfactants and was comparable with those of theoretically calculated to be 5505. The number-average other micelle-like polymeric aggregates.[37] molecular weight (Mn ) of PEDNB was 5800 as determined by GPC in Figure S4 (Supporting Information), which comAs is known, the triblock copolymer PEG-b-PEDNBpared perfectly well to the theoretically calculated molecb-PEG possesses light-cleavable o-nitrobenzyl linkages ular weight. and acid-labile β-thiopropionate linkages positioned
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Light and pH Dual-degradable Triblock Copolymer Micelles for Controlled Intracellular Drug Release
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repeatedly in the main chain of PEDNB block. The light and pH degradation of PEG-b-PEDNB-b-PEG was further investigated. The aqueous solution of PEG-b-PEDNB-b-PEG was irradiated under UV light (365 nm) for different time intervals (from 5 min to 5 h). Figure 2A showed the GPC chromatograms recorded for PEG-b-PEDNB-b-PEG with light irradiation. With increasing irradiation time, the peak was shifting towards higher elution time. At the same time, bimodal GPC chromatograms were observed with concomitant appearance of a shoulder at about 18 min, which indicated the generation of low-molecularweight products, owing to the cleavage of o-nitrobenzyl linkages (Scheme S1, Supporting Information). It is evident that after irradiation for 30 min, the degradation is not complete, which might be attributed to the relatively low intensity of the light source (8 W). However, after irradiation for 5 h, the copolymer can be completely degraded (Figure 2A). In order to investigate the pH degradation of PEG-b-PEDNB-b-PEG, the aqueous solution was stirred at pH 5.0 at regular time intervals and analyzed by GPC. As shown in Figure 2B, the number-average molecular weight of PEG-b-PEDNB-b-PEG decreased as time went on because of the cleavage of β-thiopropionate linkages (Scheme S2, Supporting Information). However, the rate of pH degradation was much slower than that of light degradation, which might be attributed to the relatively slow degradation kinetics of β-thiopropionate linkages. Furthermore, the degradation of the copolymer under UV irradiation at different pH was also investigated. The copolymer was irradiated with UV light for 1 h at pH 7.4 or 5.0. The degradation of the copolymer at pH 5.0 was a little faster than that at pH 7.4, although the distinction was not very obvious (Figure S6, Supporting Information). After light irradiation or acid treatment, the core of the triblock copolymer micelles will be degraded, which might result in the disassembly of micelles. DLS results of the micelles before and after degradation were shown in Figure 3. It should be noted that the degraded products can
be proposed to be hydrophilic because of the generation of carboxyl groups as shown in Schemes S1 and S2 (Supporting Information). As a result, no precipitations or large particles were observed after degradation. The intensity-average Dh of the micelles decreased dramatically after light irradiation with 30 min or acid treatment with 1 week, which confirmed the disassembly of the triblock copolymer micelles. Furthermore, since more low-molecular-weight products were generated after acid treatment with 1 week than that after light irradiation with 30 min as shown in Figure 2, the particle size of acid-degraded micelles is smaller than lightdegraded micelles (Figure 3). Therefore, the micelles will be disassembled after degradation, which is of great importance for drug delivery applications. 3.3. In Vitro Drug Release Study In order to evaluate the potential of the dual-degradable micelles as smart drug nanocarriers, loading and release of DOX (a potent hydrophobic anticancer drug) from PEG-bPEDNB-b-PEG micelles were investigated. The drug-loading content (DLC) was 13.1 wt% measured by fluorescent spectra. Light and pH degradation-triggered drug release behaviors of DOX-loaded micelles were presented in Figure 4. At pH 7.4 without light irradiation, only less than 20% of DOX was released within 48 h. The very slow drug release might come from the overstable micelles, which is not beneficial for cancer therapy. However, more than 50% of DOX can be released within 8 h under light irradiation at pH 7.4. It might be attributed to the very fast light degradation of the core of micelles, which could result in rapid burst release of loaded DOX. Similarly, pH degradationtriggered drug release was further investigated. Compared to the drug release behavior at pH 7.4 without light irradiation, the release of DOX was accelerated at pH 5.0 without light irradiation. We can find the relatively slow sustained release and about 40% of DOX was released within 48 h in this situation. Overall, these results show that the light and
Figure 2. GPC traces of triblock copolymer A) PEG-b-PEDNB-b-PEG after light irradiation; B) after acid treatment (pH 5.0).
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Figure 3. DLS results of the triblock copolymer micelles before degradation and after light irradiation with 30 min or acid treatment with 1 week.
pH dual-degradable triblock copolymer micelles are able to realize rapid burst release or slow sustained release of the encapsulated drugs when it is triggered by UV light or acid environment. By contrast, the drug release would be dramatically suppressed at pH 7.4 without light irradiation. 3.4. Cell Internalization and Intracellular Drug Release Studies The cellular uptake of DOX-loaded PEG-b-PEDNB-b-PEG micelles was studied in A549 cancer cells by flow cytometric analysis. This method was demonstrated by
Figure 4. In vitro drug release behavior of DOX-loaded micelles under different stimulus conditions.
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quantitative fluorometry of DOX from cell internalization. The fluorescence intensity of A549 cells increased greatly with prolonged incubation time (Figure S7, Supporting Information). The rapid enhancement of fluorescence intensity indicated that the DOX-loaded PEG-b-PEDNBb-PEG micelles can be effectively internalized by A549 cells. Furthermore, the light-triggered intracellular DOX release was visualized by CLSM. The A549 cells incubated with DOX-loaded micelles were irradiated under UV light for 30 min before measurement. The A549 cells without UV irradiation were used as a control. As shown in Figure 5, stronger intracellular DOX fluorescence was observed in the cells with UV irradiation, compared to the cells without UV irradiation. It is documented that free DOX shows stronger fluorescence compared with the DOX-loaded nanoparticles at the same DOX concentration due to the self-quenching effect of DOX.[38] Thus, the enhanced fluorescence intensity in the cells with UV irradiation suggested that the core of the micelles was degraded under UV irradiation, which accelerated the intracellular DOX release from PEG-b-PEDNB-b-PEG micelles and resulted in the rapid localization of DOX in cell nucleus. 3.5. Cytotoxicity of DOX-Loaded PEG-b-PEDNB-b-PEG Micelles In order to examine the biocompatibility of PEG-b-PEDNBb-PEG micelles, the cytotoxicity was determined for the PEG-b-PEDNB-b-PEG micelles at different concentrations in human endothelial HUVEC cells using the MTT cell proliferation assay. The viability of HUVEC cells was higher than 90% in a wide polymer concentration range from 0.1 to 1 mg mL−1 (Figure S8, Supporting Information), demonstrating that the polymeric micelles do not have acute or intrinsic cytotoxicity against normal cells. The cytotoxicity and antitumor activity of DOX-loaded PEG-b-PEDNB-b-PEG micelles with and without UV irradiation were also investigated in A549 cells using MTT assay. The free DOX was used as a control. As shown in Figure 6, the DOX-loaded micelles without UV irradiation exhibited low cellular proliferation inhibiting ability even in high DOX concentration (10 μg mL−1), probably because of the overstable micellar structure. However, the DOX-loaded micelles treated with UV irradiation exhibited much higher cytotoxicity with relatively low IC50 value (≈3.15 μg mL−1), which was much lower than that without UV irradiation (>10 μg mL−1). This was due to the rapid DOX release under UV irradiation. Additionally, in order to eliminate the possibility that the increased cytotoxicity of DOX-loaded PEG-b-PEDNB-b-PEG micelles after UV irradiation comes from the cell death under UV light or the cytotoxicity of the byproducts from the degraded PEG-b-PEDNB-b-PEG, the in vitro cytotoxicity of blank
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Light and pH Dual-degradable Triblock Copolymer Micelles for Controlled Intracellular Drug Release
Macromolecular Rapid Communications www.mrc-journal.de
Information), which showed that the excellent anticancer activity of DOX-loaded PEG-b-PEDNB-b-PEG micelles under UV irradiation was not caused by the UV light or the byproducts from the degraded PEG-b-PEDNB-b-PEG. The low intensity UV irradiation (365 nm, 8 W) showed no cytotoxicity in Figure S9 (Supporting Information), which was similar to previously reported.[40]
4. Conclusions
Figure 5. Representative CLSM images of A549 cells incubated with DOX-loaded PEG-b-PEDNB-b-PEG micelles. a) 2 h, without light irradiation; b) 2 h, with light irradiation. From top to bottom: DAPI (blue), DOX (red), and a merge of the two images.
PEG-b-PEDNB-b-PEG micelles with or without UV irradiation was also investigated by the MTT assay using A549 cells.[39] The blank PEG-b-PEDNB-b-PEG micelles with or without UV irradiation did not exhibit obvious cytotoxicity to A549 cells as shown in Figure S9 (Supporting
A novel amphiphilic ABA-type triblock copolymer PEG-bPEDNB-b-PEG was successfully synthesized by sequential thiol-acrylate Michael addition polymerization in one pot. PEG-b-PEDNB-b-PEG was designed to possess two stimuliresponsive functionalities positioned repeatedly in the main chain of the hydrophobic PEDNB block, namely, light-cleavable o-nitrobenzyl linkages and acid-labile β-thiopropionate linkages. This triblock copolymer can be degraded under UV irradiation or in mild-acid environment as indicated by GPC. DLS and TEM results verified the copolymer can self-assemble into micelles, which can be used to load anticancer drug DOX. In vitro drug release showed that DOX can be released from the micelles by two different manners, i.e., light-induced rapid burst release and pH-induced slow sustained release. The flow cytometry results indicated that DOX-loaded PEG-b-PEDNB-b-PEG micelles can be effectively internalized by A549 cells. The intracellular DOX release behaviors of DOX-loaded micelles towards A549 cells were measured by CLSM, which demonstrated that DOX-loaded micelles exhibited faster drug release in A549 cells treated with UV irradiation, compared to that without UV irradiation. Furthermore, DOX-loaded micelles with UV irradiation showed stronger inhibition of the proliferation of A549 cells than that of the nonirradiated ones. These results indicated that the novel light and pH dual-degradable triblock copolymer micelles could be served as promising drug nanocarriers for controlled intracellular drug release.
Supporting Information Supporting Information is available from the Wiley Online Library or from the author.
Figure 6. Cytotoxicity of free DOX and DOX-loaded PEG-bPEDNB-b-PEG micelles with and without UV irradiation against A549 cells after 24 h incubation.
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Acknowledgements: Financial support from the National Natural Science Foundation of China (Nos. 51333005, 21174126, 51303154, and 51025312), the National Basic Research Program of China (2011CB606203) and Research Fund for the Doctoral Program of Higher Education of China (20120101130013, 20130101120177), the Fundamental Research Funds for the Central Universities (No. 2013QNA4047) and a Project Supported by Scientific Research Fund of Zhejiang Provincial Education Department (Y201326564) are gratefully acknowledged.
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Received: March 21, 2014; Revised: April 15, 2014; Published online: May 22, 2014; DOI: 10.1002/marc.201400171 Keywords: drug release; light-degradable; self-assembly; triblock copolymer
pH-degradable;
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