Radiation-Sensitive Diselenide Block Co-polymer Micellar Aggregates

Apr 13, 2011 - low dose of γ-radiation, such as 5 Gy, which is close to the radiation dose received by patients during a single radiotherapy treatmen...
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Radiation-Sensitive Diselenide Block Co-polymer Micellar Aggregates: Toward the Combination of Radiotherapy and Chemotherapy Ning Ma,† Huaping Xu,*,† Liping An,‡ Juan Li,‡ Zhiwei Sun,§ and Xi Zhang*,† †

Key Laboratory of Organic Optoelectronics and Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084, People’s Republic of China ‡ School of Public Health, Jilin University, Changchun 130021, People’s Republic of China § Capital Medical University, Beijing 100069, People’s Republic of China ABSTRACT: We have developed a potential radiation-sensitive drug-delivery system using active diselenide-containing block co-polymer aggregates in aqueous solution that can load and release anticancer drugs. These aggregates were sensitive to even a low dose of γ-radiation, such as 5 Gy, which is close to the radiation dose received by patients during a single radiotherapy treatment. This line of research may open an avenue for the combination of radiotherapy and chemotherapy.

’ INTRODUCTION One of the most important functions of stimuli-responsive aggregates is the controllable load and release of functional species under certain stimuli.1 Among the various external stimuli, irradiation has been widely used for not only its convenience of operation but also because it requires no other chemical additives. For example, ultraviolet visible (UV vis) and near-infrared (IR) light have been employed to destroy the responsive aggregates, which realizes the load and release of functional molecules. This is especially critical to drug delivery.2 In addition, high-energy rays, such as γ-radiation, have also proven to be able to decompose or cross-link certain polymers and may be applied to induce structural changes in radiation-sensitive polymeric aggregates.3 However, it is rarely used because a high radiation dose is needed to break the covalent bonds in the polymeric backbone. The high doses of radiation present little potential for the application of this technique in biological or medical fields because they greatly exceed the dose limit that living organisms can survive. Therefore, it is desirable for new kinds of radiation-sensitive aggregates to be developed, which are able to respond to a low radiation dose similar to the radiotherapy radiation dose used on human subjects. This would provide novel drug-delivery systems for the combination of radiotherapy and chemotherapy. Organic selenium compounds have proven to be excellent candidates as stimuli-responsive materials because of their sensitivity in the presence of oxidants or reductants.4 Recently, we have reported a series of redox-responsive aggregates using selenium-containing block co-polymers, which are sensitive to oxidants in a very low concentration and are able to release loaded functional molecules.5 Among the selenium-containing polymers, the ones containing diselenide groups in the polymeric backbone are particularly sensitive to oxidation stimuli and undergo cleavage of their diselenides via r 2011 American Chemical Society

degradation upon oxidation. In this work, we attempt to use γ-radiation to destroy the aggregates formed by diselenide-containing block co-polymers in aqueous solution and release the encapsulated anticancer drugs based on the active nature of the diselenide bonds, which is shown in Scheme 1. Our study demonstrates that diselenidecontaining block co-polymers are potentially new responsive materials for use in the combination of radiotherapy and chemotherapy.

’ EXPERIMENTAL SECTION Materials. The synthesis of the PEG PUSeSe PEG block co-polymer and the preparation of their aggregates in aqueous solution have been described in our previous publication.5a For comparison to PEG PUSeSe PEG, a non-selenium-containing block co-polymer (PEG PUC6 PEG; Figure 1), in which the diselenide diol monomers were replaced by 1,6hexanediol during condensation, was also synthesized for use to experimentally verify the significance of the diselenide moieties. 1H nuclear magnetic resonance (NMR) (300 MHz, DMSO-d6) δ (ppm): 7.05 (3H, b, aromatic H), 4.04 (4H, b, NHCOOCH2), 3.50 (8H, b, OCH2CH2 of PEG), 1.62 (4H, b, NHCOOCH2CH2), 1.39 (4H, b, NHCOOCH2CH2CH2). The Mw of PEG PUC6 PEG was 12500, and Mw/Mn = 1.65, measured using gel permeation chromatography (GPC). Doxorubicin (Dox) (hydrochloride, 99.7%) was procured from Zhongshuo Pharmaceutical Co., Ltd. (Beijing, China). 2,4-Toluene diisocyanate, 30% H2O2, and other solvents were analytical-grade products purchased from the Beijing Chemical Reagent Company (Beijing, China). Instruments and Methods. The fluorescence of Dox was measured with a Hitachi F-7000 (Tokyo, Japan) spectrofluorometer, with an Received: March 15, 2011 Revised: April 4, 2011 Published: April 13, 2011 5874

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Langmuir excitation wavelength of 480 nm. The 1H NMR spectra were recorded on a JEOL JNM-ECA 300 (300 MHz) spectrometer (Tokyo, Japan). Fourier transform infrared (FTIR) spectra were obtained using a Bruker IFS 66v/s spectrometer (Ettlingen, Germany) using KBr substrates. Electronic spinning resonance (ESR) measurements were performed on a JEOL JES-FA200 apparatus, using 5,5-dimethyl-1-pyrroline N-oxide as a spin trap for free radicals. The GPC measurement was performed on a Waters 515 (Milford, MA) apparatus using polystyrene as a standard and tetrahydrofuran (THF) as an eluent. Transmission electron microscopy (TEM) images were obtained from a JEOL JEM-2010 microscope with an accelerating voltage of 120 kV, and the sample was stained by 0.2% phosphotungstic acid hydrate before observation.

Preparation of Dox-Loaded PEG PUSeSe PEG Micelles. To prepare the Dox-loaded micelles, the block co-polymer and Dox were both dissolved in N,N-dimethylformamide (DMF) at a concentration of 10 mg mL 1. Subsequently, the solution was placed in 15 mL of deionized water and then dialyzed for 72 h. The volume of the solution increased to 20 mL with the addition of deionized water, which created an aggregate solution with a concentration of 0.5 mg mL 1 for the experiments. It should be noted that the obtained micellar solution was dialyzed against deionized water until the water outside the dialysis tube exhibited negligible Dox fluorescence emission. This dialysis ensured the removal of Dox molecules that were not entrapped by the PEG PUSeSe PEG micelles. The final loading capacity of Dox was 0.055 mg mL 1, which was estimated by fluorescence spectra. The PEG PUC6 PEG micelles were prepared with the same method. γ-Radiation Induced Disassembly and Dox Release. Gy refers to the radiation energy absorbed by objects of a certain mass, 1 Gy = 1 J kg 1. The γ-ray irradiation was performed at Peking University Medical Center using a 60Co radiation source at a dose rate of 1.5 Gy min 1 (for the radiation of 500 Gy, the dose rate was 15 Gy min 1). Following irradiation, releasing measurements were conducted as soon as possible. For the release experiments, after exposure to γ-radiation of

Scheme 1. Schematic Illustration of the Disassembly and Drug Release of the Radiation-Sensitive PEG PUSeSe PEG Aggregates under Exposure to γ-Radiation

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different doses, 3 mL of Dox-loaded micellar solution, which was kept in a dialysis tube, was dialyzed against 30 mL of deionized water. The released percentage of Dox was estimated by comparing the remaining fluorescence to the original fluorescence.

Cytotoxicity Experiments for PEG PUSeSe PEG Aggregates. The human hepatocellular carcinoma cell line, HepG2, was purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. Cytotoxicity of PEG PUSeSe PEG aggregates was assessed by the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) assay. Cells were exposed to various doses of PEG PUSeSe PEG aggregates for 48 h at 37 °C. MTT indicator dye (5 mg mL 1) was added to cell wells, and the cells were incubated for 2 h in the dark. The supernatant was then removed, and 150 μL of dimethyl sulfoxide (DMSO) was added and incubated for 10 15 min to dissolve formazan crystal. Absorption was measured at 490 nm using an enzyme microplate reader (Biorad, Hercules, CA).

’ RESULTS AND DISCUSSION The diselenide-containing block co-polymer that we used here is the amphiphilic triblock co-polymer, PEG PUSeSe PEG. PUSeSe refers to the hydrophobic diselenide polyurethane block, and PEG refers to the hydrophilic poly(ethylene glycol) block. PEG PUSeSe PEG has proven to be able to form micellar aggregates in aqueous solution and load functional species in its micellar cores.5a To test if PEG PUSeSe PEG micellar aggregates are appropriate for the combination of radiotherapy and chemotherapy, Dox, an antitumor drug, was loaded into the aggregates. After adequate dialysis, unencapsulated Dox was removed and the final loading capacity was found to be 0.055 mg mL 1 (11 wt % of the block co-polymer). To investigate the radiation sensitivity of this system, the Doxloaded micellar solutions were exposed to γ-ray and then examined using TEM. The spherical micellar aggregates were observed before γ-radiation, which are shown in Figure 2a. These micellar aggregates maintained spherical structures but were slightly swollen after a radiation dose of 5 Gy, with an average diameter increase from 67 to 82 nm (Figure 2b). These results suggest that small doses of radiation do not destroy the micellar structures of the PEG PUSeSe PEG aggregates but may start to induce a more swollen state of the aggregates. When the radiation dose was increased to 50 Gy, the aggregates began to collapse into irregular aggregates. The effect of the radiation was confirmed by a stronger dose of 500 Gy; all of the spherical aggregates collapsed into irregular aggregates, indicating that the micellar structure has been completely destroyed by the radiation. A question arises whether the diselenide groups are essential for the high radiation sensitivity. To answer this question, we synthesized a triblock co-polymer PEG PUC6 PEG for the control experiment, which has a similar structure to PEG PUSeSe PEG but lacks active diselenide groups. PEG PUC6 PEG aggregates were prepared in aqueous solution in a procedure identical to that of PEG PUSeSe PEG. It was shown that the PEG PUC6 PEG aggregates were quite stable under γ-radiation, and there were no change on the size and structure of the aggregates after doses as high as 500 Gy, as shown in Figure 3. This controlled experiment clearly demonstrates that the radiation sensitivity of the PEG PUSeSe PEG aggregates can be ascribed to the introduction of the active diselenide groups in the triblock co-polymer.

Figure 1. Chemical structure of PEG PUC6 PEG. 5875

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Figure 2. TEM images of PEG PUSeSe PEG aggregates (a) before and after γ-radiation doses of (b) 5 Gy, (c) 50 Gy, and (d) 500 Gy.

Figure 3. TEM images of PEG PUC6 PEG aggregates (a) before and (b) after γ-radiation of 500 Gy.

To demonstrate that the PEG PUSeSe PEG aggregates can be used as a potential system for the combination radiotherapy and chemotherapy, we have loaded Dox molecules into the aggregates and studied its release properties induced by the γ-ray irradiation. The release of Dox from the PEG PUSeSe PEG aggregates was monitored by fluorescence measurements, and the released percentages were evaluated by the comparison of the fluorescence before and after dialysis. As seen from Figure 4a, the released percentage of Dox gradually increased with the dose of γ-radiation and was 77% when a 50 Gy radiation was applied. Figure 4b shows that the Dox molecules can be released from PEG PUSeSe PEG aggregates after γ-ray irradiation; the maximal release was reached at 8 h. As described in the Experimental Section, the Dox-loaded micellar aggregates were prepared by dialysis against deionized water to remove the unloaded Dox molecules, and thus, the aggregate showed good stability in pure water. As indicated in

Figure 4b, the loaded Dox cannot be easily diffused out without γ-ray irradiation. In other words, the drug release is induced by exposing aggregate solutions to γ-radiation. It should be noted that even with a small dose (5 Gy) of γ-radiation, the PEG PUSeSe PEG aggregates can still release about 45% of the loaded Dox molecules, although the spherical micellar structure maintains in the TEM results. This is important because this small dose of 5 Gy is close to the radiation dose that patients receive during a single radiotherapy treatment. Thus, this greatly enhances the possibility of biological and medical applications for these diselenide-containing block co-polymer aggregates. For comparison, the Dox-loaded PEG PUC6 PEG aggregates were stable to a large dose of radiation (500 Gy) and did not show any release of Dox from the aggregates during dialysis against deionized water after a 500 Gy radiation. These results indicate the significance of the diselenide groups in the aggregates for the radiation-sensitive structural change and release behaviors. 5876

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Figure 4. (a) Relationship between the release percentage of Dox and γ-radiation dose and (b) release plots of Dox from the PEG PUSeSe PEG aggregates under exposure to γ-radiation doses of 5 and 50 Gy, with plots of 0 Gy for reference. The time in this figure represents the releasing time after γ-radiation.

Figure 5. (a) ESR signals of the pure water and the PEG PUSeSe PEG aggregate solution under UV irradiation with trace TiO2 to detect the reaction between HO• radicals and diselenide block co-polymers. (b) FTIR analysis to the collected products after different doses of γ-radiation, in which the absorption band of seleninic acids at 880 cm 1 become stronger with increasing doses of γ-radiation.

To explain the mechanism behind the radiation-sensitive disassembly, we employed γ-rays to irradiate a PEG PUSeSe PEG co-polymer powder. Interestingly, we found that there was no obvious structural change to the diselenide block co-polymer after a γ-ray irradiation of 500 Gy, as revealed by NMR measurements. Therefore, it is reasonable to assume that the radiation sensitivity can be attributed to the aqueous environments of the PEG PUSeSe PEG aggregates used in our experiments. The radiation chemistry of water has been well-studied, and it is known that water can generate oxidative species, including HO•, •HO2, and H2O2, under exposure to radiation, which have concentrations proportional to the radiation doses.6 To confirm the reactions between the oxidative radicals and the PEG PUSeSe PEG copolymer, we designed a model system to generate HO• radicals in situ by UV light in the presence of TiO2 and detect the HO• signals through electronic spinning resonance (ESR) measurements.7 In the experiments, traces of TiO2 were added to the pure water and the PEG PUSeSe PEG aggregate solution, and the systems were irradiated in situ using UV lights. During this process, the HO• signals were recorded. From Figure 5a, it can be seen that the HO• radical signals were greatly weakened (∼60%) by the introduction of the PEG PUSeSe PEG aggregates, which indicated that the diselenide co-polymer absorbed the oxidative radicals and underwent a structural variation on the polymer backbone, resulting in the radiation-sensitive disassembly. Furthermore, because of the reductive nature of diselenide groups, the oxidative radicals are mostly combined by the diselenide co-polymer and the oxidation of loaded Dox has not been observed in our experiments. The mechanism of the radiation-sensitive disassembly was investigated further through FTIR analysis of the products from

PEG PUSeSe PEG aggregate solutions after varied doses of γ-radiation. The FTIR data in Figure 5b show that there was a new absorption band near 880 cm 1 created after irradiation, and its intensity increased with increasing radiation doses. Even at a very low dose of 5 Gy, the new absorption band appeared, suggesting that diselenide was partially destroyed, which helps to explain 45% release of Dox in the absence of the apparent structural change in the aggregates. The absorption band intensified for doses of 50 and 500 Gy. This absorption band, which has its peak at 880 cm 1, is attributed to a characteristic absorption of organic seleninic acids, which are common oxidation products of diselenide bonds.4a,8 After the results of the ESR are combined with FTIR investigations, we conclude that the diselenide groups have been partly oxidized into seleninic acids by the oxidative radicals generated during irradiation and the cleavage of diselenide bonds induces the partial disassembly of and drug release from the PEG PUSeSe PEG aggregates. We were curious about the role of oxygen in the solutions during irradiation. For this reason, we exposed a degassed solution to γ-radiation to investigate the influence of dissolved oxygen in the solution. It was found that the PEG PUSeSe PEG aggregates exhibit a lower release percentage (∼20% for 5 Gy and ∼39% for 50 Gy) and release rate than the normal aggregate solutions at the same radiation doses. When the releasing plots of 5 Gy are taken as an example, Figure 6a shows the different releasing behavior of PEG PUSeSe PEG aggregates with and without oxygen. These effects can be attributed to the absence of molecular oxygen, which greatly inhibits the generation of •HO2 radicals. Accordingly, most HO• radicals and H2O2 are formed during irradiation, and the concentration of the oxidative species is reduced in degassed solution, responsible for a lower release percentage of loaded Dox. 5877

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Figure 6. (a) Release behavior of Dox from the normal and degassed PEG PUSeSe PEG aggregate solutions under exposure to γ-radiation dose of 5 Gy. The time in this figure represents the releasing time after γ-radiation. (b) MTT cytotoxicity experiments of PEG PUSeSe PEG polymers of varied concentrations in aqueous solution. Pure PEG is used as a reference.

Although selenium is an essential micronutrient for humans, for a new potential drug release system, the cytotoxicity of the selenium-containing block co-polymer is still an important factor and needs to be evaluated.9 To address this problem, we used HepG2 cells to model the cytotoxicity of PEG PUSeSe PEG. From Figure 6b, it can be seen that, at high concentrations, such as 0.1 mg mL 1 and 0.01 mg mL 1, PEG PUSeSe PEG polymers had a slight inhibitory effect on the HepG2 cells, which still kept growing for 48 h after adding the polymers. At a relatively low concentration, our PEG PUSeSe PEG polymers showed little toxicity to the HepG2 cells. Interestingly, at a concentration of 1  10 4 mg mL 1, the growth of HepG2 cells was slightly accelerated, which may be related to the nutritive effects of the selenium element at low concentrations. The above results indicate that PEG PUSeSe PEG polymers may be further employed to develop new drug-delivery systems. In vivo drug-delivery studies are now underway.

’ CONCLUSION In summary, we have successfully developed a potential radiation-sensitive drug-delivery system using active diselenidecontaining block co-polymer aggregates in aqueous solution that can load and release anticancer drugs. These aggregates are responsive to a low dose of γ-radiation, such as 5 Gy, which is close to the radiation dose received by patients during a single radiotherapy treatment. We believe that selenium-containing polymers are a new kind of biomaterial, which open an avenue for the combination of radiotherapy and chemotherapy. ’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected] (H.X.); xi@mail. tsinghua.edu.cn (X.Z.).

’ ACKNOWLEDGMENT This work was financially supported by the National Basic Research Program (2007CB808000), the National Natural Science Foundation of China (21074066, 50973051, and 20974059), the Tsinghua University Initiative Scientific Research Program (2009THZ02-2), and the National Science Foundation of China (NSFC) German Research Foundation (DFG) Joint Grant (TRR 61).

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