Article pubs.acs.org/bc
Radiolytic Reduction Characteristics of Drug-Encapsulating DNA Aggregates Possessing Disulfide Bond Kazuhito Tanabe,* Takumi Asada, Takeo Ito, and Sei-ichi Nishimoto Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Katsura Campus, Nishikyo-ku, Kyoto 615-8510, Japan ABSTRACT: Radiation-responsive aggregates consisting of artificial DNA were demonstrated. We prepared DNA amphiphiles (DAMs) consisting of oligodeoxynucleotides (ODNs) as the hydrophilic part and an alkyl chain as the hydrophobic part, which were linked by a radiation-sensitive disulfide bond. DAMs assembled to form nanosized aggregates in aqueous solution that encapsulated hydrophobic dyes and drugs. X-irradiation, which triggered selective reduction at the disulfide bond in the DAMs, induced an exchange reaction, resulting in dissociation of the aggregates and release of encapsulated dyes and drugs. Among the DAMs synthesized in this study, DAM 1 possessing 5 mer ODNs showed favorable properties as a nanocarrier; for example, the large amount of drug was encapsulated in the aggregates consisting of DAM 1, and the aggregates showed the high sensitivity to radiolytic reduction. Biological experiments using living A549 cells revealed that the aggregates smoothly penetrated into the cells without the need for transfection reagents, while the drugs inside the aggregates were inert because of the encapsulation. X-irradiation enhanced the cytotoxicity because of dissociation of the aggregates and concomitant release of drugs.
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INTRODUCTION Nanosized DNA architectures have attracted much attention as medical materials. Although exogenous oligonucleotides, which are applicable to antisense and siRNA, are prone to enzymatic degradation and showed low cell permeability due to their own negative charges, recent studies revealed that aggregates comprised of oligonucleotides had different properties from individual oligonucleotides, showing nuclease resistance and enhanced cellular uptake.1−5 Therefore, aggregates of oligonucleotides are expected to be a new class of intelligent materials for gene targeting and manipulation without the need for conventional transfection reagents such as cationic polymers,6,7 liposomes,8 and viruses.9 Stimuli-responsive nanocarriers offer favorable properties for target-specific delivery of drugs.10,11 The carriers keep the toxic drugs encapsulated in their hydrophobic core, while the drugs can be readily released and exhibit their original toxicity once appropriate stimuli are applied. Thus, the drugs can be delivered to target areas highly selectively to maximize their potential and minimize side effects. Several stimuli including pH,12−14 temperature,15−18 redox reaction,19−21 and photoirradiation22−24 have been employed to activate nanocarriers and release drugs; however, precise control of the function and drug efficacy still remain major issues. Herein, we demonstrate a degradation of nanocarriers and release of drugs by means of hypoxic X-irradiation as an external stimulus. X-ray is an attractive trigger because the radiation reaction for activation of biomaterials can be regulated spatially and temporally without any additives. In addition, the high live-body permeability of X-ray is preferable for in vivo © 2012 American Chemical Society
drug targeting. Recently, we identified that radiolytic reduction of a disulfide bond located in oligodeoxynucleotides (ODNs) induced an exchange reaction of the strand at the disulfide linkage in a hypoxia-selective manner.25 Mechanistic studies revealed that reducing species such as hydrogen atoms and/or hydrated electrons (eaq−) generated from radiolysis of water molecules26,27 are most likely to be a key active species for the exchange reaction and that a chain reaction proceeded to form exchanged products with multiple turnovers. This reaction has been applied to the interstrand ligation of two flanking ODNs25 and intrastrand cyclization of stem-and-loop structured ODNs.28 These unique properties of DNA aggregates and the reaction of the disulfide bond motivated us to construct radiationresponsive drug carriers by means of ODNs possessing disulfide bond. We prepared nanocarriers consisting of DNA amphiphiles (DAMs), in which ODNs and an alkyl chain are employed as hydrophilic and hydrophobic parts, respectively, and these two parts were linked by a radiation-sensitive disulfide bond. In aqueous solution, DAMs self-assembled and formed nanosized aggregates which encapsulated hydrophobic dyes or antitumor drugs in their hydrophobic parts. The aggregates penetrated into living cells quite smoothly, and they did not show any significant cytotoxicity. The drugs in the aggregates were also inert because of the encapsulation. In contrast, aggregates containing drugs exhibited enhanced Received: June 5, 2012 Revised: July 23, 2012 Published: August 22, 2012 1909
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solution containing 2-methyl-2-propanol (1.8 mM) and phosphate Na buffer (100 mM, pH 7.0) as described above. The resulting solutions were purged with argon for 10 min and then irradiated in a sealed glass ampule at ambient temperature with an X-ray source (6.0 Gy min−1). Before or after the irradiation, the solution was subjected to fluorescence measurements. Just before the measurements of fluorescence, the solutions were filtered through a 0.45 μm filter. Assessment of Cytotoxicity toward A549 Cells. A549 cells were cultured in Dulbecco’s Modified Eagle’s Minimum Essential Medium (DMEM) containing 10% fetal bovine serum (FBS). The cells were seeded into 96-well plates (2000 cells/ well) and cultured at 37 °C in a well-humidified incubator with 5% CO2 and 95% air (aerobic condition) for 24 h. The cells were then incubated with the various concentrations of DAM 1 under aerobic conditions for 2 h. After the incubation, the medium containing DAM 1 was replaced by the fresh one, and the cells were further incubated for 48 h at 37 °C. After incubation, WST 8 was added to the cells and the cell viability assay was performed using a Microplate Reader (BIO-RAD). Assessment of Cellular Penetration Using DAM 1 Aggregates Encapsulating Nile Red. To form the aggregate, DAM 1 (100 μM) in aqueous solution was added to nile red (1 μM) in acetonitrile. After the removal of the solvent in vacuo, the resulting mixture was dissolved in DMEM. A549 cells were seeded into 96-well plates (10000 cells/well) and incubated at 37 °C for 24 h. The cells were then incubated with the DAM 1 aggregate encapsulating nile red under hypoxic conditions for 2 h. After the incubation, the medium containing DAM 1 was replaced by phosphate buffer and images were taken by microscope. (images under hypoxic conditions). The images were acquired with a microscope system (BZ-8000, KEYENCE) equipped with 560 nm excitation filter, 595 nm dichroic mirror, and 630 nm detection filter. A detector of the microscope was a CCD camera. Radiation-Induced Cytotoxicity of DAM 1 Aggregate Encapsulating Camptothecin. To form the aggregate, DAM 1 (100 μM) in phosphate buffer (100 mM, pH 7.0) was added to camptothecin (200 nM) in acetonitrile. After the removal of the solvent in vacuo, the resulting mixture was dissolved in water to assess the radiation-induced cytotoxic effect. A549 cells were seeded into 96-well plates (2000 cells/well) and incubated at 37 °C for 24 h under hypoxic conditions (0.3% O2). For the hypoxic treatment, the cells were treated in a hypoxic chamber, Invivo2 400 (Ruskinn). The cells were then incubated with the DAM 1 aggregate encapsulating camptothecin under hypoxic conditions for 2 h. After incubation, the medium containing DAM 1 was replaced by the fresh one, and the cells were X-irradiated at a dose of 6 and 9 Gy. When Xirradiation was conducted, the plates were kept under hypoxic conditions using an Anaeron pack system (Mitsubishi Gas Chemical Company Inc.). After irradiation, the cells were incubated for 48 h under aerobic conditions. Then, the cell viability assay was performed as described above.
cytotoxicity upon hypoxic X-irradiation due to degradation of the aggregates and release of the drugs.
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EXPERIMENTAL PROCEDURES General Procedures. All starting materials and reagents were purchased from Tokyo Kasei Kogyo (Tokyo, Japan), Wako (Tokyo, Japan), and Aldrich Chemical (Milwaukee, WI). All other solvents, purchased from Wako, were GR grade or dry grade and used without further purification. The ESI-MS spectra were recorded on a Exactive (Thermo) spectrometer. The organic reactions were carried out in oven-dried glassware under an argon atmosphere with magnetic stirring. Fluorescence spectra were recorded on a Shimadzu RF-5300PC spectrofluorophotometer with a 1 cm quartz cell. Hypoxic cell culture conditions were made by means of Russkin Invivo 400. DAMs were synthesized by automated DNA synthesis using Applied Biosystems model 3400 DNA/RNA synthesizer. Syntheses of DAMs. Octadecanol (43.3 mg, 160 μM) was dissolved in dry CH2Cl2 (1 mL), and diisopropylethylamine (41.4 mg, 320 μmol) and N,N′-diisopropylmethylphosphonamidic chloride (39.5 mg, 200 μmol) were added to the solution. The resulting mixture was stirred at ambient temperature for 2 h. After the reaction, the mixture was filtered off and used for automated DNA synthesis without further purification. For the preparation of DAM 1−4, aqueous NH3 solution was used for the cleavage of ODNs from support. On the other hand, K2CO3 in MeOH was used for the cleavage of DAM 5 from support. Synthesized DAMs were purified by reversed phase HPLC to give DAM 1−5. The purity and concentration of the DAMs were determined by complete digestion by AP, P1, and phosphodiesterase I at 37 °C for 12 h. Identities of synthesized ODNs were confirmed by ESI-TOF mass spectrometry (DAM 1: calcd 1065.30, found [M − 2H]2− 1065.31. DAM 2: calcd 1825.89, found [M − 2H]2− 1825.43. DAM 3: calcd 2230.56, found [M − 3H]3− 2230.11. DAM 4: calcd 1945.76, found [M − 5H]5− 1946.66. DAM 5: calcd 1058.31, found [M − 2H]2− 1058.26. Measurement of Fluorescence Spectra. To form the aggregate, indicated concentrations of DAMs in phosphate buffer (100 mM, pH 7.0) were added to nile red or pyrene (10 μM) in acetonitrile. After the removal of the solvent in vacuo, the resulting mixture was dissolved in water to form the aggregate and measure the fluorescence spectra. We measured the fluorescence spectra by means of the excitation wavelength at 580 and 340 nm for nile red and pyrene, respectively. Mesurement of Dynamic Light Scattering (DLS). The size distribution of aggregates was determined by Nano series Nano-ZS Zetasizer (Malvern Instrument). The aggregates (DAM 1, 60 μM; DAM 2, 30 μM; DAM 3, 30 μM; DAM 4, 30 μM) were prepared in aqueous solution containing phosphate Na buffer (100 mM, pH 7.0) as described above. After the filtration using 0.45 μm filter, the measurements of DLS were conducted at 25 °C. Radiolytic Reduction of DAMs. To establish hypoxia, aqueous solutions of DAMs (30−60 μM) in 100 mM phosphate Na buffer (pH 7.0) containing 2-methyl-2-propanol (50 eq. to DAM) were purged with argon for 10 min and then irradiated in a sealed glass ampule at ambient temperature with an X-ray source (6.0 Gy min−1). After the irradiation, the solution was immediately subjected to HPLC analysis. Radiolytic Reduction of the Aggregates Encapsulating Pyrene Dye. Aggregates of DAM 1 (36 μM) encapsulating pyrene dye (10 μM) were formed in aqueous
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RESULTS AND DISCUSSION DAMs possessing various lengths of ODNs, and alkyl chains were prepared by automated DNA synthesis using the conventional phosphoramidite method. The crude DAMs were purified by reversed-phase HPLC, and incorporation of the alkyl chain into the ODN was confirmed by enzymatic digestion and ESI-TOF mass spectrometry. We employed simple oligothymidines as hydrophilic parts to avoid 1910
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Scheme 1. Syntheses and Structures of DNA Amphiphiles (DAMs) Used in This Studya
a
Reagents and conditions: (a) N,N-diisopropylmethylphosphonamidic chloride, diisopropylethylamine, CH2Cl2; (b) automated DNA synthesis.
Figure 1. Formation of aggregates from DAM 1 and encapsulation of nile red. (A) Fluorescence spectra of nile red (10 μM) in aqueous solution containing phosphate Na buffer (100 mM, pH 7.0) in the presence of DAM 1 (20 μM, black), octadecane (20 μM, red), or oligodeoxynucleotides dT5 (20 μM, green). The fluorescence spectra were measured at 580 nm excitation. (B) Plot of fluorescence intensity of nile red vs concentration of DAM 1 to determine critical aggregation concentration (CAC).
complicated function of other bases and base sequence in aggregate formation and radiolytic reduction. The DAMs used in this study are summarized in Scheme 1. We initially measured the fluorescence emission of nile red in the presence of DAMs to characterize their aggregation properties in aqueous solution. Nile red is an intrinsically water-insoluble fluorescent dye, and therefore it shows no fluorescence in aqueous suspension. On the other hand, encapsulation of nile red into the aggregate consisting of amphiphilic molecules to dissolve the dyes results in a fluorescence emission even in aqueous solution.11 Thus, we evaluated the aggregation of DAMs by monitoring the fluorescence emission. As shown in Figure 1, we observed strong fluorescence of nile red around 630 nm in the presence of DAM 1. The evidence that addition of corresponding ODN units (dT5) or alkane (octadecane) to nile red did not enhance the fluorescence emission indicates that DAM 1 formed aggregates in aqueous solution to encapsulate hydrophilic nile red inside its hydrophobic part. All of the other DAMs showed similar behavior in the fluorescence emission. We next estimated the critical aggregation concentration (CAC) of DAMs by monitoring the fluorescence emission with increasing concentration of DAMs (Figure 1B). We observed a sudden increase in fluorescence intensity in the concentration range 2.3−8.4 μM, which was assigned as the CAC of each DAM. Among the DAMs possessing a phosphotriester group at hydrophobic parts (DAM 1−4), DAMs possessing a long ODN chain showed relatively small CAC values, while DAMs possessing a short ODN had high CAC values (Table 1). On the other hand, the CAC of DAM 5, which had phosphodiester group at a hydrophobic part, was estimated to be 33.8 μM. These results indicate that balance between hydrophilicity and hydrophobicity in DAMs is responsible for the formation of stable aggregates. We further verified the formation of
Table 1. Critical Aggregation Concentrations (CAC) of DAMs CAC (μM)
DAM 1
DAM 2
DAM 3
DAM 4
DAM 5
8.4
4.2
2.3
2.5
33.8
aggregates from DAMs 1−4 and measured their size in aqueous solution by means of dynamic light scattering (DLS). Figure 2 shows representative spectra of DAMs in 100 mM phosphate buffer (pH 7.0). The formation of two sizes of aggregate was observed for each DAM. The DAMs possessing shorter ODNs as a hydrophilic part predominantly formed
Figure 2. Size of aggregates consisted of DAMs and encapsulation of nile red by larger aggregates. (A) Size of aggregates consisting of DAM 1 (60 μM, black), DAM 2 (30 μM, blue), DAM 3 (30 μM, green), and DAM 4 (30 μM, red). DLS experiments were conducted using aqueous solution of DAMs containing phosphate Na buffer (100 mM, pH 7.0). (B) Fluorescence spectra of nile red (10 μM) in aqueous solution containing phosphate Na buffer (100 mM, pH 7.0) in the presence of DAM 1 (20 μM) before (black) or after (red) filtration using 100 nm filter. The fluorescence spectra were measured at 580 nm excitation. 1911
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Figure 3. Radiolytic reduction of aqueous solution of DAMs under hypoxic conditions. (A) HPLC profiles for the reaction of DAM 1 (60 μM) in the hypoxic X-ray radiolysis of phosphate Na buffer solution (100 mM, pH 7.0) containing 2-methyl-2-propanol (3 mM) at ambient temperature. (B) X-ray irradiation of DAM 1 to form DAM 1a via strand exchange reaction. (C) G values for the decomposition of DAMs in the X-ray radiolysis.
large aggregates with a diameter of ca. 110 nm, while the main aggregates consisting of DAMs with long ODNs formed small aggregates with a diameter of ca. 15 nm. It is well-known that the sizes of the hydrophobic part and the hydrophilic part have potent influence on the size and shapes of the aggregates.29 Although the correct forms of DAM aggregates are unclear at present, it is most likely that the chain length of ODNs significantly affects the size of the aggregates. To confirm which size of aggregate contributes to the encapsulation of nile red, we measured the fluorescence spectra of aggregates consisting of DAM 1 after filtration using a filter with 100 nm pore size. As shown in Figure 2B, a marked decrease in the fluorescence emission was observed for the filtered sample, indicating that encapsulation of nile red is attributed to the larger aggregates. We also confirmed that the amount of nile red encapsulated by DAM 1 and DAM 2 was larger than that for DAM 3 and DAM 4, which is consistent with the size of aggregates formed in the aqueous solution. To characterize the reaction properties, we performed Xradiolytic reduction of DAMs 1−4 in argon-purged aqueous solutions containing 2-methyl-2-propanol. The scavenger 2methyl-2-propanol was added to prevent possible side reactions derived by oxidizing hydroxyl radicals (OH•), which are one of the active species generated by the radiolysis of water.26 Thus, reducing hydrated electrons (eaq−) and hydrogen atoms (H•) act as the major active species under these radiolysis conditions. Figure 3A shows a representative profile for the radiolytic reduction of DAM 1. Hypoxic irradiation of DAM 1 produced a single new product, DAM 1a, the yield of which increased as a function of increasing radiation dose. To identify the product, we purified DAM 1a and measured the mass spectrum. The molecular weight of the product was identical to the 10 mer ODNs possessing a disulfide bond, indicating that hypoxic Xirradiation of DAM 1 induced a change of chemical structure via intramolecular exchange of disulfide. The G values were estimated to be 258 nmol/J for the decomposition of DAM 1. Similar reductive exchange reactions upon hypoxic X-irradiation were confirmed for DAM 2, DAM 3, and DAM 4 with longer ODNs, although their reaction efficiencies were lower than that of DAM 1 because of the lower accessibility of reducing species to the target disulfides in DAMs 2−4, which showed increased
electrostatic screening effects by negative charges on the ODN phosphate units (Figure 3C). In this study, we developed a series of radiolytically degradable amphiphiles consisting of ODNs and alkyl chains. Of these, DAM 1 possessing 5 mer ODNs seems to have favorable properties for the construction of radiation-activated nanocarriers because DAM 1 formed the largest aggregate to encapsulate nile red as a phantom drug and showed the highest reaction efficiency upon hypoxic X-irradiation. In light of these observations, we next characterized the basic properties of DAM 1 aggregates against living cells and their ability to release drugs under irradiation conditions to apply DAM 1 aggregates as a radiation-activated drug carrier. We then evaluated the radiolytic reduction of DAM 1 aggregates, which encapsulated a fluorescent dye. We used pyrene as a phantom drug instead of nile red because nile red was unstable during hypoxic Xirradiation. As shown in Figure 4, strong fluorescence of pyrene was observed when DAM 1 was added to the aqueous solution. Thus, pyrene dye was encapsulated in the DAM 1 aggregates. In contrast, X-irradiation of the aggregates under hypoxic conditions led to a significant decrease in fluorescence, indicating that DAM 1 underwent an exchange reaction of disulfide to form DAM 1a, and thereby the encapsulated pyrene
Figure 4. Radiolytic reduction of DAM 1 aggregate encapsulating pyrene. Fluorescence spectra of pyrene (10 μM) in the presence of DAM 1 (36 μM) in phosphate Na buffer solution (100 mM, pH 7.0) containing 2-methyl-2-propanol (1.5 mM). The spectra were measured after hypoxic X-irradiation (0 Gy, solid line; 360 Gy, dotted line). 1912
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Figure 5. Biological experiments of DAM 1 aggregates encapsulating CPT using A549 cells. (A) Cytotoxicity of DAM 1 against A549 cells. A549 cells were incubated with indicated concentrations of DAM 1 under aerobic conditions for 48 h. To calculate the cell viability in each condition, WST 8 counts (OD450) for each concentration of DAM 1 were compared to those in minimal concentration. Results are shown with the mean ± SD (n = 5). (B) Emission images of A549 cells as incubated with DAM 1 aggregates (100 μM) encapsulating nile red (1 μM). The cells were incubated with the aggregate for 2 h at 37 °C, and then the medium was replaced by PBS. After the replacement, the images were immediately taken by means of microscopy (excitation at 560 nm and emission at 630 nm). (C) Radiation-induced cyototoxicity of CPT encapsulated in DAM 1 aggregate against A549 cells under hypoxic conditions. A549 cells were cultured in the presence (+) or absence (−) of DAM 1 (100 μM) and CPT (200 nM) and treated with X-ray (0, 6, or 9 Gy) under hypoxic conditions. Results are shown with the mean ± SD (n = 4).
X-irradiation of the cells, to which CPT-encapsulating aggregates were administered, resulted in a significant decrease in cell viability, although A549 cells were practically resistant to 9 Gy radiation under hypoxic conditions in the presence of empty aggregates. We also confirmed that a lower dose of 6 Gy led to a decrease in cell survival rate (sample 4). These results strongly suggest that DAM 1 aggregates preferentially release toxic CPT via radiolytic reduction in hypoxic cells and thereby lead to enhanced cytotoxicity, consistent with the chemical reactivity upon X-irradiation. Thus, it is reasonably concluded that DAM 1 aggregates act as radiation-degradable drug carriers.
was released from the aggregates, the structure of which could not be maintained under radiation conditions. The amount of the released pyrene during X-irradiation up to 360 Gy was estimated to be 293 nM according to the relationships between fluorescence intensity and concentration of pyrene. On the basis of the above reaction characteristics, an attempt was also made to demonstrate the radiolytic onset of drug potency using a human cell line of lung carcinoma A549 and an antitumor agent, camptothecin (CPT), as an encapsulated drug. Before an evaluating the radiolysis of the aggregates in living cells, we assessed the cytotoxic properties and cellular penetration of DAM 1. For the assessment of cytotoxicity, A549 cells were cultured for 48 h in the presence of various concentrations of DAM 1 and were subsequently subjected to a cell viability assay (Figure 5A). We confirmed that almost all of the cells had a high survival rate even in the presence of DAM 1 up to 200 μM, suggesting that DAM 1 had low cytotoxicity in this concentration range. The cellular penetration of DAM 1 aggregates containing nile red as a phantom drug was also evaluated. As shown in Figure 5B, clear fluorescence of nile red was observed from the cytoplasm of A549 cells when the cells were incubated with aggregates for 2 h. Thus, DAM 1 aggregate smoothly penetrated into the cells. We subsequently investigated the radiation-dependent cytotoxic effect of drug-encapsulating DAM 1 aggregates. After administration of CPT-containing DAM 1 aggregates to A549 cells and incubation for 2 h to allow penetration into the cells, the medium was replaced with fresh medium to avoid undesirable activation of aggregates outside the cells. We then exposed A549 cells to X-radiolysis under hypoxic conditions. As shown in Figure 5C, a slight cytotoxic effect in the presence of 200 nM CPT, which was encapsulated in the aggregate, was observed under the conditions without X-irradiation (sample 2). However, given that the IC50 value of CPT against A549 cells is 59.4 nM, it is suggested that encapsulation of drugs by DAM 1 aggregates effectively suppressed the onset of the effect of CPT and that collapse of the aggregates to release CPT in reductive cytoplasm of the cells was negligible. It is striking that
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CONCLUSION In conclusion, we have synthesized a series of DNA amphiphiles, DAMs, as radiation-activated drug carriers. The DAMs had an alkyl chain as the hydrophobic unit and ODNs as the hydrophilic unit, and these two functional units were connected by an X-ray-sensitive disulfide bond. The DAMs selfassembled to form nanosized aggregates, which encapsulated hydrophobic dyes and active drugs in their hydrophobic parts. Among the amphiphiles, DAM 1 possessing 5 mer ODNs showed good properties for drug encapsulation and radiolytic activation, and thereby we characterized the radiolytic release of an antitumor agent, camptothecin (CPT), which was encapsulated in the aggregates consisting of DAM 1. Xirradiation of A549 cells, to which DAM 1 aggregates with CPT were administered, resulted in an enhancement of cytotoxicity because of the radiolytic release of CPT from the aggregates. Thus, DAM 1 aggregates are promising as radiation-activated nanocarriers that are applicable to drug delivery and targeting. In vivo experiments to characterize the performance of the DAM 1 aggregates are in progress.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +81-75-383-2505. Fax: +81-75-383-2504. E-mail:
[email protected]. 1913
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Notes
(19) Takae, S., Miyata, K., Oba, M., Ishii, T., Nishiyama, N., Itaka, K., Yamasaki, Y., Koyama, H., and Kataoka, K. (2008) PEG-Detachable polyplex micelles based on disulfide-linked block catiomers as bioresponsive nonviral gene vectors. J. Am. Chem. Soc. 130, 6001− 6009. (20) Stevenson, M., Ramos-Perez, V., Singh, S., Soliman, M., Preece, J. A., Briggs, S. S., Read, M. L., and Seymour, L. W. (2008) Delivery of siRNA mediated by histidine-containing reducible polycations. J. Controlled Release 130, 46−56. (21) Napoli, A., Valentini, M., Tirelli, N., Muller, M., and Hubbell, J. A. (2004) Oxidation-responsive polymeric vesicles. Nature Mater. 3, 183−189. (22) Nishiyama, N., Iriyama, A., Jang, W.-D., Miyata, K., Itaka, K., Inoue, Y., Takahashi, H., Yanagi, Y., Tamaki, Y, Koyama, H., and Kataoka, K. (2005) Light-induced gene transfer from packaged DNA enveloped in a dendrimeric photosensitizer. Nature Mater. 4, 934−941. (23) Goodwin, A. P., Mynar, J. L., Ma, Y., Fleming, G. R., and Fréchet, J. M. J. (2005) Synthetic micelle sensitive to IR light via a two-photon process. J. Am. Chem. Soc. 127, 9952−9953. (24) Jiang, J., Tong, X., and Zhao, Y. (2005) A new design for lightbreakable polymer micelles. J. Am. Chem. Soc. 127, 8290−8291. (25) Tanabe, K., Kuraseko, E., Yamamoto, Y., and Nishimoto, S. (2008) One-electron reductive template-directed ligation of oligodeoxynucleotides possessing disulfide bond. J. Am. Chem. Soc. 130, 6302−6303. (26) Buxton, G. V., Greenstock, C. L., Helman, W. P., and Ross, A. B. (1988) Critical review of rate constants for reactions of hydrated electrons, hydrogen atoms and hydroxyl radicals in aqueous solution. J. Phys. Chem. Ref. Data 17, 513−886. (27) Spinks, J. W. T., Woods, R. J. (1990) An Introduction to Radiation Chemistry, Wiley-Interscience, New York. (28) Tanabe, K., Matsumoto, E., Ito, T., and Nishimoto, S. (2010) Radiolytic cyclization of stem-and-loop structured oligodeoxynucleotide with neighboring arrangement of α,ω-bis-disulfides. Org. Biomol. Chem. 8, 4837−4842. (29) van Hest, J. C. M., Delnoye, D. A. P., Baars, M. W. P. L., van Genderen, M. H. P., and Meijer, E. W. (1995) Polystyrene− Dendrimer amphiphilic block copolymers with a generation-dependent aggregation. Science 268, 1592−1595.
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was partly supported by the Strategic Promotion Program for Basic Nuclear Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
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