Letter pubs.acs.org/macroletters
Facile Preparation and Radiotherapy Application of an Amphiphilic Block Copolymer Radiosensitizer Kaijie Zhao,† Wendong Ke,† Wei Yin,† Junjie Li,† Ming Qiang,*,‡ and Zhishen Ge*,† †
CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei 230026, Anhui China ‡ Department of Oncology, 105 Hospital of People’s Liberation Army, Hefei 230031, Anhui, China S Supporting Information *
ABSTRACT: Radiosensitizer plays an important role in the cancer radiotherapy for efficient killing of hypoxic cancer cells at a low radiation dose. However, the commercially available small molecular radiosensitizers show low efficiency due to poor bioavailability in tumor tissues. In this report, we develop a novel amphiphilic block copolymer radiosensitizer, metronidazole-conjugated poly(ethylene glycol)-b-poly(γ-propargyl-Lglutamate) (PEG-b-P(PLG-g-MN)), which can be selfassembled into core−shell micelles (MN-Micelle) with an optimal size of ∼60 nm in aqueous solution. In vitro cytotoxicity evaluation indicated that MN-Micelle sensitized the hypoxic cancer cells more efficiently under radiation with the sensitization enhancement ratio (SER) of 1.62 as compared with that of commercially available sodium glycididazole (GS; SER = 1.17) at the metronidazole-equivalent concentration of 180 μg/mL. Upon intravenous injection of MN-Micelle into the tumor-bearing mice, high tumor deposition was achieved, which finally suppressed tumor growth completely after electron beam radiation at a low radiation dose of 4 Gy. MN-Micelle with outstanding performance as an in vivo radiosensitizer holds great potentials for translation into radiotherapy application. and elevated levels of interstitial fluid pressure in tumor tissues leading to low bioavailability of hypoxic cancer cells.15−18 Therefore, more efficient and safer radiosensitizers are still desirable for maximizing radiotherapy and minimizing lesions to the normal cells. An important class of successful electron-affinic radiosensitizers, nitroimidazoles, attracted great attention.2,5,19 Early success of 2-nitroimidazoles (e.g., misonidazole) had activity in all solid murine tumor models.20,21 However, they were rarely used clinically due to their severe neurotoxicity. Notably, 5-nitroimidazole derivatives showed good performance as assessed in the clinical use. For example, nimorazole has been routinely used for the treatment of neck and head cancers in Demark.22,23 Sodium glycididazole (GS; Scheme S1) has been approved to treat neck and head cancers, esophageal cancer, or lung cancer in China.24−26 However, rapid drug metabolic rate and lower tumor accumulation, as well as the dense matrix structure and high interstitial fluid pressure in tumor tissues limit the bioavailability of radiosensitizers particularly in hypoxic cells which are frequently distant from tumor capillaries. Thus, further development of more efficient 5-nitroimidazole-based radiosensitizers may promote radio-
R
adiotherapy as one of most widely used tumor treatment modalities was used to treat more than 50% cancer patients around the world.1 During radiation therapy, the ionizing radiation, including X-ray, electron beam, and γ-ray, is locally applied to the tumor sites, which need to penetrate healthy tissues. The radiation with enough energy can kill tumor cells and simultaneously cause severe damage to the exposed healthy cells.2−4 Moreover, in the hypoxic tumor tissues, the tumor cells usually exhibit high resistance ability against radiation.2−6 Thus, radiosensitizers were frequently used to enhance the radiotherapy efficiency under a low radiation dose. The radiosensitizers usually work through two pathways. First, the nanoparticles with heavy metal atom in the tumor can concentrate the radiation energy within tumor tissue and increase the production efficiency of toxic reactive oxygen species.7−12 The safety concerns of the heavy metals limited the wide use of this method. Therefore, no related clinical translation was progressed based on this method. Another method is using the electron-affinic molecules as oxygenmimetic species which can fix the radiation damage to the cells, or using some molecules to reduce the repairing ability of the damaged cells.2,13,14 To date, the radiosensitizers clinically used or in the progress of clinical trials belong to this type. However, in vivo enhancement of radiotherapy efficacy based on small molecular radiosensitizers is limited particularly in the hypoxic tumors, which may be attributed to the dense matrix structure © XXXX American Chemical Society
Received: March 14, 2017 Accepted: May 2, 2017
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DOI: 10.1021/acsmacrolett.7b00196 ACS Macro Lett. 2017, 6, 556−560
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ACS Macro Letters therapy efficacy against the hypoxic tumors at a low irradiation dose. Nanoparticles with proper sizes and optimized surface properties usually showed preferential accumulation in tumor tissues via enhanced permeability and retention (EPR) effect.27−29 For construction of radiosensitizer nanoparticles, herein, we for the first time design and synthesize amphiphilic block copolymers, poly(ethylene glycol)-b-poly(γ-propargyl-Lglutamate) conjugating 5-nitroimidazole (PEG-b-P(PLG-gMN)) via click chemistry (Scheme 1A). The block copolymer Scheme 1. (A) Synthetic Routes Employed for the Preparation of the Amphiphilic Block Copolymer Radiosensitizer; (B) Schematic Illustration for Formation of Polymeric Radiosensitizer Micelles (MN-Micelle) and Application in Radiotherapy
Figure 1. (A) 1H NMR spectrum recorded for the amphiphilic block copolymer PEG-b-P(PLG-g-MN). (B) GPC traces obtained for the polymers amino group-terminated PEG (PEG-NH2, Mn = 5 kDa, Mn/ Mw = 1.06), PEG-b-PPLG (Mn = 17.7 kDa, Mn/Mw = 1.07), and PEGb-P(PLG-g-MN) (Mn = 29.5 kDa, Mn/Mw = 1.09).
shoulders in the positions of PEG-b-PPLG precursor or PEGNH2 initiator indicates well-defined structure of the final amphiphilic block copolymer (Figure 1B). To self-assemble into appropriate nanoparticles for in vivo radiotherapy application, the final block copolymer with degrees of polymerization (DPs) of 113 and 60 for PEG and P(PLG-gMN), respectively, was denoted as PEG113-b-P(PLG-g-MN)60 and used for the following evaluation. The amphiphilic block copolymer PEG113-b-P(PLG-g-MN)60 can self-assemble into core−shell micelles in aqueous solutions upon addition into water from organic solvent, which was denoted as MN-Micelle. First, we determined the critical micelle concentration (CMC) of the block copolymer using pyrene as a probe, which is as low as 9.0 × 10−3 mg/mL (Figure 2A). Next, we characterized the nanoparticle properties of MN-
with proper block lengths can be self-assembled into micelles with small size (∼60 nm) in aqueous solution, which were explored to be used as radiosensitizers with the advantages over conventional small molecular radiosensitizers including low toxicity, high radiosensitization efficiency, longer circulation in blood, and high tumor accumulation in tumor tissue (Scheme 1B). For preparation of the amphiphilic block copolymer, PEG-bP(PLG-g-MN), azide group-functionalized metronidazole (1(2-azidoethyl)-2-methyl-5-nitro-1H-imidazole) (MN-N3)30 and alkynyl moieties-containing PEG-b-poly(γ-propargyl-L-glutamate) (PEG-b-PPLG)31 were first synthesized according to the previously reported routes. Subsequently, efficient click reaction between PEG-b-PPLG and MN-N3 was performed (Scheme 1A). FT-IR analysis of PEG-b-P(PLG-g-MN) indicated that the signals of alkynyl and azide moieties at the wavenumber of ∼2100 cm−1 disappeared completely after click reaction (Figure S1). The quantitative conjugation was confirmed by 1H NMR analysis through comparing the integrals of methylene peaks adjacent to nitroimidazole moiety (h and g) and PEG peak (a; Figure 1A). GPC traces show a shift to higher molecular weight (MW) after conjugation of MN-N3. Low MW distribution (Mw/Mn = 1.09) without
Figure 2. (A) Fluorescence intensity ratios of pyrene excitation bands (I339/I332) as a function of the concentration of block copolymer PEGb-P(PLG-g-MN) in aqueous solution. Pyrene concentration was fixed at 5 × 10−7 M. The inflection points of the curves were taken as CMCs. (B) and (C) DLS and TEM characterization of MN-Micelle at the polymer concentration of 0.5 mg/mL. (D) Time-dependent size change of MN-Micelle in the presence of 150 mM NaCl or DMEM with 10% FBS. Mean ± s.d., n = 3. 557
DOI: 10.1021/acsmacrolett.7b00196 ACS Macro Lett. 2017, 6, 556−560
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ACS Macro Letters
significantly enhanced DNA damage after radiation. Further cytotoxicity evaluation under hypoxic condition revealed that electron beam radiation showed high cytotoxicity after treatment by MN-Micelle and GS when the metronidazoleequivalent concentrations were higher than 60 μg/mL at a 5 Gy dose (Figure 3C). Notably, both MN-Micelle and GS are nontoxic without radiation, regardless of hypoxic or normal cell culture conditions (Figures 3C and S3). Radiation dosedependent cell survival fractions can also be detected in the presence of radiosensitizers (Figure S4). MN-Micelle showed better radiosensitization ability with the sensitization enhancement ratio (SER) of 1.62 as compared to that of small-molecule GS (SER = 1.17) at the metronidazole-equivalent concentration of 180 μg/mL because MN-Micelle showed higher stability in aqueous solution and better cellular internalization. Under normoxic condition, no significant difference of cytotoxicity was observed upon addition of MN-Micelle or GS, which was likely ascribed to the fact that oxygen is the best radiosensitizer (Figures S4 and S5). To investigate the in vivo radiotherapy sensitization efficacy of MN-Micelle, we established murine hepatic carcinoma H22 tumor models. IVIS Lumina system was used to observe the biodistribution and deposition of MN-Micelle loading 1,1′dioctadecyl-3,3,3′,3′-tetramethylindo-tricarbocyanine iodide (DiR) in tumor tissue. As shown in Figure 4A, DiR-loaded
Micelle in aqueous solution. The size of MN-Micelle obtained from dynamic laser light scattering (DLS) is approximately 61 nm with relatively low particle size distribution (PDI 0.122; Figure 2B). Transmission electron microscope (TEM) images showed the average particle size of 41 nm which is smaller than that from the DLS result (Figure 2C). The inconsistence should be ascribed to the dry state of the TEM samples and low contrast of PEG shells. Moreover, stability of MN-Micelle was evaluated through monitoring the size changes of the micelles in the aqueous solution with 150 mM NaCl or in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fatal bovine serum (FBS; Figure 2D). The results revealed that MN-Micelle can maintain stable at least within 1 week. The physicochemical properties of MN-Micelle are favorable for the in vivo application via systemic administration. The low CMC value ensures that MN-Micelle can maintain micellar structures in highly diluted condition. The suitable particle size and high stability in salt or serum-containing solutions facilitate long blood circulation and uniform distribution in the tumor tissues especially in the hypoxic regions distant from tumor capillaries.32,33 Under hypoxic conditions, cancer cells exhibit high resistance ability against radiation and the addition of radiosensitizer would fix the damage of radiotherapy.2,5 Nile red-loading MNMicelle was first added into cell culture medium to study the uptake of HeLa cells (Figure S2). The strong fluorescence intensity inside cells indicated efficient cellular internalization of the micelles after incubation for 4 h. Next, we evaluated the capability of MN-Micelle to sensitize the radiotherapy by comparing with the commercially available GS. HeLa cells were cultured in AnaeroPack-Anaero for 24 h to mimic the hypoxic environment34 and then treated with phosphate buffered saline (PBS), GS, or MN-Micelle, followed by electron beam radiation at a 5 Gy dose. Comet assay was used to investigate the DNA damage of the cells (Figure 3A,B). PBS control group showed very little DNA damage indicating that HeLa cells are resistant against radiation under hypoxic condition. In sharp contrast, HeLa cells treated by GS or MN-Micelle exhibited
Figure 4. (A) IVIS imaging of DiR-loaded MN-Micelle in one representative mouse at different times postinjection. (B) Tumor growth curves upon electron beam radiotherapy at a 4 Gy dose on day 1, 4, and 7. Different formulations were injected before treatment by radiotherapy at the metronidazole-equivalent concentration of 1.4 mg/ mL. (C, D) Representative tumor images and average tumor weights collected from the mice at day 17 post treatment. Mean ± s.d., n = 5, *P < 0.05, ***P < 0.005 (t-test).
MN-micelle showed remarkable accumulation at 1 h postinjection. At 2 h, the tumors showed highest fluorescence intensity which can be maintained for at least 48 h without obvious decrease, suggesting that MN-Micelle can deposit and retain efficiently in tumor tissues for radiosensitization. To further evaluate the antitumor efficacy of MN-Micelle through radiosensitization, H22 tumor-bearing mice were treated by electron beam radiation at a 4 Gy dose for each treatment after systemic administration of radiosensitizers GS or MN-Micelle at the metronidazole-equivalent concentration
Figure 3. (A, B) Comet assay for HeLa cells after radiation of electron beam at a 5 Gy dose in the presence of various formulations (a: control, b: PBS + Radiation, c: GS + Radiation, d: MN-Micelle + Radiation) under hypoxic condition. Mean ± s.d., n = 10, **P < 0.01, ***P < 0.005 (t-test). (C) Cytotoxicity treated by GS or MN-Micelle at various metronidazole-equivalent concentrations under hypoxic condition with and without radiation at a 5 Gy dose. Mean ± s.d., n = 4. 558
DOI: 10.1021/acsmacrolett.7b00196 ACS Macro Lett. 2017, 6, 556−560
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of 1.4 mg/mL (Figure 4B). Notably, the radiotherapy was performed on days 1, 4, and 7, respectively. GS was intravenously injected within 1 h before each radiotherapy according to the manufacturer’s protocol while MN-Micelle solution was injected at 4 h before radiotherapy. The tumor growth curves revealed that the radiotherapy treated by MNMicelle significantly suppressed the tumor growth and the tumor sizes were reduced obviously at day 17. For PBS control and GS group, the tumor sizes showed a 12-fold and 7-fold increase after 17 days, respectively. Correspondingly, the tumor weights in mice treated by MN-Micelle and radiation were significantly lower than those in PBS and GS groups (Figure 4C,D). Moreover, no significant body weight loss and main organ toxicity were observed for mice treated with GS and MNMicelle (Figures S6 and S7). The excellent tumor growth suppression of MN-Micelle-sensitized radiation should be primarily owing to the optimized physicochemical properties suitable for deposition and distribution in tumor tissue as well as efficient radiosensitization capability to kill cancer cells. In summary, we prepared a novel amphiphilic block copolymer radiosensitizer, PEG-b-P(PLG-g-MN), which can be used to self-assemble into core−shell micelles in aqueous solution. The optimized physicochemical properties of the obtained micellar nanoparticles achieved high deposition and prolonged retention in tumor tissues. As compared with commercially available small molecular radiosensitizer GS, MNMicelle showed significantly improved radiosensitization capability, which achieved high-efficiency tumor growth suppression at a low radiation dose. These remarkable advantages endow MN-Micelle with great potentials to be translated into clinical applications as a radiosensitizer.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.7b00196. Materials, characterization methods, polymer synthesis, micelle preparation and characterization, in vitro cytotoxicity, body weight change, and H&E staining (PDF).
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Zhishen Ge: 0000-0002-2668-6974 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge financial support from National Natural Scientific Foundation of China (NNSFC) Project (21674104) and the Fundamental Research Funds for the Central Universities (WK3450000002).
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REFERENCES
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