Letter www.acsami.org
Diselenide-Containing Hyperbranched Polymer with Light-Induced Cytotoxicity Chenxing Sun,† Shaobo Ji,† Feng Li,* and Huaping Xu* Key Laboratory of Organic Optoelectronics and Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084, China S Supporting Information *
ABSTRACT: A light-induced cytotoxicity system was fabricated using active diselenide/porphyrin-containing hyperbranched polymer aggregates in aqueous solution through emulsification. When the nanoparticles were irradiated with visible light, 1O2 was produced by the porphyrin photosensitizers in the system, which cleaved the diselenide bonds in the polymer chains and disassembled the nanosystem. Interestingly, the oxidized products exhibited cytotoxicity to the MDA-MB 231cell line without using extra anticancer drugs, which endowed the system with potential visible light-induced antitumor activity. In combination with photodynamic therapy, it is greatly anticipated that better anticancer efficacy can be achieved with this system. KEYWORDS: diselenide, hyperbranched polymer, singlet oxygen, chemotherapy, stimuli response
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To reduce the quenching of 1O2 and enhance diselenide bond oxidation after light irradiation, we herein synthesized a selenium/porphyrin-containing hyperbranched polymer (PSePor) using stepwise polymerization to build a light-responsive self-cleaving system. This polymer had a good response to visible light and could highly improve the use of singlet oxygen. After irradiation, the diselenide bonds could be oxidized to seleninic acid. Beyond expectations, the produced seleninic acid possessed anticancer activity. This work presents a lightinduced-cytotoxicity system that is a potential platform for cancer therapy (Scheme 1). A diselenide-containing amphiphilic hyperbranched polymer was designed and synthesized. Meso-tetra(p-hydroxyphenyl) porphine, a porphyrin derivative, and diselenide-containing 11,11′-diselanediylbis(undecan-1-ol) were utilized as the monomers. In a solution of tetrahydrofuran (THF), they were polymerized with a slight excess of toluene-2,4diisocyanate (TDI) and poly(ethylene glycol) (PEG) monomethyl ether (Mw = 5000) was added as terminating groups ends (Scheme 2). The Mw value of PSe-Por was measured using gel permeation chromatograph (GPC) in a N,Ndimethylformamide (DMF) solution (Mw = 4.7 × 104 g/mol, Mn = 3.3 × 104 g/mol, PDI = 1.42, Figure S2b). The total concentration of porphyrin in the polymer was determined to be 18.6 μg/mg from a fluorescent method, as shown in Figure S2c.
hemotherapy plays an important role in cancer treatment. Numerous controlled drug delivery nanosystems have been explored to overcome the limitations of traditional chemotherapic drugs, including poor water solubility and systematic toxicity. Among them, benefiting from various selfassembled structures, stimuli-responsive polymers have been extensively investigated and developed.1−5 Stimuli-responsive polymers are designed to change their physical or chemical properties when triggered by external stimuli, such as temperature, 6−9 pH, 10−13 redox conditions, 14−16 and light.17−23 However, the drug loading capacities of most stimuli-responsive polymer nanocarriers and contribution of the nanocarriers to the overall therapy efficacy need to be improved. Therefore, it is of significance to design stimuliresponsive polymers that itself can independently exhibit anticancer efficacy when stimuli are introduced. Organic selenium compounds have unique redox properties, and, thus, can be excellent candidates for stimuli-responsive materials.24−26 Diselenide bonds have been used as functional groups in drug delivery systems that can, respond to both oxidation and reduction. Singlet oxygen (1O2) can be produced by photosensitizers, such as porphyrin derivatives and indocyanine green. In addition, 1O2 has been widely applied for constructing light-responsive materials due to its low systemic toxicity. In our previous work, we found that diselenide-containing polymers and compounds were sensitive to 1O2, which could be produced by porphyrin derivatives when irradiated with visible light.27,28 However, in that work, the singlet oxygen had to diffuse into the nanoparticles before reaching the diselenide bonds, which hindered its utilization efficiency. © XXXX American Chemical Society
Received: February 17, 2017 Accepted: April 5, 2017 Published: April 5, 2017 A
DOI: 10.1021/acsami.7b02367 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
Scheme 1. Schematic Illustration of the Formation of the PSe-Por Micelles; After Light Irradiation, the Oxidization Product has Anticancer Activity
Scheme 2. Synthetic route for selenium/porphyrin-containing hyperbranched polymers
Figure 1. Ability of the polymer solution to produce 1O2. (a) SOSG regent detection. When irradiated with visible light, the nanoparticles solution could produce 1O2 to oxidize the SOSG reagents, followed by the enhancement of the fluorescence intensity, while the dark control group remained almost unchanged. (b) ESR results. Under visible-light irradiation, singlet oxygen signal increased with the increase in the illumination time.
solution, which was consistent with the results from DLS (Figure S 3d). In our previous work, we proved that diselenide bonds were sensitive to singlet oxygen, which could be produced using photochemistry during photodynamic therapy. Here, we used meso-tetra(p-hydroxyphenyl) porphine as a singlet oxygen photo generator to produce 1O2 under visible light irradiation. The ability of PSe-Por, which contains porphyrin groups in the polymer chain, to produce 1O2 was tested. Singlet Oxygen Sensor Green (SOSG, excitation/emission 488 nm/525 nm)
Emulsification method was used to prepare the PSe-Por nanoparticles in aqueous solution with poly(vinyl alcohol) (PVA) as the stabilizer (Figure S3a). Dynamic laser light scattering (DLS) and transmission electron microscopy (TEM) were employed to characterize the size distribution and the morphology of the nanoaprticles, respectively. The size distribution of the aggregates with a core−shell structure was approximately 308 nm, as shown in Figure S3b, c. TEM images showed that the PSe-Por formed spherical micelles in aqueous B
DOI: 10.1021/acsami.7b02367 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
Figure 2. Morphology and chemical changes of the nanoparticles before and after light irradiation. (a) Diameter distribution changes. TEM images of the PSe-Por aggregates (b) before and (c) after light irradiation for 1 h. (d) XPS results of the nanosystem before and after light irradiation showing changes in the diselenide bonds. The binding energy peak of Se 3d shifted from 55.8 to 58.9 eV, indicating oxidation of the diselenide bonds. (e) FTIR spectra showing the existence of a new peak at approximately 872 cm−1 attributed to the vibrations of Se−O, indicating that the oxidization product might be selenite.
reagent was selected as the probe to detect 1O2, which can be easily oxidized by 1O2 and subsequently enhances the fluorescence intensity. SOSG reagent was first added into the PSe-Por nanoparticle solution with a concentration of 2.5 μM. Then, the mixture was irradiated with white light (5000 lux) for a certain time. After that, the fluorescence intensity of the nanosystem was recorded. As shown in Figure 1a, the fluorescence intensity increased with the extension of the irradiation time in the experimental group, while the dark control basically remained unchanged. 1O2 is a kind of oxygen radical and could also be detected using electron spinresonance spectroscopy (ESR). ESR measurements were taken under visible light (400−800 nm) irradiation. The results showed enhanced 1O2 signals as the irradiation time increased (Figure 1b). Under visible-light irradiation, the polymer could produce 1O2 continuously. To further investigate the light-response behavior of the nanosystem after 1O2 generation, we exposed the nanoparticles solutions to visible light (xenon lamp, 10 A, wavelength >420 nm) for different time and then examined using DLS and TEM. To eliminate the influence of temperature, the experiments were performed in an isothermal water bath. As shown in Figure 2a, the average diameter changed from 308 to 220 nm after 1 h of irradiation, indicating that the nanoparticles partially dissociated. Further study of the hydrodynamic particle size values (Rh) of the nanoparticles upon light exposure indicated that secondary self-assembly of particles should occur in our experiments (Figure S3e). The TEM was also employed to characterize the morphology change of the nanoparticles. After light irradiation, the nanoparticles disassembled, and during
drying for TEM sample preparation, parts of nanoparticles adhered with each other to form irregular aggregates (Figure 2b, c). We predicted that cleavage of the diselenide bonds might be the reason for the above morphology change. To confirm this assumption, was employed GPC to characterize the molecular weight of the hyperbranched polymer. After visible-light irradiation for 1 h, the peak corresponding to the polymer decreased (Figure S3f). This result suggested that most of the polymers were degraded into low-molecular-weight products after light irradiation, thus breaking the nanoparticles. The detailed chemical changes after visible light irradiation were then investigated. The degradation of the polymer was attributed to the oxidative cleavage of diselenide bonds in the presence of 1O2. To characterize the product after oxidization, XPS and FTIR were employed. As shown in Figure 2d, a new peak for the Se 3d binding energy appeared at 58.9 eV, whereas part of the binding energy remained at 55.8 eV. According to the XPS handbook,29 55.8 eV was close to the peak for diselenide bonds and 58.9 eV was close to the R-SeO(OH) peak. This shift indicated partial oxidization of the diselenide bonds in the polymer and oxidative products should be seleninic acid. Further, the aggregates before and after oxidization were processed using freeze-drying and then characterized using FTIR spectroscopy. According to our previous study, for seleninic acid, a peak belonging to the vibration of Se−O should have appear at approximately 870− 880 cm−1. From our results, after 40 min of light irradiation, a new peak appeared at approximately 872 cm−1, which further confirmed the existence of Se−O (Figure 2e). With these results, we deduced that the chemical changes from the visibleC
DOI: 10.1021/acsami.7b02367 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
Figure 3. (a) Intracellular distributions of the nanoparticles (scale bar = 200 μm). The viability of the MDA-MB 231 cells treated with PSe-Por nanoparticles (b) before and (c) after irradiation for 40 min. The results are presented in mean ± SD, and n = 5 replicates per experiment (*: significant difference, P < 0.05).
S4c). Hence, the nanosystem was quite stable under the reduction conditions. The long PEG chains may have prevented the GSH from approaching the diselenide bonds. The nanoparticles were confirmed to be able to enter cells and remain stable under cellular reduction conditions. Next, the biocompatibility of the system was characterized. Interestingly, with low cell toxicity of the hyperbranched polymer itself, the light-induced oxidization product could kill MDA-MB 231 cells. The nanoparticles solution was first irradiated with visible light for 40 min, then diluted into different concentrations with DEME as experimental groups. After 24 h of incubation with MDA-MB 231 cells and normal cells, the in vitro toxicity of the nanoparticles was evaluated by using a cell counting KIT-8 (CCK-8) assay. L-02 cells (normal a liver cell line) were used for toxicity study as shown in Figure S5. The nanoparticles had no significant cytotoxicity to L-02 cells. The results in Figure 3b indicated that the PSe-Por nanoparticles were almost nontoxic to MDA-MB 231 cells at given concentrations ranging from 10 μg/mL to 200 μg/mL. However, after light irradiation, the PSe-Por nanoparticles exhibited cytotoxicity (Figure 3c). At a concentration of 100 μg/mL, almost half of the MDA-MB 231 cells were killed. These results were unexpected. The porphyrin we used here was almost nontoxic to MDA-MB 231 cells (Figure S6). The only change after light irradiation except for the morphology was the oxidation of the diselenide bonds. Thus, the cell toxicity might have been caused by the production of seleninic acid. To verify this hypothesis, we employed a model molecule,11hydroxyundecane-1-seleninic acid, RSeO(OH). This molecule has a similar structure as the oxidization products of the nanosystem. 11,11′-Diselanediylbis(undecan-1-ol) (DSeOH)
light-induced morphology changes of the nanoparticles were as follows: the hydrophobic diselenide bonds were oxidized to hydrophilic seleninic acids after light irradiation, and the polymer chains were cleaved into small amphiphilic parts, causing the change of aggregates formation. After measuring the light of response the system, its bioactivity was also characterized. First, the intracellular distribution of the PSe-Por nanoparticles was observed usingconfocal laser scanning microscopy to show their ability to cross the cell membrane. MDA-MB 231 human breast cancer cells were chosen for the experiments. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI). The PSePor nanoparticles were used directly without adding other fluorescent groups, as porphyrin itself is a fluorescent material (excitation/emission 467 nm/663 nm). After incubation with MDA-MB 231 cells for 0.5 and 3 h, the fluorescent signals of the nanoparticles and DAPI were recorded. As shown in Figure 3a, signal of the PSe-Por nanoparticles only appeared in the cytoplasm and increased with longer incubation time. Hence, the nanoparticles could be swallowed by the MDA-MB 231 cells. Because diselenide bonds are also sensitive to reductants, such as glutathione (GSH), we wondered if the nanoparticles could be reduced by intracellular GSH (approximately 1−10 mM) and cause the system to disassemble. To answer this question, the stability of the nanosystem under reduction conditions (GSH 10 mM) were explored. DLS, TEM, and XPS was used to characterize the nanoparticles. The size distribution of the nanosystem was almost same with GSH, even after 1 week, and the TEM images were also consistent with the DLS results (Figure S4a, b). Additional XPS results showed that the Se 3d binding energy remained basically unchanged (Figure D
DOI: 10.1021/acsami.7b02367 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Figure 4. Cytotoxicity of DSeOH and RSeO(OH). (a) DSeOH exhibited significant nontoxicity to the MDA-MB 231 cells, whereas (b) at a concentration of approximately 0.1 mmol/mL, RSeO(OH) killed half of the cancer cells. Control experiments were done without adding any chemical reagents. c) Flow cytometry analyses of the cells treated with various doses of RSeO(OH), 0.01 mmol/L and 0.05 mmol/L, after 24 h: UL = dead cells, UR = late apoptotic cells, LL = live cells, and LR = early apoptotic cells.
We have designed and prepared a selenium/porphyrincontaining hyperbranched polymer using stepwise polymerization, which can self-assemble into nanoparticles through emulsification. Under visible-light irradiation, these aggregates continuously produced 1O2, and cleaved the diselenide bonds in the polymer chain. The oxidation product exhibits certain cytotoxicity. Further experiments indicated that the seleninc acid exhibited excellent anticancer activity toward MDA-MB 231 cells with a low IC50 value of 2.84 μg/mL. This system showed that with a potential light-triggered antitumor activity, in combination with photodynamic therapy, better anticancer efficacy can be achieved.
was chosen as a model molecule before oxidation. These two chemical reagents were first dissolved into DEME and then diluted into various concentrations from 0.25 mmol/L to 0 mmol/L (1 mmol/L is equal to 28.4 μg/mL). After incubation with MDA-MB 231 cells for 24 h, a CCK-8 assay was used to evaluate the toxicity of these two molecules. As shown in Figure 4a, all the cell viabilities of DSeOH groups were more than 95%, indicating that DSeOH was almost nontoxic to the MDAMB 231 cells at the given concentrations. In contrast, almost half of the MDA-MB 231 cells were killed when exposed to RSeO(OH) at a concentration of 0.1 mmol/L (2.84 μg/mL), as shown in the Figure 4b. The cytotoxicity of the oxidization product was significantly enhanced, which was consistent with the results using the hyperbranched polymers. A549 human lung cancer cells were also employed to prove the universal cytotoxicity of seleninic acid. The IC50 value to A549 cells was about 7.1 μg/mL, for the tolerance of A549 cells were higher than MDA-MB 231 cells (Figure S7). We subsequently performed a flow cytometric assay 24 h after incubation with two given concentrations, i.e., 0.1 and 0.05 mmol/L (Figure 4c). Annexin-V/PI assay was used to prove that the dead cells died of apoptosis. As the statistical data show, the cancer cells presented a higher apoptosis rate in the higher concentration seleninic acid group. It is reasonable to think that the seleninic acid groups played an important role in killing the tumor cells.30 All the results indicated that the apoptosis after light irradiation with PSe-Por nanoparticles might have been caused by the seleninic acid. The hyperbranched polymer exhibited light-induced cell toxicity, enabling potential applications in combination with light-triggered antitumor activity and photodynamic therapies.
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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/acsami.7b02367. NMR spectrum of the seleninic acid, TEM and DLS results of the PSe-Por nanoparticles, stability of the nanoparticles in GSH, and cytotoxicity of the seleninic acid (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Huaping Xu: 0000-0001-5131-7188 Author Contributions †
C.S. and J.S. contributed equally.
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DOI: 10.1021/acsami.7b02367 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces Notes
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the National Science Foundation for Distinguished Young Scholars (Grant 21425416), the National Basic Research Program of China (Grant 2013CB834502), the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (Grant 21421064), the National Natural Science Foundation of China (Grant 91427301), Natural Science Foundation of Beijing for Young Scholars (Grant 2164064), and China Postdoctoral Science Foundation (Grant 2016M590083).
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