Facile Synthesis of Multifunctional Germanium ... - Wiley Online Library

FULL PAPER DOI: 10.1002/asia.201402227

Facile Synthesis of Multifunctional Germanium Nanoparticles as a Carrier of Quercetin to Achieve Enhanced Biological Activity Yan-Jie Guo, Fen Yang, Lu Zhang, Jiang Pi, Ji-Ye Cai, and Pei-Hui Yang*[a] Abstract: A simple method for preparing quercetin surface-functionalized germanium nanoparticles (Qu-GeNPs) with enhanced antioxidant and anticancer activity is reported. Spherical germanium nanoparticles (GeNPs) were capped by quercetin (Qu) with a mean particle size of approximately 33 nm and were characterized by TEM, AFM, UV-visible absorption spectroscopy, FTIR, and XRD measurements. The in vitro drug release of Qu from

the Qu-GeNPs indicated that Qu could principally be distributed around tumor tissues rather than in the normal section and Qu-GeNPs were internalized by MCF-7 cells. Their biological activity test results indicated that these QuGeNPs possessed stronger hydroxylKeywords: antitumor agents · drug delivery · germanium · nanoparticles · quercetin

Introduction

GeNPs that they produced from the decomposition of Ge[NACHTUNGRE(SiMe2)2]2 in hexadecylamine at 300 8C, which were isolated in 80–90 % yield, were used to facilitate cell imaging and photothermal therapy. The authors found that the GeNPs were able to function as biomarkers for cell signaling, were sufficiently nontoxic, and were active for photothermal behavior upon excitation with near-infrared (NIR) radiation at low laser powers. Yin et al. synthesized waterdispersable multifunctional GeNPs in chitosan (CS) colloid.[10] The CS-GeNPs were functionalized by loading doxorubicin (Dox) and conjugating with folic acid (FA) to improve their effectiveness of drug-targeted delivery. Doxloaded FA-CS-GeNPs showed good anticancer targeting and photothermal effects toward MCF-7 cells. Tilley and coworkers used the allylamine-capped GeNPs that they produced from the reduction of GeCl4 with various metal hydrides to study their application as optical probes for imaging HepG2 cells.[11] The GeNPs transfected into the cytosol and, upon excitation with a mercury lamp, exhibited blue fluorescence. The authors also studied the effect of the GeNPs on mitochondrial activity and determined that the 50 %-inhibitory toxicity concentration (TC50) was 100 mg mL 1. This indicated that at nanoparticle concentrations of 100 mg mL 1, toxicity was relatively low, although this TC50 value is lower than that for UV exposure of CdSe quantum dots in HeLa cells. Lin and co-workers also studied the effect on cell toxicity of allylamine-capped GeNPs synthesized using the method of Warner and Tilley, and found that the water-soluble GeNPs were toxic to cells at concentrations greater than 3 mm.[12] The authors found differences in how nanoparticles produced by different methods impacted cell toxicity, and they also suggested a pathway by which the GeNPs induced cell death and acted as a possible antag-

Nanoparticles of elemental germanium have excited scientists and engineers because of their size-dependent optical properties and their potential applications in optoelectronics, biological imaging and therapeutics, and energy conversion and storage. To further develop these applications and to gain deeper insight into their size-dependent properties, robust and facile synthetic methods are needed to controllably synthesize germanium nanoparticles (GeNPs). GeNPs potentially offer a lower-toxicity and more environmentally friendly alternative to related narrow-bandgap II/VI, III/V, and IV/VI semiconductor nanoparticles.[1] However, analogous to early work on group II/VI, III/V, and IV/VI semiconductor nanoparticles, the emphasis in solution syntheses of GeNPs has mostly focused on obtaining size and aspectratio control of pure nanocrystalline materials.[2–8] To date, reports on the biological applications of germanium nanoparticles have been much less mature. Several relevant examples of biological applications of GeNPs are highlighted below and include biological imaging and therapeutics. Boyle and co-workers reported the synthesis of approximately 3–5 nm phospholipid-encapsulated water-soluble germanium nanocrystals that were used as biomarkers for cell signaling in RBL-2H3 cells in vitro.[9] The

[a] Y.-J. Guo, F. Yang, L. Zhang, J. Pi, Prof. J.-Y. Cai, Prof. P.-H. Yang Department of Chemistry, Jinan University Guangzhou 510632 (P.R. China) Fax: (+ 86) 2085223039 E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/asia.201402227.

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scavenging effects and proliferative inhibition effect on MCF-7 cancer cells than quercetin, thus suggesting that the strategy to use GeNPs as a carrier of Qu could be an efficient way to achieve enhanced antioxidant and anticancer activity. In addition, Qu-GeNPs possessed a high apoptotic induction effect in cancer cells, especially in high dosages, and could arrest MCF-7 cells in the S phase.

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onist to counteract the toxic effects of nanoparticle exposure. For germanium nanoparticles to be used in biological applications, water solubility is vital. Thus the development of synthetic methods for producing germanium nanoparticles with controlled sizes and modified surfaces, and with low toxicity, is important for the biological applications of germanium nanoparticles. There are numerous reports on methods to prepare germanium nanoparticles. Many of these early reports required specialized reagents and equipment or involved high temperatures, toxic precursors, and organic byproducts.[13, 14] Solution-phase synthetic chemistry methods offer better control over the size and shape of the nanoparticles, but often they do not provide the good crystallinity required for many applications and they require the use of long-chain ligands and surfactants to stabilize the particle surface and control growth.[15–27] Recently, some new synthetic methods for water-dispersable GeNPs have been reported. Although Jing et al. reported the synthesis of GeNPs from GeO2 as a nontoxic and inexpensive precursor, they did not obtain dispersed GeNPs.[28] Therefore it is important to select an appropriate stabilizer to keep GeNPs dispersable. Wu et al. synthesized water-dispersable GeNPs from GeO2 powders, and polyvinylpyrrolidone (PVP) molecules were used as the surface stabilizer.[29] Nevertheless, it was difficult to further biological applications, because PVP did not provide the functional amino or carboxyl groups. Yin et al. synthesized chitosan-capped water-dispersable GeNPs.[10] For biological applications, CS-GeNPs were functionalized by loading DOX and conjugating with FA. The resulting Dox-loaded FA-CS-GeNPs were capable of effectively killing MCF-7 human cancer cells. However, the multistep reaction reduced the drug-loading efficiency. Therefore, the development of a new one-pot synthetic method to prepare waterdispersable, biocompatible GeNPs for biological applications is still a challenge. Quercetin (Qu), a phenolic compound and topical antioxidant, has superior antioxidant potency relative to many other well-known antioxidant molecules owing to the optimized number and distinctive positions of the free hydroxyl groups in this molecule.[30, 31] At the same time, quercetin has a wide range of biological activity, including delayed ultraviolet-radiation-mediated oxidant injury and cell death by scavenging oxygen radicals, thereby protecting lipids against peroxidation to terminate the chain-radical reaction, and it enhances apoptosis mediated by death receptors in glioma cells.[32–37] These activities of quercetin make it a promising candidate for the treatment and prevention of various cancers. However, the insolubility of quercetin in water results in poor absorption, low bioavailability, and inability to cross the blood–brain barrier, thereby limiting its potential clinical application in the treatment of cancer. Furthermore, the solvent used (e.g., dimethylsulfoxide) may cause hemolysis and liver and kidney damage.[38] Therefore, development of novel preparations that enhance the solubility of quercetin and improve its bioactivity in inhibiting tumor growth is of great importance.

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Pei-Hui Yang et al.

In this paper, water-dispersable quercetin surface-functionalized germanium nanoparticles (Qu-GeNPs) were synthesized by means of wet-chemical reduction. GeNPs could be capped with Qu molecules, which effectively prevented GeNPs from gathering and being oxidized. At the same time, the insolubility of quercetin in water and its bioavailability were improved. Furthermore, the study showed that Qu-GeNPs possessed stronger hydroxyl-scavenging effects and a higher proliferative inhibition effect on MCF-7 cancer cells than quercetin. Our present work provides a design for a drug-delivery system by using GeNPs as a carrier of Qu to achieve enhanced antioxidant and anticancer activity.

Results and Discussion Morphology and Stability of Qu-GeNPs In the present study, we demonstrated a simple method to synthesize Qu-functionalized GeNPs through conjugation of Qu to the surface of the nanoparticles. As shown in Scheme 1, GeNPs were capped with Qu molecules to form

Scheme 1. Schematic illustration of the preparation of Qu-GeNPs.

more compact and stable globular nanocomposites. The morphology and chemical composition of Qu-GeNPs were characterized using various spectroscopic and microscopic methods. Figure 1 shows the TEM images of the GeNPs with the absence (Figure 1a, b) and presence (Figure 1c, d)

Figure 1. TEM images of a, b) GeNPs and c, d) Qu-GeNPs.

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of the capping agents Qu, which clearly revealed that QuGeNPs presented a disperse and homogeneous spherical structure with an average diameter of about 35 nm, whereas GeNPs in aqueous solutions were poorly aggregated owing to their high surface energy, thus resulting in significant precipitation. These results suggest that the presence of Qu is a key factor in regulating and controlling the size of the GeNPs. The AFM image demonstrates the topographic morphology of the Qu-GeNPs in Figure 2a and b. We calculated the size distribution of the nanoparticles from the AFM image using ImageJ software. We made the calculations using a large number of nanoparticles (100 in the AFM images) so that the size-distribution curve would have as little error as possible. The size distribution of the nanoparticles is shown in Figure 2d. The largest nanoparticles are in the range of 32–33 nm.

Figure 3. a) z potential of GeNPs and Qu-GeNPs and b) stability of QuGeNPs in aqueous and PBS (pH 7.4) solutions.

aggregation of the nanoparticles began to take place (Figure 3b). In contrast, under physiological conditions (PBS, pH 7.4), Qu-GeNPs remained stable at least for 120 h. The hydrodynamic diameter was distributed between 85 and 120 nm, and no aggregation and precipitation were observed. The high stability of Qu-GeNPs under physiological conditions supports their future applications in medicine.[39] Chemical Composition and Structure of Qu-GeNPs In Figure 4a, the UV-visible absorption spectrum shows that the peaks of Qu appeared at around 269 and 397 nm, whereas the Qu-GeNPs presented characteristic absorption peaks of Qu (274 nm) and structural shoulders near 510 nm, respectively. The growth of a layer of Qu on the core of GeNPs was redshifted by 5 nm on account of the increase in the refractive index of the surrounding medium, which indicated that the Qu molecules were functionalized on the surface of GeNPs. A little hump at around 510 nm (2.43 eV) might be due to the first direct transition at the L coordinate of the Brillouin zone in germanium nanocrystals, which agrees well with previous reports.[4] No photoluminescence was observed from the GeNPs solutions, which suggests the possibility of surface trap states that are inhibiting direct bandgap recombination. Qu-GeNPs were further characterized by FTIR to confirm that the formation of chemical bonds between Qu and Ge occurred during the synthesis reaction. As shown in Figure 4b, the FTIR spectrum of Qu-GeNPs resembles those of Qu and GeNPs, thus giving clear evidence that Qu and GeNPs form parts of the nanocomposite. In the spectrum of Qu, the peaks at 1665 cm 1 were assigned to the stretching

Figure 2. AFM images of a, b) Qu-GeNPs. c) Height profile and d) size distribution of Qu-GeNPs corresponding to the AFM image.

Stability is an important issue for the use of nanoparticles in future applications. To examine the effects of Qu on the surface properties and stability of GeNPs, we measured the z potential and size distribution of GeNPs and Qu-GeNPs. As shown in Figure 3a, the z potential of GeNPs was 11.8 mV, which decreased to 36.5 mV after the surface decoration with Qu on account of the fact that Qu as a phenolic compound has many free hydroxyl groups and it is easy to lose hydrogen ions in the weak alkaline environment, thus negatively charging itself, thereby explaining the higher stability of Qu-GeNPs than GeNPs. Furthermore, we used a Zetasizer Nano-ZS particle analyzer to test the changes in size distribution of Qu-GeNPs under both aqueous and physiological conditions. The results revealed that Qu-GeNPs remained stable for up to 108 h in aqueous solutions. However, when the reaction time increased to 120 h, the sizes of the Qu-GeNPs increased dramatically, and the

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elemental composition analysis employing EDS showed the presence of a strong signal from the Ge atoms, together with a C and O atom signal from Qu molecules, whereas the Cu atom signal was attributed to the supporting grid. No clear peaks for other elements or impurities were observed. The presence of the O atom indicates that Qu has been conjugated to the surface of GeNPs. pH-Mediated Drug Release of Qu In Vitro The in vitro drug release of Qu from Qu-GeNPs in DMEM cell culture medium with 10 % serum at pH values of 7.4 and 4.7 was investigated to simulate the normal body blood and acidic environments. Figure 5 Figure 4. a) UV/Vis absorption spectrum and b) FTIR spectra of Qu and Qu-GeNPs. c) XRD spectra and shows the release profiles of d) EDS analysis of Qu-GeNPs. Qu from the nanoparticles into the culture medium. The cumulative release of Qu mainly occurred in the first 12 h under vibration of C=O, whereas the peak at 1614 cm 1 corretwo pH systems, which reached 52.9 % at pH 4.7 and 35.9 % sponded to the stretching vibration of C=C. The peak at at pH 7.4 within 12 h, respectively. Thereafter, the cumula1381 cm 1 corresponded to the stretching vibration of C tive release of Qu reached 79.1 % at pH 4.7 and 44.9 % at OH, but the peaks at 1166 cm 1 were assigned to the pH 7.4 after 48 h, respectively. This initial burst of Qu restretching vibration of C-O-C. The appearance of the above lease could be partly attributed to the weak bonds or the adpeaks in the spectrum of Qu-GeNPs confirmed the presence sorption of drugs onto the surface of the nanoparticles.[41] of Qu on the surface of GeNPs. The IR absorption spectrum of the Qu-GeNPs exhibits the characteristic peaks at 885, These results also demonstrated that the release process at 707, and 619 cm 1; this showed the formation of a Ge O pH 7.4 was much slower than that at pH 4.7. One of possible reasons was the low solubility of Qu at pH 7.4 than that at bond,[40] which clearly indicated the interaction between Qu pH 4.7. Interestingly, a faster release of Qu has been oband GeNPs. Moreover, a redshift was observed for the served under acidic conditions (pH 4.7), which is exactly groups of C=O, C=C, C OH, and C-O-C in Qu-GeNPs, what we expected. Qu was able to be principally distributed which could be due to the formation of Ge O bonds. around tumor tissues in an acidic microenvironment rather Figure 4c shows a powder XRD pattern for a representative sample of as-synthesized Ge nanoparticles, which was confirmed by the appearance of intense sharp reflections in the XRD pattern centered at approximately 27, 45, 53, 66, and 738, characteristic of the (111), (220), (311), (400), and (331) crystal planes of diamond-structured Ge, which is in good agreement with the standard value (JCPDS card no. 04-0545) within experimental error. It is worth noting that no characteristic peaks from other crystalline forms, such as GeO2 (source material) or GeO, are detected in the XRD pattern, which definitely proves that the as-synthesized GeNPs have not been oxidized during the synthesis process (in an aqueous system under ambient conditions). This is assumed to be the case since the surfaces have been capped with Qu, and this effectively protects the synthesized Figure 5. In vitro release profiles of Qu from Qu-GeNPs in DMEM cell GeNPs from being oxidized. Therefore, Qu played an imculture medium with 10 % serum. The Qu concentrations were deterportant role in stabilizing GeNPs. As shown in Figure 4d, an mined by HPLC analysis.

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than in the normal section. Taken together, these results demonstrate that GeNPs hold promise as a pH-mediated release delivery vehicle for potential cancer therapy. In Vitro Cellular Uptake of Qu-GeNPs An important factor that usually contributes to nanomaterialbased drug cytotoxicity is cellular uptake. Nanomaterials tend to accumulate in cancer cells as “nanocarriers” for chemotherapeutics. However, this passive strategy has limitations owing to its random delivery mode.[42] To understand the cellular uptake and intracellular activity of Qu-GeNPs, we loaded rhodamine B onto Qu-GeNPs as a fluorescence marker for intracellular fluorescence imaging combined with specific fluorescence imaging of lysosomes. Firstly, to investigate whether Qu-GeNPs could be internalized by MCF-7 cells, the lysosomes were stained by LysoTracker Red as described previ- Figure 6. Fluorescence microscopy images showing the internalization of Qu-GeNPs and colocalization of Quously.[43] As shown in Figure 6, GeNPs (red) and lysosomes (green) in MCF-7 cells after having been internalized by MCF-7 cells: a) 30, both Qu-GeNPs and lysosomes b) 60, c) 90, and d) 120 min. were observed in MCF-7 cells, as evidenced by the location of the red and green fluoresas antioxidant and anticancer activity. It has been reported cence, respectively; the red fluorescence from the nanopartithat the free-radical scavenging ability of quercetin is highly cles penetrated into the MCF-7 cells in a time-dependent directed toward hydroxyl radical (OHC).[45] In this paper, the manner. Most of the nanoparticles were internalized into antioxidant activity of Qu and Qu-GeNPs against hydroxyl the cytoplasm of the cells. The colocalization of Qu-GeNPs radical (OHC) was investigated. As shown in Figure 7, the in(red) with lysosomes (green) produced a yellow fluoreshibitory effect of the Qu and Qu-GeNPs on OHC was concence in merged images. With as little as 30 min of treatcentration-related, and the suppression ratio increased as ment, Qu-GeNPs were internalized by MCF-7 cells and parthe sample concentration increased in the range of 20– tially colocalized with lysosomes (Figure 6a). After 60 min of incubation, more Qu-GeNPs aggregates were observed in MCF-7 cells, and most of these Qu-GeNPs were inside the lysosome vesicles (Figure 6b). The amounts of Qu-GeNPs in MCF-7 cells increased upon extending the incubation time (Figure 6c, d). These results demonstrated that Qu-GeNPs could be internalized by MCF-7 cells and then preferentially be transported into lysosome vesicles, which was consistent with the results obtained by Pi et al.[44] Antioxidant Activity of Qu-GeNPs Since Qu exhibits a wide range of biological activity, it is worth studying other potential aspects of Qu-GeNPs, such

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Figure 7. Antioxidant activities of Qu and Qu-GeNPs.

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200 mm. Figure 7 shows that when the concentration of QuGeNPs was 200 mm, the scavenging rate of hydroxyl radicals reached 89 %, which was significantly higher than that of Qu (49 %) at the same concentration. The antioxidant activity of Qu and Qu-GeNPs against hydroxyl radicals is comparable under the same experimental conditions. Therefore, Qu-GeNPs showed a higher suppression effect toward hydroxyl radicals than that of free Qu. The value of the hydroxyl inhibition rate of the Qu-GeNPs was close to a known antioxidant, such as ascorbic acid,[46] which inhibited hydroxyl oxidation by 45 % (50 mm). The antioxidative activities of Qu-GeNPs could be attributed to three main reasons: First, the arrangements of the outer electrons of the Ge atom are 4s24p2, which could make unpaired electrons become easily trapped by Ge atoms, and furthermore would make it possible for Ge to scavenge free radicals.[47] Second, Qu has been reported to be an excellent antioxidant.[30, 31] Third, the introduction of a radical-sensitive Ge O bond effectively enhanced the antioxidative activity of Qu-GeNPs. On the basis of these results, our strategy to assemble antioxidant ligands on the surface of GeNPs could be a highly efficient way to enhance the antioxidant activity.

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GeNPs in MCF-7 cells, with a viability of 10.08 % during treatment with Na2GeO3 (500 mm). The cell morphology changes were also used to assess the anticancer activity of Qu-GeNPs over 48 h. Microscopic and AFM images of MCF-7 cells treated with Qu-GeNPs indicated the decrease in the intensity of MCF-7 cells and significant morphological changes of MCF-7 cells after treatment with Qu-GeNPs (see the Supporting Information). The cytotoxicity of Qu-GeNPs, Qu, and GeNPs toward MCF-7 cells was investigated to examine the synergistic interaction between Qu and GeNPs as analyzed by the isobologram method.[48] The IC50 values for Qu-GeNPs, Qu, and GeNPs were found to be 348.6, 458.1, and 666.4 mm, respectively (Figure 8b). The results of the isobologram analysis revealed that the growth inhibitory effects between Qu and GeNPs in the Qu-GeNP system were weakly synergistic, as evidenced by the location of the data point in the isobologram being below the line that defined an additive effect. The combination index (CI) of the IC50 value of the QuGeNPs was found to be 0.94, which further confirmed the weak synergism between Qu and the GeNPs.[48] Taken together, our results clearly demonstrate that the strategy to use GeNPs as a carrier of Qu could be a efficient way to enhance its anticancer efficacy. Apoptosis or programmed cell death is an essential process of cell death during embryonic and postnatal tissue remodeling as well as some pathological conditions. Apoptosis is characterized by cytoplasmic and nuclear condensation, membrane blebbing, DNA fragmentation, and the formation of apoptotic bodies. The significant biochemical characteris-

Induction of Apoptotic Cell Death by Qu-GeNPs

The cytotoxicity of Na2GeO3, Qu-GeNPs, Qu, and GeNPs toward MCF-7 cells over 48 h were evaluated by MTT assay, and the results are shown in Figure 8a. The viability of MCF-7 cells was reduced in a dose-dependent manner, and reached just 21.6 % for exposure to Qu-GeNPs (500 mm), which was significantly decreased relative to that of MCF-7 cells treated with Qu and GeNPs. The results suggest that using GeNPs as a carrier of Qu could achieve enhanced anticancer activity. The main reason was that GeNPs as a carrier of Qu were then internalized by MCF-7 cells; the release of the drug by the highly acidic pH of the lysosomal compartment within the cells improved the drug concentrations of quercetin within the cells. Another reason was that after Qu-GeNPs were internalized by MCF-7 cells, the release of Qu from Qu-GeNPs took place under acidic conditions, and the oxidation of Ge nanoparticles ultimately led to dissolution of the particles and possibly resulted in cell toxicity.[9] This is Figure 8. a) Cell viability of MCF-7 cells treated with Na GeO , GeNPs, Qu, and Qu-GeNPs at various concenbecause the oxidation product trations. Cells were incubated with nanoparticles for 482h. b) 3Isobologram analysis of the antiproliferative efis Na2GeO3, which showed fects of Qu and GeNPs on MCF-7 cells. c) Flow cytometric analysis of Qu-GeNPs induced apoptosis in MCFa higher toxicity than Qu- 7 cells using annexin-V-FITC/PI.

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tic of cell apoptosis is DNA fragmentation. Thus, we performed an in vitro apoptotic detection assay, PI-flow cytometric analysis, to determine whether apoptosis was involved in the cell death induced by the Qu-GeNPs in MCF-7 cells. Figure 8c shows the representative DNA histograms from PI staining cells, which revealed that exposure of MCF-7 cells to different concentrations of QuGeNPs for 48 h resulted in a marked dose-dependent increase in the proportion of apoptotic cells, as reflected by the increased sub-G1 populations. Qu-GeNPs (500 mm) increased the percentage of apoptotic cells to 52.12 %. The results strongly suggested that Figure 9. Effect of Qu-GeNPs on the cell cycle of MCF-7 cells over 48 h as detected by PI-based flow cytomeQu-GeNPs possess a high apop- try. totic induction effect in cancer cells, especially in high dosages. To further detect the alterations in cell cycles induced by especially in high dosages, and could arrest MCF-7 cells in Qu-GeNPs, the MCF-7 cell cycle was investigated by flow the S phase. Taken together, our results suggest that the cytometry using PI staining after 48 h of treatment with Qustrategy of using GeNPs as a carrier of Qu could be a highly GeNPs. It was found that the percentage of cells in the S efficient way to achieve enhanced antioxidant and anticancphase increased from 22.5 % to 23.7, 26.8, and 31.0 % after er activity. This superior biological activity could be ascribed treatment with 0, 100, 300 and 500 mm Qu-GeNPs, respecto two points. Firstly, the use of GeNPs as a carrier of Qu tively (Figure 9). This suggests that treatment with Quwould be beneficial to distribute Qu around tumor tissues, GeNPs could arrest MCF-7 cells in the S phase. It is beand therefore the bioavailability and bioactivity of Qu lieved that the information obtained from the present work would be improved. Secondly, after Qu-GeNPs were interwould ultimately be helpful in developing new potential annalized by MCF-7 cells, the oxidation of GeNPs would ultitioxidants and new therapeutic reagents for some diseases. mately lead to dissolution of the particles and possibly result in cell toxicity. Furthermore, Qu-GeNPs might be candidates for further evaluation as a chemotherapeutic agent for human cancers, especially breast carcinoma. Conclusion In summary, a facile method for one-pot synthesis of GeNPs in an aqueous solution of Qu was investigated. Spherical GeNPs were capped by Qu through physical adsorption and the formation of Ge O bonds. The studies on in vitro drug release revealed that faster release of Qu was observed under acidic conditions, which is exactly what we expected. Qu could thus principally be distributed around tumor tissues with an acidic microenvironment rather than in the normal section. Therefore, GeNPs hold promise as a pHmediated release delivery vehicle for potential cancer therapy. Qu-GeNPs could be internalized by MCF-7 cells and then preferentially transported into lysosome vesicles. Moreover, Qu-GeNPs exhibited significantly enhanced antioxidative and anticancer activities, showing both stronger hydroxyl-scavenging effects and a proliferative inhibition effect on MCF-7 cancer cells than quercetin. In addition, Qu-GeNPs possessed a high apoptotic induction effect in cancer cells,

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Experimental Section Reagents All of the chemical reagents were analytically pure or better and used as received without further purification. GeO2 (analytically pure) was purchased from Sinopharm Chemical Reagent Beijing Co., Ltd. (China). Quercetin (spectral pure) was purchased from FeiBo Biological Technology Guangzhou Co., Ltd. (China). NaBH4, the reducing agent, was obtained from Aladdin Chemical Co., Ltd. (China). H2O2 and FeSO4 were purchased from Guangzhou Chemical Co., Ltd. (China). Thiazolyl blue tetrazolium bromide (MTT) and the Cell Cycle and Apoptosis Analysis Kit were purchased from Sigma. Aqueous solutions were prepared with freshly deionized water (18.2 m specific resistance) obtained with a Pall Cascada laboratory water system. Preparation of Quercetin-Stabilized GeNPs (Denoted as Qu-GeNPs) Qu-GeNPs were synthesized by a wet-chemical method as described below.[29] Firstly, NaBH4, as a reducing agent to reduce GeIV to Ge0, was

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dissolved in icy cold distilled water ( 4 8C) to form a 0.26 m homogeneous solution and then stored in a refrigerator ( 4 8C). Subsequently, GeO2 (0.013 g; a source material of Ge) and quercetin (0.076 g; stabilizer) were dissolved completely in an NaOH solution (20 mL, 0.15 m) to result in a clear yellow solution, and then the pH of the solution was adjusted by 0.5 m HCl solution to be 7.5. Then, under continuous magnetic stirring, a solution of NaBH4 (2 mL) was added into a flask. Qu-GeNPs were prepared by reducing the Na2GeO3/Qu suspension in water bath at 60 8C for 4 h. Ultimately, a red-brown solution was formed and was dialyzed in a 4000 Da dialysis bag against deionized water for 2 days to remove excess amounts of Na2GeO3/Qu. The resultant red brown solution was freeze-dried, and a dark brown powder of Qu-GeNPs was obtained for further characterization. Inductively coupled plasma (ICP) and UV/Vis analyses could be used to quantify the concentration of elemental Ge in GeNPs and adsorbed drug fractions. Also, such estimates could then be used to determine the equivalent concentrations of free quercetin for all subsequent comparative experiments between free quercetin and GeNP-loaded quercetin (antioxidant activity, cytotoxicity, and so on).

Cell Lines and Cell Culture Adherent MCF-7 cells, grown to confluence in DMEM culture medium, were maintained under standard cell-culture conditions at 37 8C in a humidified atmosphere of 95 % air and 5 % CO2 prior to examination. Human breast carcinoma cell line MCF-7 cells were obtained from the Life Science Research Institute of the Cell Resource Center, Shanghai, China. MTT Assay The cytotoxicity of the synthesized Qu-GeNPs was evaluated using an MTT assay. MCF-7 cells were seeded in 96-well tissue culture plates at a density of 5 103 cells per well and incubated with Qu-GeNPs at different concentrations at 37 8C for 48 h. After treatment, 10 mL per well of MTT solution (5 mg mL 1 phosphate-buffered saline) was added to the well and incubated for another 4 h. To dissolve the formazan salt that was formed, the medium was aspirated and replaced with 150 mL per well of DMSO. The cell-growth conditions were reflected by the color intensity of the formazan solution. Absorbance at 570 nm was taken on a 96-well microplate reader (TECAN, Switzerland). Cytotoxicity (IC50) data were analyzed using the GraphPad computer software.

Characterization of Qu-GeNPs

Flow Cytometric Analysis

The as-prepared products were characterized by using microscopic and spectroscopic methods. Briefly, TEM samples were prepared by dispersing the powder onto a holey carbon film on copper grids. The micrographs were obtained with a Hitachi (H-7650) transmission electron microscope operated at an accelerating voltage at 80 kV. The AFM system was a BioScope Catalyst (Bruker, Germany), and the AFM probe was a Tap 150Al-G Silicon, which was purchased from the Budget Sensor Company (Bulgaria). The z potential and size distribution of the nanoparticles were measured by photon correlation spectroscopy (PCS) with a Nano-ZS instrument (Malvern Instruments Limited). Absorption spectra were recorded with a Cary 5000 UV-visible spectrophotometer (Varian technology Co., LTD). Fourier transform infrared spectroscopy (FTIR) spectra of the samples were recorded with an Equinox 55 IR spectrometer in the range 4000–400 cm 1 using the KBr-disk method. Powder X-ray diffraction (PXRD) data were collected with a Bruker D8 Advance X-ray diffractometer equipped with CuKa radiation. Energy-dispersive X-ray spectroscopy (EDS) was carried out during TEM measurements and was used to examine the elemental composition of the QuGeNPs powder.

Cell-cycle distribution was monitored by flow cytometric analysis as previously described.[51] Briefly, the treated cells were harvested and washed with phosphate buffer solution (PBS). Cells were treated with RNase A (5 mL) and stained with propidium iodide (PI) for 4 h in darkness after being fixed with 70 % ethanol at 4 8C overnight. Data acquisition was performed with a Beckman Coulter Epics XL MCL flow cytometer (Miami, Florida, USA). The cell-cycle distribution was analyzed using BD FACSAria (USA). The proportion of cells in G0/G1, S, G2M phases was represented as a DNA histogram. Apoptotic cells with hypodiploid DNA content were estimated through quantifying the sub-G1 peak in the cellcycle pattern. For each experiment, 10 000 events per sample were recorded.

Acknowledgements This work was supported by the National Natural Science Foundation of China (grant nos. 21071064, 21375048).

In Vitro Drug Release of Qu The assay procedure was developed by the steps described below.[49] An aliquot (10 mg) of the Qu-GeNPs was suspended in Dulbecco’s Modified Eagle’s Medium (DMEM; 10 mL) with 10 % serum and with constant shaking at 37 8C in a hard glass tube. At a specific time following incubation, a specific amount of medium was taken out from the vial with pipette and the same volume of fresh medium was replaced. All samples were analyzed with a HPLC system (Agilent 1100) with a model UV1000 UV detector, and the detection wavelength was set at 397 nm. The column used was a m-Bondapak C18 (3.9 300 mm, Grom, Germany). The mobile phase was methanol/H3PO4 (0.3 %) (55:45 v/v), and the flow rate was adjusted to 1.0 mL min 1.

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Scavenger Measurements of Hydroxyl Radical (OHC) The hydroxyl radical (OHC) in aqueous media was generated by the Fenton system.[50] The solution of the tested complexes was prepared with N,N-dimethylformamide (DMF). The assay mixture (5 mL) contained the following reagents: safranin (28.5 mm), ethylenediaminetetraacetic acid (EDTA)–FeII (100 mm), H2O2 (44.0 mm), the tested compounds (20–200 mm), and a phosphate buffer (67 mm, pH 7.4). The assay mixtures were incubated at 37 8C for 30 min in a water bath, after which the absorbance was measured at 520 nm. All the tests were run in triplicate and expressed as the mean. Ai was the absorbance in the presence of the tested compound; A0 was the absorbance in the absence of tested compounds; Ac was the absorbance in the absence of tested compound, EDTA–FeII, H2O2. The suppression ratio was calculated on the basis of (Ai A0)/ACHTUNGRE(Ac A0) 100 %.

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