Zinc Oxide Nanoparticles as Adjuvant To Facilitate Doxorubicin

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Zinc Oxide Nanoparticles as Adjuvant to Facilitate Doxorubicin Intracellular Accumulation and Visualize pHresponsive Release for Overcoming Drug Resistance Juan Liu, Xiaowei Ma, Shubin Jin, Xiangdong Xue, Chunqiu Zhang, Tuo Wei, Weisheng Guo, and Xing-Jie Liang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00311 • Publication Date (Web): 12 Apr 2016 Downloaded from http://pubs.acs.org on April 17, 2016

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Molecular Pharmaceutics

Zinc Oxide Nanoparticles as Adjuvant to Facilitate Doxorubicin Intracellular Accumulation and Visualize pH-responsive Release for Overcoming Drug Resistance Juan Liu,† Xiaowei Ma, †,* Shubin Jin, Xiangdong Xue, Chunqiu Zhang, Tuo Wei, Weisheng Guo, Xing-Jie Liang* Chinese Academy of Sciences Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, and Laboratory of Controllable Nanopharmaceuticals, National Center for Nanoscience and Technology of China, Beijing 100190, China. KEYWORDS: Zinc oxide nanoparticles, Doxorubicin, pH-responsive, Drug resistance, Drug delivery.

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ABSTRACT

Multidrug-resistance (MDR) of cancer is a challenge to effective chemotherapeutic interventions. The stimuli-responsive drug delivery system (DDS) based on nanotechnology provides a promising approach to overcome MDR. Through the development of a doxorubicin delivery system based on znic oxide nanomaterials, we have demonstrated MDR in breast cancer cell line can be significantly circumvent by a combination of efficient cellular uptake and a pHtriggered rapid drug release due to degradation of nanocarriers in acidic environment. Doxorubicin & zinc oxide nanoparticles, compared with free doxorubicin, effectively enhanced the intracellular drug concentration by simultaneously increased cell uptake and decreased cell efflux in MDR cancer cells. The acidic environment-triggered release of drug can be tracked real-time by the doxorubicin fluorescence recovery from its quenched state. Therefore, with the combination of therapeutic potential and the capacity to track release of drug in cancer cells, our system holds great potential in nanomedicine by serving dual roles of overcoming drug resistance and tracking intracellular drug release from the DDS.


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1. Introduction Chemotherapy is the most widely used frontline strategies for cancer therapy in clinic.1, 2 However, a key challenge to successful cancer therapy is the appearance of multidrug-resistance (MDR). MDR mainly results from decreased cell uptake and increased efflux of drug. The mechanism of MDR is usually related with the overexpressed drug efflux proteins.3, 4 Recently, for purpose of overcoming MDR, considerable attempt have been devoted to construct stimuliresponsive multifunctional nanoparticle-based drug delivery system (DDS) that can not only deliver drug into cells, but also release the drug in a site- or time- specific way.5-7 In general, an optimal DDS for overcoming MDR is required to release drugs into cytoplasm rapidly and thoroughly, leading to a sufficiently high intracellular drug concentration to exceed drug efflux and threshold concentration to inhibit the proliferation of drug-resistant cancer cells and kill them.8,

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During the past decade, in order to achieve controllable drug release, various

nanomaterials responding to different stimulis, such as physical stimulis (electrical, electrochemical, temperature, light, ultrasonic, and magnetic),10-12 chemical stimulis (ionic, redox, and pH),13-16 and biological stimulis (glucose, enzymes, and inflammation)17-19 have been developed as effective DDSs.20 Among different types of stimuli-responsive DDS, pH-triggered drug release system has been paid more attention to because of its universal applicability, which is expected to stabilize drug at natural environment, and rapidly achieve drug release once the pH of microenvironment reaches the trigger point.9, 14 The pH values in inflammatory tissues and tumors are significantly lower than them in normal tissues and blood because of the high speed of glycolysis in cells. More importantly, late endosome and lysosome represent more acidic, the pH of which is in range of


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4.5~5.5.21,

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The lower pH is of particular importance, since most of the anticancer drugs

delivered by nanocarriers are internalized into cells through endocytosis pathway and are trapped in endosome and lysosome after entering the cells. PH-sensitive linkers are commonly employed to connect nanocarriers and drugs, and pH-sensitive molecules containing ionizable chemical groups are used to form nanostructures for drug encapsulation.23-25 However, although pHresponsive DDS is of valuable practical importance for cancer therapy, the biodegradability of nanocarriers remains a controversial problem, especially for inorganic materials such as mesoporous silica nanoparticles, carbon nanotubes, and gold nanomaterials.26 Znic oxide (ZnO) nanostructured materials, as a novel type of low toxicity and low cost material, are comparatively insoluble at physiological environment, but can dissolve as non-toxic ions at acidic environment, such as the late endosome and lysosome of the tumor cells.27-30 Because of this excellent property, ZnO materials are explored as multifunctional nanocarriers to facilitate the drug delivery and release process.31 Therefore, inspired by the features of ZnO materials, we developed a novel DDS by simple reprecipitation method to load anticancer drug within the ZnO nanostructures in this study. Doxorubicin (DOX), which is an effective chemotherapeutic drug, was selected as the drug model, because it has red fluorescence for easily detection, and cancer resistance and cardiotoxicity of DOX limited its further success in clinic.32 These nanoparticles can significantly overcome drug-resistance by the combination of increased drug intracellular uptake and a pH-responsive release of drug in the acidic organelles (Figure 1). In addition, after the DOX was precipitated into the nanoparticles, its fluorescence was switched off due to the “π-π stacking” of rigid planar structure.33 Therefore, the intracellular trafficking from the endosome to the lysosome and the pH-responsive DOX release from the nanoparticles could be effectively detected and directly visualized by fluorescence signal recovery.34-36


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2. Materials and Methods 2.1 Preparation of Doxorubicin & Zinc Oxide Nanoparticles Briefly, 27.4 mg zinc acetate dihydrate was dissolved in 72mL ethanol at 50°C, and mixed with 28mL ethanol containing 8.0 mg sodium hydroxide. The mixture was stirred at 55°C for 2h via refluxing. Another 27.4 mg acetate dihydrate was dissolved in 72mL ethanol at 50°C, and mixed with the above 100mL reaction solution. 28mL ethanol containing 8.0 mg sodium hydroxide and 20.0 mg doxorubicin was added into the mixture with a slow speed of 10 mL/h. And then the reaction mixture was stirred strongly at 80°C for another 10h via refluxing. After the reaction vessel was cooled down to the room temperature, the DOX loaded zinc oxide nanoparticles (ZD NPs) samples were separated by centrifugation, and then washed by ethanol. Finally, they were dried in a vacuum oven overnight, prior to characterization. ZnO NPs was prepared by the same method without loading doxorubicin. 2.2 Characterization of Nanoparticles The morphology of ZD NPs was determined using transmission electron microscope (TEM, Tecnai G2 20 STWIN, Philips, Netherlands) and scanning electron microscope (SEM, Hitachi S4800). Particles’ size and zeta potential were measured using dynamic light scattering (DLS, Zetasizer 5000, Malvern, Worcestershire, U.K.). 2.3 Cell Culture Human breast cancer cell sensitive (MCF-7S) and resistant (MCF-7R) to doxorubicin, were respectively cultured in DMEM and RPMI1640 medium, which are supplemented with 10% fetal bovine serum and 1% antibiotic solution (penicillin and streptomycin), in the humidified atmosphere containing 5% CO2 at 37°C.


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2.4 Cellular uptake and subcellular localization of ZD NPs The cellular uptake and the subcellular localization of ZD NPs were investigated by confocal laser scanning microscopy (CLSM, Carl Zeiss, Germany). MCF-7R and MCF-7S cells were seeded in the confocal microscopy dishes, and then incubated for 24 h at 37°C. Then cell culture medium was removed, the cells were incubated with free DOX and ZD NPs with equal DOX concentration at 10 µg/mL at 37°C for 1h and 4h. After that, the cells were washed by PBS, stained by LysoTracker deep red, and observed by confocal laser scanning microscopy. The uptake of the nanoparticles was also measured by the quantitative flow cytometry analysis. Cells were seeded in 6-well plates, and incubated for 24h at 37°C. Then the free DOX and ZD NPs were used to treat the cells as described above. Finally, the cells were treated with trypsin-EDTA, and then collected, followed by the analysis of Attune® acoustic focusing cytometer (Applied Biosynthesis, Invitrogen, Germantown, MD). 2.5 Drug efflux inhibition of ZD NPs MCF-7R cells were incubated with 10 µg/mL free DOX or ZD NPs for 4h, then the medium containing drug was removed, and washed by PBS solution for three times, followed by incubating with fresh complete medium for another 0h, 1h, 3h, 6h or 8h. At end, cells were washed by PBS, harvested by trypsin-EDTA, and analyzed by flow cytometry using an Attune® acoustic focusing cytometer. 2.6 Cytotoxicity studies MCF-7R and MCF-7S cells were seeded in a 96-well plate, preincubated for 24h at 37°C, and then treated with free DOX, ZD NPs for 24h at doxorubicin concentrations of 0.001 to 100 µg/mL. The medium containing drug was replaced with 100 µl serum free medium containing


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MTT at 0.5 mg/mL, and after 3h, the medium containing MTT was replaced by 100 µl DMSO. Finally, the absorbance was determined at 570 nm with the reference wavelength of 630 nm by microplate reader (Infinite M200, Tecan, Durham, USA). Cells without treatment were used as control. All experiments were repeated with four times. The cytotoxicity of ZnO NPs was tested as the method discribed above.

3. Results and Discussions 3.1 Preparation and Characterization of ZD NPs The ZD NPs were prepared directly in ethanol with reprecipitation method, and dried in vacuum oven overnight, prior to characterization. The morphology of ZD NPs at different pH (7.5 and 5.0) was determined by TEM and SEM. At pH 7.5, TEM micrograph of ZD NPs and its magnified image in Figure 2A showed ZnO and DOX assembled into nano-assemblies which are highly porous in nature. From the SEM micrographs in Figure 2B, the ZD NPs were in spherical shape, with the diameter of ~ 40 nm. At the same time, the size distribution was determined by DLS. They had a good dispersion, which is in a good agreement with DLS result in Figure S1. All of these results proved the potential application of these porous assemblies as a novel drug carrier. At the same time, the instant decomposition of ZD NPs at low pH was verified by analyzing TEM and SEM micrographs. We readjusted the pH of aqueous solution of ZD NPs to 5.0 by adding acetate buffer and immediately observed the samples by TEM and SEM. The NPs could readily be found at pH 7.5 as depicted, whereas at pH 5.0, we couldn’t find any intact nanostructures in Figure 2C and D. The results were a convincing evidence of instant decomposition of ZD NPs in mildly acidic environment, precisely because of the rapidly decomposition of ZnO.


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Conventionally, drugs are encapsulated into nanocarriers by covalent or non-covalent interactions. In this study, Zeta potential analysis of ZnO NPs represented that the negative charge of -14.4 mV, while the surface charge of ZD NPs still showed a negative charge of -7.6 mV, but the value decreased due to the loading of positively charged DOX (Figure S2). The results indicated that DOX was loaded into ZnO nanostructures by electrostatic interactions, which are non-covalent interactions. The fluorescence spectra of the nanoparticles was also measured. What's worth mentioning is that the fluorescence intensity of ZnO NPs and DOX markedly decreased after formation of ZD NPs. Figure 3A showed that the luminescence of ZnO NPs solution was green under UV irradiation, while the luminescence of DOX solution was red. The absorption peak of ZnO NPs was about 330nm, and the wavelength of emission was around 510nm. The absorption peak of DOX was around at 477nm and the red emission wavelength was around 590 nm. After the formation of ZD NPs, no matter the excitation wavelength was 330 nm or 477 nm, ZD NPs always showed no fluorescence. By rights, the photoluminescence emission band of DOX in ZD NPs should be enhanced significantly, which could be explained by the fluorescence resonance energy transfer (FRET) effect. However, in our system, we concluded that ZnO transferred its energy to DOX, and leaded to the fluorescence decrease of ZnO. However, the transferred energy didn’t cause the fluorescence increase of DOX, because “π−π stacking” interaction between doxorubicin molecules leaded to fluorescence quenching of DOX. This was a kind of energy transfer relay, which has been discussed in detail in our previous work.37 We hypothesized that the DOX fluorescence should be recovered after it is released from ZD NPs. 3.2 pH-Responsive Drug Release from ZD NPs


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The degradation of ZnO nanomaterials will cause the DOX release from ZD NPs. The quenched fluorescence would recover, once DOX was released, following the decompositon of ZnO. To demonstrate this, the pH-responsive behavior of ZD NPs was also validated by fluorescence spectroscopy. Firstly, to study the degradation of nanocarrier, we adjusted the pH level of ZnO NPs aqueous solution. Figure 3B illustrated the green fluorescence of ZnO disappeared when pH value of ZD NPs solution was altered from 7.5 to 5.0, suggesting the decomposition of ZnO. In contrast, the red fluorescence of DOX instantly recovered when the pH value dropped with the slowly addition of acetate buffer (Figure S3 and Figure 3C). These results confirmed that the DOX molecules had been released from ZD NPs. Exposure to pH 6.0, which mimicks the extracellular tumor environment, caused 60% drug release. When the pH was reduced to 5.0, mimicking endosomal/lysosomal environment, burst drug release was observed due to the instant decomposition of ZnO. Based on the above results, a self-responsive DDS (ZD NPs) loaded with the anticancer drug — DOX was successfully fabricated, which is a pHresponsive, degradable system and suitable for cancer therapy. 3.3 In Vitro Drug Delivery and Release of ZD NPs Here, the breast cancer cell line, MCF-7R, which is resistant to DOX, was selected to test the efficiency of ZD NPs in overcoming MDR. MCF-7R cells were overexpressed of MDR1 gene and P-gp protein, indicating their great drug-resistance property.15 MCF-7S cells, which is sensitive to DOX were also selected to test and verify the anticancer activity of ZD NPs. Before the in vitro experiments, we checked the stability of ZD NPs in the serum containing medium. As shown in Figure S4, no aggregation or precipitation of ZD NPs was observed after incubating with serum containing medium for 24 h, indicating the good stability of ZD NPs.


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As we described above, after the DOX was delivered and released from ZD NPs, the recovered fluorescence could be real-time observed to achieve the dynamic trafficking of drug release. Subsequently, confocal microscopy was used to evaluate the ability of ZD NPs for effectively delivery and controlled release of drug in cells. MCF-7R, and MCF-7S cells were treated with ZD NPs for 1 h and 4 h. Subcellular localization of free DOX, and ZD NPs in MCF7R are shown in Figure 4A, MCF-7S in Figure S5. In the free DOX treated group, we can only observe very faint red fluorescence after 1 h of treatment, However, in cells treated with ZD NPs, the fluorescence of DOX is already bright after 1 h. The trend is also obvious after 4 h of treatment, indicating after the DOX loaded into ZnO NPs, the nanostructure could significantly promote the internalization of drug into cancer cells, and ZD NPs entered the cells more rapidly than free DOX. In ZD NPs treated cells, the DOX fluorescence appeared in both of the lysosomes and cytoplasm. We conclude that after the ZD NPs entered into the endosome or lysosome of cancer cells, DOX could be easily released from the nanostructures and enter the cytoplasm, due to their pH sensitive property. In addition, the uptake behavior of ZD NPs was time-dependent, and the fluorescence increased while the treated time increased. Further, we made a quantitative measurement using flow cytometry. All the cell lines treated with ZD NPs showed a significant right shift using flow cytometry analysis, indicating the enhanced cellular uptake. As shown in Figure 4C, almost all the cells represented increased fluorescence, suggesting their ability of internalizing free DOX and ZD NPs, and nearly 100% of cells showed increased fluorescence.. However, a big difference of mean fluorescence intensity (MFI) between free DOX and ZD NPs could be found, especially in MCF-7R cells (Figure 4D). The cell uptake of free DOX by MCF-7R was markedly lower than that by MCF7S (Figure S6), due to P-gp induced decreasing cell uptake and increasing cell efflux. The MFI


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in ZD NPs treated cells was 11-fold higher than free DOX treated cells after 1h, indicating that ZD NPs can successfully escape from the P-gp pumps and rapidly enter MCF-7R cells, leading to significantly enhanced accumulation of DOX. 3.4 Decreased drug efflux of ZD NPs on resistant cancer cells It has been well established that decreased cell uptake and increased cell efflux are the main mechanisms leading to drug resistance, which is associated with over-expressed P-gp on drugresistant cancer cells. In above discussion, we have confirmed that the ZD NPs could enhance the cellular uptake of drug. To further determine whether ZD NPs helped to avoid the P-gp caused drug efflux, drug efflux of MCF-7R cells was detected. MCF-7R cells were treated with medium containing free DOX and ZD NPs for 4h at equal DOX concentration at 10 µg/mL respectively, then replaced by fresh medium for 1h, 3h, 6h, and 8h. The result in Figure 5 showed that, most of free DOX was pumped out of the MCF-7R cells within 1h, whereas in ZD NPs treated cells, little DOX was pumped out even after 8h. These results demonstrated that DOX loaded into ZD NPs can effectively circumvent the efflux of P-gp. Overall, our system significantly enhanced cellular uptake of DOX, decreased the efflux of drug, and hence increase the drug effective concentration in drug-resistant tumor cells. 3.5 In vitro cytotoxic activity Once it was verified that, the ZD NPs were able to effectively deliver and controlled release DOX in cancer cells, the cytotoxicity of ZD NPs and free DOX was investigated using MTT assay. After the cells were incubated in the medium containing free DOX or ZD NPs, we found that compared to free DOX, ZD NPs were more toxic to the all the cell lines. As depicted in Figure 6, ZD NPs showed a lower IC50. More importantly, the bigger difference of IC50 between free DOX and ZD NPs were found on MCF-7R, suggesting that ZD NPs were able to efficiently


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overcome the drug resistance. The IC50 of ZD NPs against MCF-7R cells is 0.5 µg/mL, while free DOX could only kill less than 10% resistant cells even at the maximum DOX concentration of 100 µg/mL. Therefore, we concluded as follows, the developed nanosystems could effectively overcome drug resistance. In addition, we also evaluated the cytotoxicity of ZnO NPs. The result, shown in Figure S7, indicated that they only had little toxicity to MCF-7R and MCF-7S cells at the concentration corresponding to DOX, which is possibly attributed to the Zn2+ released from the NPs after degradation. Hence, lower IC50 of ZD NPs was contributed by the toxic effect of the nanocarrierss themselves, but mainly by enhanced cellular internalization and retention of DOX. To further illustrate the mechanism of drug resistance circumvented by ZD NPs, we used LysoSensor Green DND-189 to probe the pH value of lysosome in the drug-resistant and sensitive cells. The results in Figure 6 revealed that MCF-7R cells showed a lower lysosomal pH level than MCF-7S cells. It could be concluded that, more acidic microenvironment of lysosomes in drug-resistant cancer cells was helpful for degradation of ZD NPs, thereby contribute to circumvention of DOX-resistance. This gives an explanation why the bigger difference in IC50 is observed between free DOX and ZD NPs on the resistant cells.

4. Conclusion Due to their unique properties, nanoparticles-based drug delivery system has been payed much attention as a candidate with great promise for successful clinical cancer therapy. However, MDR continues remains to be a key barrier. In our study, we developed a innovative pHresponsive DDS based on ZnO nanomaterials by simple reprecipitation method to load the anticancer drug DOX within the ZnO nanostructures with simple chemistry. The resulting ZD


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NPs can bypass the P-gp induced drug efflux, while and therefore increase the drug accumulation in resistant cells. More importantly, ZnO could dissolve to zinc ions in response to pH and to achieve the degradation of nanocarriers once the nanoplatform enters the endosome/lysosome, leading to a rapid intracellular drug release. Our results have demonstrated that the delivery of DOX with this system significantly inhibit the proliferation of cancer cells, especially drug-resistant cells, owing to the high intracellular drug concentration. On the other hand, it is worth noting that this system can easily achieve the real-time tracking of the drug release in cancer cells. Therefore, the ZD NPs served dual roles by overcoming drug resistance and probing the intracellular drug release from DDS. In summary, this work opens up a new insight to the development of responsive DDS with simple method to reverse the multidrug resistance of cancer.


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Figure 1. Schematic presentation showed the formation of ZD NPs and their combined effect of intensive cellular internalization and a pH-triggered intracellular drug release for overcoming drug resistance. The drug acid-triggered released from ZD NPs with the fluorescence recovery (Fluorescence “ON”), and then entered into nucleus to execute its anticancer function.


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Figure 2. Characterization of ZD NPs at different pH (7.5 and 5.0). TEM images of ZD NPs (A: pH7.5; C: pH5.0); SEM images (B: pH7.5; D: pH5.0).


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Figure 3. (A) Fluorescent behaviors of ZnO NPs, free DOX, and ZD NPs. Fluorescence spectra of ZnO NPs, free DOX, and ZD NPs was on the left. ZnO NPs λex = 330 nm; Free DOX λex = 477 nm; ZD NPs λex = 330 nm and λex = 477 nm. Bright photographs and fluorescence photographs of ZnO NPs (1), free DOX (2), and ZD NPs (3) were on the right. (B) Fluorescence spectra of ZnO NPs at pH 7.5 and 5.0 excited at 330nm. Photograph showed the fluorescence of ZnO NPs at different pH. The fluorescence of ZnO NPs disappeared at acid condition due to the dissolution of ZnO. (C) Fluorescence spectra of ZD NPs at pH 7.5 and 5.0 excited at 477nm. Photograph showed the fluorescence of ZD NPs at different pH. The recovered red fluorescence was suggesting the acid-triggered release of doxorubicin.


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Figure 4. Subcellular spatial distribution and intracellular uptake of different DOX formations. (A) Confocal images of the distribution of free DOX and ZD NPs. Scale bars are 10µm. (B) The quantitative uptake of ZD NPs. (C) Percentages of cells with appeared fluorescence. (D) MFI of cells. Control cells were without treatment (**, p < 0.01).


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Figure 5. Inhibition of doxorubicin efflux by ZD NPs in drug-resistant cells. (A) Quantitative analysis of free DOX efflux. (B) Quantitative analysis of ZD NPs efflux. (C) MFI of cells. (D) Percentages of MCF-7R cells with decreased fluorescence.


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Figure 6. In vitro cytotoxicity of free DOX, or ZD NPs against drug-resistant cells (A, MCF7R) and sensitive cells (B, MCF-7S) determined by MTT assay. The pH value of lysosome was measured by the confocal microscopy (C) and flow cytometry (D, E).


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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * Corresponding author: CAS Key Laboratory for Biological Effects of Nanomaterials & Nanosafety, National Center for Nanoscience and Technology, No.11, First North Road, Zhongguancun, Beijing 100190 (China), Fax: (+86) 10-62656765, Tel: (+86) 10-82545569, Email: [email protected], [email protected]. Author Contributions † J. Liu and X.W. Ma contributed equally to this paper.

ACKNOWLEDGMENT This work was supported by the Chinese Natural Science Foundation key project (31430031) and National Distinguished Young Scholars grant (31225009), State High-Tech Development Plan (2012AA020804 and SS2014AA020708). The authors also appreciate the support by the "Strategic Priority Research Program" of the Chinese Academy of Sciences，Grant No. XDA09030301 and support by the external cooperation program of BIC, Chinese Academy of Science, Grant No. 121D11KYSB20130006.


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Table of Contents


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Figure 1. Schematic presentation showed the formation of ZD NPs and their combined effect of intensive cellular internalization and a pH-triggered intracellular drug release for overcoming drug resistance. The drug acid-triggered released from ZD NPs with the fluorescence recovery (Fluorescence “ON”), and then entered into nucleus to execute its anticancer function. 56x39mm (300 x 300 DPI)



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Figure 2. Characterization of ZD NPs at different pH (7.5 and 5.0). TEM images of ZD NPs (A: pH7.5; C: pH5.0); SEM images (B: pH7.5; D: pH5.0). 78x74mm (600 x 600 DPI)


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Figure 3. (A) Fluorescent behaviors of ZnO NPs, free DOX, and ZD NPs. Fluorescence spectra of ZnO NPs, free DOX, and ZD NPs was on the left. ZnO NPs λex = 330 nm; Free DOX λex = 477 nm; ZD NPs λex = 330 nm and λex = 477 nm. Bright photographs and fluorescence photographs of ZnO NPs (1), free DOX (2), and ZD NPs (3) were on the right. (B) Fluorescence spectra of ZnO NPs at pH 7.5 and 5.0 excited at 330nm. Photograph showed the fluorescence of ZnO NPs at different pH. The fluorescence of ZnO NPs disappeared at acid condition due to the dissolution of ZnO. (C) Fluorescence spectra of ZD NPs at pH 7.5 and 5.0 excited at 477nm. Photograph showed the fluorescence of ZD NPs at different pH. The recovered red fluorescence was suggesting the acid-triggered release of doxorubicin. 54x36mm (300 x 300 DPI)



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Figure 4. Subcellular spatial distribution and intracellular uptake of different DOX formations. (A) Confocal images of the distribution of free DOX and ZD NPs. Scale bars are 10µm. (B) The quantitative uptake of ZD NPs. (C) Percentages of cells with appeared fluorescence. (D) MFI of cells. Control cells were without treatment (**, p < 0.01). 131x211mm (300 x 300 DPI)


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Figure 5. Inhibition of doxorubicin efflux by ZD NPs in drug-resistant cells. (A) Quantitative analysis of free DOX efflux. (B) Quantitative analysis of ZD NPs efflux. (C) MFI of cells. (D) Percentages of MCF-7R cells with decreased fluorescence. 69x58mm (600 x 600 DPI)



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Figure 6. In vitro cytotoxicity of free DOX, or ZD NPs against drug-resistant cells (A, MCF-7R) and sensitive cells (B, MCF-7S) determined by MTT assay. The pH value of lysosome was measured by the confocal microscopy (C) and flow cytometry (D, E). 86x89mm (300 x 300 DPI)


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Table of Contents 35x24mm (300 x 300 DPI)


Zinc Oxide Nanoparticles as Adjuvant To Facilitate Doxorubicin

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