H2O2-Responsive MB–BSA–Fe(III) Nanoparticles as Oxygen Generators for MRI-Guided Photodynamic Therapy
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The local tumor hypoxia has become one of the main obstacles for photodynamic therapy (PDT).To break through this barrier, the fabrication of O2-evolving nanoplatform has been more significant. Herein, the uniform methylene blue (MB)–bovine serum albumin (BSA)–Fe(III) nanoparticles (MB–BSA–Fe(III) NPs) were successfully synthesized by biomineralization approach. MB–BSA–Fe(III) NPs show high loading capacity of MB, and Fe(ΙΙΙ) has excellent catalytic performance towards abundant H2O2 in cancer cells to generate O2, overcoming tumor hypoxia. All these combination effects remarkably increase the therapeutic efficiency of PDT, resulting in almost complete destruction of cancer cells. Most importantly, MB–BSA–Fe(III) NPs exhibit T2- magnetic resonance imaging, which have the potential for precise visualization of the tumor location. Our results demonstrate that MB–BSA–Fe(III) is a novel H2O2-responsive and O2-evolving nanoplatform and it has great potential for clinical applications such as cancer diagnosis, treatment or drug delivery.
KeywordsPhotodynamic therapy Biomineralization H2O2-responsive O2 generator Photosensitizer Magnetic resonance imaging
For the past few decades, cancer has turned into one of the leading causes of modality, and more effort has to be devoted to seek a practicable approach to treat cancer [1, 2]. Photodynamic therapy (PDT) has been proven effective for treating different types of tumor [3, 4] because of its noninvasive nature, less inflammatory side effect, negligible drug resistance and low systemic toxicity . PDT produces toxic reactive oxygen species (ROS) such as 1O2 through using a nontoxic photosensitizer (PS) and specific wavelength light in the presence of oxygen, and induces cell death . However, tumor hypoxia greatly limits the efficiency of PDT . The design and synthesis of nanomaterials with an efficient O2-evolving property and high loading capacity of PS are of great significance but still challenging.
Recently, multifunctional nanoparticles fabricated by employing biomineralization approach have attracted much attention in biomedical and multimodal imaging applications . Protein has abundant active groups, which offer great possibility to integrate organic molecules and trigger the formation of metal ions into ion complex at mild temperature [9, 10]. In addition, albumin-mediated nanomaterials showed good biocompatibility, high diagnostic agent delivery capacity, robust stability, excellent water solubility and good reproducibility, and have potential for medical diagnosis and treatment . Consequently, it is sensible to prepare multifunctional nanomaterials through biomineralization approach.
2 Experimental Section
2.1 Chemicals and Reagents
Bovine serum albumin (BSA), 1,3-diphenylisobenzofuran (DPBF), methyl thiazolyl tetrazolium (MTT), methylene blue (MB), FeCl3 and NaOH were purchased from Aladdin Reagents Company (Shanghai, China). Ethanol, H2O2 (30%) were obtained from Beijing Chemical Reagents Company (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM) was purchased Thermo Scientific (Beijing, China). [Ru(dpp)3]Cl2 (RDPP), calcein acetoxymethyl ester (Calcein AM), propidium iodide (PI) and 2,7-dichlorofluorescein diacetate (DCFH-DA) were obtained from Sigma–Aldrich (MO, America). Standard fetal bovine serum (FBS) was purchased from Tianjin Haoyang Biological Manufacture Co. Ltd. (Tianjin, China). Dimethyl sulfoxide (DMSO) was obtained from Tianjin Concord Technology. The deionized (DI) water was generated using a Millipore–Milli-Q system (Billerica, MA). All the chemicals were used without additional purification.
The morphology of MB–BSA–Fe(III) NPs and BSA–Fe(III) NPs was investigated by transmission electronic microscope (TEM, H-600 electron microscope, Hitachi, Japan). Fourier transform infrared (FTIR) spectra were measured on a Nicolet 520 FTIR spectrometer (Nicolet) equipped with a germanium attenuated total reflection (ATR) accessory, DTGS KBr detector, and KBr beam splitter (Thermo Fisher Scientific, USA). UV-1700 spectrophotometer (Shimadzu, Japan) was utilized to obtain UV–Vis absorption spectra. The concentration of metallic element was measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES, Thermo Scientific Xseries 2, Thermo Fisher Scientific, USA). The portable dissolved oxygen meters (JPBJ-608, INESA, China) were utilized to monitor the concentration of dissolved oxygen.
2.3 Preparation of MB–BSA–Fe(III) NPs and BSA–Fe(III) NPs
To prepare MB–BSA–Fe(III) NPs, BSA (1 g) was dissolved in deionized water (36 mL), and 1 mL of FeCl3 aqueous solution (100 mM) was added into the above mixture under vigorous stirring. Then, MB aqueous solution (0.5 mg mL−1, 1 mL) was added dropwise into the mixture, then, NaOH solution (2.0 M) was applied to regulate pH (pH 12) and the mixture was stirred at 25 °C for 12 h in dark. Finally, the mixture was further dialyzed (membrane cutoff Mw: 100 KD) against deionized water for 24 h to remove excess precursors. The preparation process of BSA–Fe(III) NPs is similar to MB–BSA–Fe(III) NPs, and only the MB was not required during the preparation process. The precise concentration of MB loaded onto MB–BSA–Fe(III) NPs was detected by the UV–Vis spectrophotometer. The exact concentrations of Fe were detected by ICP–AES.
2.4 Extracellular Generation of O2
25 mL of MB–BSA–Fe(III) NPs aqueous solution (3 mg mL−1, [MB] = 5.89 µg mL−1) was added into 25 µL of H2O2 (30%) under vigorous stirring at room temperature. The dissolved oxygen meter was inserted into the above solution to detect the concentration of O2 every 10 s. MB–BSA–Fe(III) NPs aqueous solution without the addition of H2O2 served as control.
2.5 Extracellular 1O2 Detection
DPBF was employed to evaluate the 1O2 generation of MB–BSA–Fe(III)-NPs by UV–Vis absorption spectra. 10 µL of DPBF solution (10 mM in ethanol) was added into 890 µL of ethanol solution containing different concentrations of H2O2 (0, 40, 80 and 100 µM). Then, the mixture was transferred into a 10 mm-cuvette. The cuvette was irradiated with a 650 nm laser for 10 min (0.1 W cm−2) after the addition of MB–BSA–Fe(III) NP aqueous solution (2 mg mL−1, [MB] = 3.92 µg mL−1, 100 µL). The absorbance of DPBF at 410 nm was recorded every 2 min. The group without H2O2 and laser illumination served as control.
2.6 In Vitro Dark Cytotoxicity of MB–BSA–Fe(III) NPs
HeLa cells (5 × 103 cells per well) were seeded onto 96-well plates and incubated in 5% CO2 at 37 °C for 24 h. Then, 200 µL of MB–BSA–Fe(III)NP solution with different concentrations (0, 0.2, 0.4, 1, 1.5, 2 mg mL−1 in culture medium) were added into each well, and the cells were continued to be cultured for another 24 h. Cells were washed twice with PBS, and then 100 µL of MTT solution (0.5 mg mL−1 MTT in PBS, pH 7.4) was added into each well. Then, cells were incubated for another 4 h and the PBS was abandoned. The intracellular formazan crystals were dissolved by adding 100 µL of DMSO. The absorbance was recorded at 570 nm by a plate reader, and the cell viability was determined by comparing cells with control group.
2.7 The Detection of Intracellular O2
The intracellular production of O2 was measured with RDPP by confocal laser fluorescence scanning microscope (CLSM). HeLa cells (2.5 × 104 cells per dish) were seeded onto culture dishes and incubated at 37 °C in 5% CO2 for 24 h. Then, the cells were incubated with 1.5 µM RDPP suspended in medium for another 4 h. After that, each culture dish was washed with PBS for three times and further incubated with 1 mL of MB–BSA–Fe(III) NP solution (1.2 mg mL−1 in culture medium, [MB] = 2.4 µg mL−1), BSA–Fe(III) NP solution (1.2 mg mL−1 in medium) and free MB solution (2.4 µg mL−1) for 24 h. The cells without treatment served as control group. Finally, the cells were washed with PBS for three times, and fluorescence images were obtained with CLSM using an excitation of 488 nm and the emission of 600–700 nm.
2.8 The Generation of Intracellular 1O2
Intracellular 1O2 were detected by DCFH-DA. HeLa cells (2.5 × 104 cells per dish) were seeded onto culture dishes in 5% CO2 at 37 °C for 24 h. Then, cells were treated with 1 mL of MB–BSA–Fe(III) NP solution (1.2 mg mL−1 in medium, [MB] = 2.4 µg mL−1), BSA–Fe(III) NPs solution (1.2 mg mL−1 in medium) and free MB solution (2.4 µg mL−1) for 24 h. Finally, the cells were washed with PBS for three times and illuminated with a 650 nm laser (0.1 W cm−2) for 15 min. The cells without treatment acted as control. After 24 h, the cells were washed with PBS for three times and incubated with 1 mL of DCFH–DA solution (0.2 µM in PBS) for 30 min. Fluorescence of the cells were imaged with CLSM using an excitation of 488 nm, and the emission was collected between 510 and 560 nm.
2.9 In Vitro Photodynamic Therapy Effect
HeLa cells (2.5 × 103 cells per well) were seeded onto 96-well plates and incubated in 5% CO2 at 37 °C for 24 h. Then, the cells were treated with free MB (2.4 µg mL−1) and MB–BSA–Fe(III)–NPs (1.2 mg mL−1, [MB] = 2.4 µg mL−1) medium solutions. After 24 h, the cells were washed with PBS for three times, and 200 µL of fresh medium was added into each well. The cells were irradiated with the 650 nm laser at 0.1 W cm−2 for 15 min. The cells without treatment served as control. After 24 h, the cell viability was detected by MTT assay and investigated on a CLSM after co-staining with Calcein AM (40 nM) and PI (4.5 μM) for 20 min.
2.10 In Vitro MRI
MB–BSA–Fe(III) NPs were dissolved in 1 mL of deionized water with different concentrations in terms of the molar concentration of Fe (0.002, 0.004, 0.008, 0.016, 0.032, 0.064 mM). The relaxation time (T2) and transversal phantom images of MB–BSA–Fe(III)-NP solutions were recorded by a 3.0 T clinical MRI scanner (Philips, The Netherlands).
3 Results and Discussion
3.1 Synthesis and Characterization of MB–BSA–Fe(III)-NPs
3.2 Catalase-Like Activity of MB–BSA–Fe(III) NPs
In recent years, nanozymes have appealed to growing researchers, so the construction of artificial enzyme has great importance. Several studies have reported that Fe(III) with catalase-like activity can decompose H2O2 to generate O2 . Here, to verify the catalase-like activity of MB–BSA–Fe(III) NPs, we next measured the time-dependent production of O2 in the MB–BSA–Fe(III) NPs solution upon addition of 1 mM H2O2 with an oxygen meter (Fig. 1f). More and more amount of O2 was detected with time relative to the control suspension without H2O2, suggesting that MB–BSA–Fe(III) NPs can greatly enhance the generation of O2.
3.3 Photodynamic Effect of MB–BSA–Fe(III) NPs
3.4 CAT-Like Property and Synergistic Photodynamic Efficacy of MB–BSA–Fe(III) NPs in HeLa Cells
3.5 Imaging Properties of MB–BSA–Fe(III) NPs
In summary, we have successfully synthesized MB–BSA–Fe(III) NPs by biomineralization approach, which could cleave H2O2 to generate O2, overcoming tumor hypoxia and thus enhancing PDT efficiency. Moreover, MB–BSA–Fe(III) NPs have great biocompatibility, uniform morphology and high loading capacity of MB. Most importantly, MB–BSA–Fe(III) NPs exhibit excellent T2-contrast effect in MRI, which shows great potential for precise visualization of the tumor location. The above combination effects significantly increase the therapeutic efficiency of PDT, leading to destruction of almost all cancer cells. This proof of concept might provide a novel H2O2-responsive and O2-evolving nanoplatform for more efficient PDT, radiotherapy sensitization and increasing the effect of chemotherapy.
This work was financially supported by the National Natural Science Foundation of China (21675149, 21505130, 21705146), the Science and Technology Development Program of Jilin Province (20150519014JH, 20170414037GH, 20170520133JH), and the Key Research Program of Frontier Sciences, CAS (QYZDY-SSW-SLH019).
- 6.Ge J, Lan M, Zhou B, Liu W, Guo L, Wang H, Jia Q, Niu G, Huang X, Zhou H, Meng X, Wang P, Lee C, Zhang W, Han X. A graphene quantum dot photodynamic therapy agent with high singlet oxygen generation. Nat. Commun. 2011;5:4596–603.Google Scholar