Citation

Yuzhen Wang, Yujun Song, Guixian Zhu, Dechen Zhang, Xuewu Liu. Highly biocompatible BSA-MnO₂ nanoparticles as an efficient near-infrared photothermal agent for cancer therapy[J]. Chin. Chem. Lett., 2018, 29(11): 1685-1688  

Yuzhen Wang^a, Yujun Song^b, Guixian Zhu^c, Dechen Zhang^c, Xuewu Liu^c    

Cancer has become one of the leading diseases threatening human health [1, 2]. Current cancer therapies including surgery, chemotherapy and radiotherapy, have their own limitations, such as low therapy efficiency and high side effects [3]. Photothermal therapy (PTT) is an emerging technology for cancer treatment through conversion of near infrared (NIR) light into hyperthermia. It has attracted much attention due to simple, noninvasive, highly selective characteristics which destroy cancer cells without affecting surrounding healthy tissues as the PTT agent and nearinfrared light are localized in tumor sites [4].

To date, a variety of NIR-activated materials have been reported for photothermal cancer therapy. Gold nanostructures, such as Au nanorod [5-7], Au nanocage [8] or Au nanoshells [9], showed high light extinction coefficients and photothermal conversion efficiency in the NIR region owing to strong surface plasmon resonance (SPR) absorption. However, these materials are costly and tend to undergo irreversibly morphology changes as well as photothermal conversion diminishes gradually upon NIR repeated irradiation [10]. Carbon-based nanomaterials, such as carbon nanotube [11-13] and grapheme [14, 15], suffer from tedious synthesis, purification and modification procedures. Furthermore, the size cannot be well controlled. The other kinds of copper sulphide (CuS) [16, 17], WS₂ nanosheets [18], and naphthalocyanine [19] also demonstrated to be useful for promising photothermal agents, but their longterm toxicity remains concerns.

Manganese dioxide (MnO₂) has attracted tremendous interests in a wide variety of fields including nanoelectronics [20, 21], sensor [22, 23], molecules adsorption [24] and catalysis [25]. More recently, the biomedical applications of MnO₂ in bioanalysis, cell imaging, and drug delivery as a result of their appealing physicochemical properties, have been reported and expanded rapidly [26-31]. However, research on a NIR photothermal response of MnO₂ was ignored. In addition, the biological applications of MnO₂ still face challenges: On one hand, hydrophobic properties of MnO₂ make it not stable in aqueous media and physiological environments, resulting in that surface modification is surely required; on the other hand, few functional groups available on the surface of MnO₂ make the further bioconjugation difficult and very complex. Therefore, in order to endow MnO₂ with great colloidal stability in physiological solutions and muti-functionalities (e.g., tumor targeting ability), there is a need to develop a simple method to synthesize and functionalize MnO₂. Albumin, as the most abundant serum protein, is widely used to templated synthesize nanoparticles [32] and enhance the biocompatibility of various types of nanoparticles by coating on their surface [33]. Furthermore, albumin coating can facilitate post-synthesis surface modifications with functional ligands. Herein, we report the one-step synthesis of bovine serum albumin (BSA)-stabilized MnO₂ nanoparticles (BSA-MnO₂ NPs) with good dispersion and high biocompatibility, which serve as a novel class of effective photothermal agent under the laser irradiation.

For the synthesis of well-stable BSA-MnO₂ NPs dispersions, we employed a facile, one-pot method by simply mixing manganese permanganate (KMnO₄) directly with BSA for overnight until the color of the solution changed from fuchsia to brown, indicating that all the KMnO₄ was reduced to MnO₂. In this synthesis procedure, BSA served as a reducing reagent as well as a protective coating in consideration of the aggregation tendency of hydrophobic MnO₂ itself. Transmission electron microscopy (TEM) image (Fig. 1a) revealed that the resultant BSA-MnO₂ NPs have a spherical structure with an average size distribution of around 5 nm. The BSA-MnO₂ NPs exhibited high uniformity, revealed from the low polydispersity index (PDI) measured by dynamic light scattering (DLS) (inset in Fig. 1a). In the biomedical applications, the stability of photothermal agents under physiological conditions is the first issue to be considered. Therefore, we examined the dispersion of BSA-MnO₂ NPs in H₂O, phosphate-buffered saline (PBS) and cell culture medium (DMEM containing 10% of fetal bovine serum). We found that the prepared BSA-MnO₂ NPs exhibited a remarkable physiological stability and were well distributed in these matrixes without any sedimentation even after 1 week storage at room temperature (Figs. S2 and S3 in Supporting information). As shown in Fig. S4 (Supporting information), there was no obvious change of size distribution for BSA-MnO₂ NPs dispersed in PBS buffer and culture medium due to the strong hydrophilicity of MnO₂ NPs functionalized with BSA. Meanwhile, the zeta potential of BSAMnO₂ NPs was negative (-35 mV in H₂O, -14.9 mV in PBS and -17 mV in the medium), making it suitable for further use in biological systems (Fig. S5 in Supporting information).

Figure 1

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Fig. 1. (a) TEM image of BSA-MnO2 NPs. Inset is the size distribution of BSA-MnO2 NPs measured by DLS. (b) UV-vis-NIR spectra of various concentrations of BSA-MnO2 NPs in water. (c) Temperature changes at different concentration of BSA-MnO2 NPs during exposure to NIR light (810 nm, 15 J/cm2) over a period of 6 min. Water and 1% BSA aqueous solution were used as controls. (d) The temperature elevation of BSA-MnO2 NPs over 5 irradiation cycles.

The optical properties of our BSA-MnO₂ NPs were then carefully examined. As illustrated in Fig. 1b, UV-vis-NIR spectra showed that BSA-MnO₂ NPs had a strong absorbance extending from UV to NIR regions. The absorbance intensity increases and the peak of maximum absorbance red shifts with increasing of BSA-MnO₂ NPs concentration. As seen in Fig. S6 (Supporting information), some MnO₂ particles are self-assembled into an ordered particle matrix leading to the formation of a larger particle with the increasing of MnO₂ NPs concentration. This may cause maximum absorbance peaks shift to higher wavelength. Fig. S7 (Supporting information) displayed that there was a good linear relationship between the absorbance at 810 nm and the concentration of BSA-MnO₂ NPs, further verifying the great dispersity of BSA-MnO₂ NPs.

BSA-MnO₂ NPs exhibited an intense optical absorption with a broad peak covering a wavelength range of the visible and NIR region of the spectrum. The absorption in NIR range enables the potential of BSA-MnO₂ NPs as the photothermal agent to efficiently convert NIR light into heat. The photothermal effect in the presence of BSA-MnO₂ NPs was investigated by monitoring the temperature of 1 mL BSA-MnO₂ NPs dispersed in water at various concentrations ranging from 50 μg/mL to 1600 μg/mL irradiated by a NIR laser (810 nm, 15 J/cm²) for 6 min. Pure water and 1% BSA aqueous solution were used as a negative control. As depicted in Fig. 1c, the temperatures of all the BSA-MnO₂ NPs solution increased with the irradiation time, and the temperature increased more rapidly with increasing the concentration of BSA-MnO₂ NPs (Fig. S8 in Supporting information). In particular, at concentration of 200 μg/mL and under irradiation for 6 min, the temperature raised from room temperature up to 43.0 ℃, which is the sufficient temperature increase to induce cellular death [34, 35]. In comparison, the control experiment of pure water and 1% BSA without BSAMnO₂ NPs demonstrates that the temperature is only increased by 4.2 ℃. These results indicated that the BSA-MnO₂ NPs can rapidly and efficiently convert the 810 nm laser energy into thermal energy and could act as an efficient photothermal agent. In addition to good photothermal conversion capability, photostability of BSA-MnO₂ NPs is another important factor needing to be further investigated. To demonstrate this, the solution of BSAMnO₂ NPs was irradiated upon 5 continuous laser on/off cycles. As shown in Fig. 1d, there is no decline of maximum temperature elevation observed during the experiment. Furthermore, the solutions also keep their colloidal stability before and after exposure (Fig. S9 in Supporting information). As showed in previous studies, Au nanorod-based PTT agents suffered a serious decrease in the temperature elevation and their morphology changed a lot with increase of irradiation times [10]. These combined results indicated that the synthesized BSA-MnO₂ NPs possessed desirable efficiency and stability on the photothermal conversion and can be served as an excellent candidate for PTT.

It has been proved that the cellular uptake of photothermal materials can enhance phtothermal efficiency [36]. Therefore, we labelled BSA-MnO₂ NPs with fluorescent dyes to evaluate the cell uptake and internalization of BSA-MnO₂ NPs toward the human lung cancer cell line (A549). The amino-group in BSA coating allowed BSA-MnO₂ NPs to be conjugated with fluorescein isothiocyanate (FITC) (excite 492/emit 518 nm). After incubation with FITC-conjugated BSA-MnO₂ NPs for different time (1, 3, 6 and 24 h), A549 cells were thoroughly washed with PBS buffer and then fixed and treated with 4', 6-diamidino-2-phenylindole (DAPI) to label nuclei. As shown in Fig. 2, intracellular fluorescence increases with the incubation time, indicating effective and time-dependent cell uptake of BSA-MnO₂ NPs. A relative low-intensity fluorescence at 1 h incubation was observed, indicating that few BSA-MnO₂ NPs entered cells. However, after 24 h incubation, large accumulation of BSA-MnO₂ NPs occurs in the cells, as revealed by dramatic increase of fluorescence intensity. This high cellular uptake facilitates photothermal ablation of cancer cells.

Figure 2

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Fig. 2. Fluorescence images of A549 cells treated with FITC-labelled BSA-MnO2 NPs for (a) 1 h, (b) 3 h, (c) 6 h and (d) 24 h, respectively.

To investigate the localized tumor photothermal therapy effect of BSA-MnO₂ NPs, A549 cells cultured in 24-well plates were first incubated with 40 μg/mL BSA-MnO₂ NPs solution for 6 h (400 μL per well). After exposure to 810 nm laser for 5 min at 15 J/cm², costaining with calcein acetoxymethyl ester (Calcein AM) and propidium iodide (PI) was used to differentiate live or dead cells, respectively. As shown in Fig. 3, only green fluorescent signal was observed in the entire well when cells were treated with laser alone (Fig. 3a) or BSA-MnO₂ NPs without laser (Fig. 3b), indicating all the cells kept survival. This means that the used 810 nm laser and BSA-MnO₂ NPs are safe. In contrast, after treated with BSAMnO₂ NPs plus laser exposure (Fig. 3c), cells within the laser spot were substantially killed while most cells outside retained survival, suggesting that BSA-MnO₂ NPs could mediate localized hyperthermia to ablate the cancer cells in vitro.

Figure 3

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Fig. 3. Fluorescence microscopy images of A549 cells stained with calcein AM and PI after treated with (a) laser only, (b) BSA-MnO2 NPs only, and (c) BSA-MnO2 NPs and laser irradiation for 5 min. The white dot line represents laser spot.

Dedicated to biomedical applications, we further quantitatively evaluated their cytotoxicity of BSA-MnO₂ NPs on normal cells using an MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay. Human umbilical vein endothelial cells (HUVECs) and human breast epithelial cell line (MCF-10A) were selected here to represent normal cell lines. After incubating HUVECs and MCF-10A cells in the presence of BSA-MnO₂ NPs over a wide concentration range from 20 μg/mL to 320 μg/mL for 24 h, the cell viability did not change significantly compared to that of cells without BSA-MnO₂ NPs (Fig. 4a and Fig. S10a in Supporting information). These results demonstrate that BSA-MnO₂ NPs is biocompatible and non-cytotoxic even at a high concentration over 300 μg/mL, which is attributed that the coating of BSA can effectively lower the cytotoxicity of MnO₂. In contrast, when the A549 cancer cells were treated with BSA-MnO₂ NPs and NIR irradiation (810 nm, 15 J/cm², 5 min) simultaneously, the cell viability was sharply decreased after 24 h incubation in a concentration dependent manner (Fig. 4b). For example, at a concentration of 60 μg/mL, more than 65% of A549 cells were killed. The photothermal cytotoxicity of BSA-MnO₂ NPs on MCF-7 and A375 cells were also evaluated (Fig. S11 in Supporting information). Less than 10% of A375 cells are remained alive with BSA-MnO₂ NPs concentration only at 20 μg/mL. All these results demonstrated that BSA-MnO₂ NPs could be a promising photothermal therapeutic agent on cancer cells.

Figure 4

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Fig. 4. (a) Cell viability of HUVEC cells with 24 h exposure to various concentrations of BSA-MnO2 NPs. (b) Cell viability of A549 cells after photothermal treatment with different concentrations of BSA-MnO2 NPs upon NIR laser irradiation (810 nm, 15 J/cm2, 5 min). Cells without laser irradiation and BSA-MnO2 NPs as a control.

In summary, BSA-MnO₂ nanoparticles have been successfully prepared from a facile one-step route using BSA as both template and reducing agent, and applied for in vitro photothermal ablation of cancer cells. The BSA coating also is a stabilizer offering the asprepared BSA-MnO₂ NPs high water dispersibility, good biocompatibility as well as colloidal stability in different physiological environments. We reported for the first time that the synthesized BSA-MnO₂ nanoparticles displayed high absorbance in NIR region with superior NIR photothermal efficiency and high photothermal stability. More importantly, the BSA-MnO₂ nanoparticles exhibited low cytotoxicity on cells even at the high tested concentration greater than 300 μg/mL. Furthermore, in vitro fluorescence staining and MTT assay verify that photothermal antitumor activity could be realized by a low concentration of BSA-MnO₂ nanoparticles under 810 nm laser irradiation.

The authors acknowledge financial support from the following sources: National Natural Science Foundation of China (No. 81601632) and the Natural Science Foundation of Jiangsu Province (No. SBK2016041233), the Fundamental Research Funds for Central Universities (No. 021314380067), Thousand Talents Program for Young Researchers, NIH (No. 1R21CA190024-01), DOD (No. W81XWH-12-1-0414) and Houston Methodist Research Institute.

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.cclet.2017.12.004.