Tumor reoxygenation for enhanced combination of radiation therapy and microwave thermal therapy using oxygen generation in situ by CuO nanosuperparticles under microwave irradiation

Abstract

As known, radiation therapy (RT) can exacerbate the degree of hypoxia of tumor cells, which induces serious resistance to RT and in turn, is the greatest obstacle to RT. Reoxygenation can restore the hypoxic state of tumor cells, which plays an important role in reshaping tumor microenviroment for achieving optimal therapeutic efficacy. Herein, we report for the first time that microwave (MW)-triggered IL-Quercetin-CuO-SiO₂@ZrO₂-PEG nanosuperparticles (IQuCS@Zr-PEG NSPs) have been used to achieve an optimal RT therapeutic outcomes by the strategy of upregulating tumor reoxygenation, i.e. hypoxic cells acquire oxygen and return to normal state. Methods: We prepared a promising multifunctional nanosuperparticle to upregulate tumor reoxygenation by utilizing CuO nanoparticle to generate oxygen under MW irradiation in the tumor microenvironment. The IQuCS@Zr-PEG NSPs were obtained by introducing CuO nanoparticles, MW sensitizer of 1-butyl-3-methylimidazolium hexafluorophosphate (IL), radiosensitizer of Quercetin (Qu) and surface modifier of monomethoxy polyethylene glycol sulfhyl (mPEG-SH, 5k Da) into mesoporous sandwich SiO₂@ZrO₂ nanosuperparticles (SiO₂@ZrO₂ NSPs). The release oxygen by IQuCS@Zr-PEG NSPs under MW irradiation was investigated by a microcomputer dissolved oxygen-biochemical oxygen demand detector (DO-BOD) test. Finally, we used the ^99mTc-HL91 labeled reoxygenation imaging, Cellular immunofluorescence, immunohistochemistry, and TUNEL experiments to verify that this unique MW-responsive reoxygenation enhancer can be used to stimulate reshaping of the tumor microenvironment. Results: Through experiments we found that the IQuCS@Zr-PEG NSPs can persistently release oxygen under the MW irradiation, which upregulates tumor reoxygenation and improve the combined tumor treatment effect of RT and microwave thermal therapy (MWTT). Cellular immunofluorescence and immunohistochemistry experiments demonstrated that the IQuCS@Zr-PEG NSPs can downregulate the expression of hypoxia-inducible factor 1α (HIF-1α) under MW irradiation. The ^99mTc-HL91 labeled reoxygenation imaging experiment also showed that the oxygen generated by IQuCS@Zr-PEG NSPs under MW irradiation can significantly increase the reoxygenation capacity of tumor cells, thus reshaping the tumor microenvironment. The high inhibition rate of 98.62% was achieved in the antitumor experiments in vivo. In addition, the IQuCS@Zr-PEG NSPs also had good computed tomography (CT) imaging effects, which can be used to monitor the treatment of tumors in real-time. Conclusions: The proof-of-concept strategy of upregulating tumor reoxygenation is achieved by MW triggered IQuCS@Zr-PEG NSPs, which has exhibited optimal therapeutic outcomes of combination of RT and MWTT tumor. Such unique MW-responsive reoxygenation enhancer may stimulate the research of reshaping tumor microenvironment for enhancing versatile tumor treatment.

Keywords: microwave; radiation therapy; reoxygenation; thermal therapy; tumor.

Scheme 1

(A) A schematic diagram of the synthesis of IQuCS@Zr-PEG NSPs is not drawn to scale. (B) Schematic diagram of IQuCS@Zr-PEG NSPs with oxygen generation upregulating tumor reoxygenation for enhanced combination of radia-microwave thermal therapy by MW irradiation.

Figure 1

Characterization of physical and chemical properties of the IQuCS@Zr-PEG NSPs. (A) SiO₂ nanoparticles. (B) SiO₂@ZrO₂ NSPs. (C) CuO-SiO₂@ZrO₂ NSPs. (D) Lattice picture of CuO nanoparticles in CuO-SiO₂@ZrO₂ NSPs. (E) Original image for taking a mapping of CuO-SiO₂@ZrO₂ NSPs. (F) Zr element. (G) O element. (H) Cu element. (I) Si element. (J) FT-IR spectrum of IQuCS@Zr-PEG NSPs. (K) The EDS diagram of the IQuCS@Zr-PEG NSPs.

Figure 2

Evaluation of the ability of CuO-SiO₂@ZrO₂ NSPs to generate oxygen, the expression of HIF-1α and ^99mTc-HL91 labeled reoxygenation imaging experiment. (A) Picture of dissolved oxygen indicators for qualitative determination of oxygen production capacity of bare PBS (pH=5.5), dH₂O, CuO-SiO₂@ZrO₂ NSPs+dH₂O, CuO-SiO₂@ZrO₂ NSPs+PBS, CuO-SiO₂@ZrO₂ NSPs+MW+dH₂O and CuO-SiO₂@ZrO₂ NSPs+MW+PBS. (B) The dissolved oxygen concentration of the solution was quantitatively determined by a microcomputer DO-BOD detector. (C) Immunofluorescence staining of nucleus and HIF-1α after treatment with the IQuCS@Zr-PEG NSPS ((DAPI (blue) and anti-HIF-1 aipha antibody (green)). (D) Quantitative analysis of the region of interest was performed using radioactive counting methods. (E₁-E₅) The small animal SPECT scanning detects the reoxygenation status of the different groups, T indicates tumor, B indicates bladder.

Figure 3

The cell experiment and in vitro MW heating experiment results of the IQuCS@Zr-PEG NSPs. (A) The viability of human lung adenocarcinoma A549 cells co-incubated with the IQuCS@Zr-PEG NSPs at different concentrations were determined by MTT assay (n=5). (B) The viability of human lung adenocarcinoma A549 cells under different treatments or different concentrations of IQuCS@Zr-PEG NSPs for 24 h (n=5). (C) The viability of human lung adenocarcinoma A549 cells under different treatments or different concentrations of IQuCS@Zr-PEG NSPs for 48 h (n=5). (D) Corresponding to (E) image of the FLIR thermal image. (E) Temperature-raising of different concentration of the IQuCS@Zr-PEG NSPs saline solution under the irradiation by MW. (F) The highest temperature rise chart was corresponding to (E). Analysis of statistical (*indicates P < 0.05, ** indicates P < 0.01 and *** indicates P < 0.001).

Figure 4

Evaluation of in vivo treatment experiments of the IQuCS@Zr-PEG NSPs. (A) FLIR map of mice in the IQuCS@Zr-PEG+RT+MW group and the IQuS@Zr-PEG+RT+MW group after MW irradiation per minute. (B) Body weight changes every two days during the 14 days of treatment in each mouse. (C) After 14 days, the mice were sacrificed, and the residual condition of the tumor after treatment in each group of mice. (D) The relative tumor volumes (Vt/V0) of each mouse within 14 days after treatment. (E) H&E stained sections of the tumor tissues were treated for 14 days after treatment in each group. The scale bar is 50 µm. Analysis of statistical (n=4, * indicates P < 0.05, ** indicates P < 0.01 and *** indicates P < 0.001).

Figure 5

Experimental results of TUNEL and immunohistochemistry. (A-E) Figures A to E were the TUNEL results of the tumor tissues in the control group, Qu+RT group, IQuCS@Zr-PEG+RT group, IQuS@Zr-PEG+RT+MW group, and IQuCS@Zr-PEG+RT+MW group, respectively (magnification 200×). (F-J) Figures F to J were the immunohistochemistry results of the tumor tissues in the control group, Qu+RT group, IQuCS@Zr-PEG+RT group, IQuS@Zr-PEG+RT+MW group, and IQuCS@Zr-PEG+RT+MW group, respectively (magnification 200×). The scale bar is 50 µm.

Figure 6

The CT imaging effect of IQuCS@Zr-PEG NSPs in vitro and in vivo. (A,B) The CT effect of different concentrations of SiO₂@ZrO₂ NSPs and CuO-SiO₂@ZrO₂ NSPs aqueous solutions in vitro. (C) After injection of 50 mg/kg of the IQuCS@Zr-PEG NSPs into tail vein of mice, the CT imaging results in vivo were measured at 0, 3, 6, 9 and 24 h, respectively.