Calcium phosphate engineered photosynthetic microalgae to combat hypoxic-tumor by in-situ modulating hypoxia and cascade radio-phototherapy

Abstract

Rationale: Hypoxia is one of the crucial restrictions in cancer radiotherapy (RT), which leads to the hypoxia-associated radioresistance of tumor cells and may result in the sharp decline in therapeutic efficacy. Methods: Herein, living photosynthetic microalgae (Chlorella vulgaris, C. vulgaris), were used as oxygenators, for in situ oxygen generation to relieve tumor hypoxia. We engineered the surface of C. vulgaris (CV) cells with calcium phosphate (CaP) shell by biomineralization, to form a biomimetic system (CV@CaP) for efficient tumor delivery and in-situ active photosynthetic oxygenation reaction in tumor. Results: After intravenous injection into tumor-bearing mice, CV@CaP could remarkably alleviate tumor hypoxia by continuous oxygen generation, thereby achieving enhanced radiotherapeutic effect. Furthermore, a cascade phototherapy could be fulfilled by the chlorophyll released from photosynthetic microalgae combined thermal effects under 650 nm laser irradiation. The feasibility of CV@CaP-mediated combinational treatment was finally validated in an orthotropic breast cancer mouse model, revealing its prominent anti-tumor and anti-metastasis efficacy in hypoxic-tumor management. More importantly, the engineered photosynthetic microalgae exhibited excellent fluorescence and photoacoustic imaging properties, allowing the self-monitoring of tumor therapy and tumor microenvironment. Conclusions: Our studies of this photosynthetic microsystem open up a new dimension for solving the radioresistance issue of hypoxic tumors.

Keywords: Chlorella vulgaris; chlorophyll; photosynthetic microalgae; phototherapy; radiotherapy; tumor hypoxia.

Scheme 1

Schematic illustration of biomineralized C. vulgaris mediated PAI/FLI guided synergistic therapy. Mechanistic representative diagram of the self-enhanced radio-phototherapy: (i) CV@CaP as a photosynthetic system produced large amounts of oxygen in situ to modulate tumor hypoxia. (ii) The alleviation of tumor hypoxia further improved the RT efficacy. (iii) X-ray induced chlorophyll releasing as photosensitizer, generating ROS under 650 nm laser, in combination with thermal effects to realize phototherapy.

Figure 1

Biomineralization of C. vulgaris. (A) Bright (top) and fluorescence images (bottom) of native C. vulgaris cells (inset: a large-scale culture of CV). (B) Schematic illustration of the CaP mineralization process of C. vulgaris, experiencing the precipitation of calcium phosphate. (C) SEM images of the bare C. vulgaris cells (left) and the mineralized CV@CaP cells (right), scale bar = 5 µm, top insert: enlarged images of the cell, scale bar = 0.5 µm. (D) EDS analysis of CV@CaP, evidencing the presence of Ca and P. (E) DLS of CV and CV@CaP. (F) FTIR patterns of CaP, CV and CV@CaP. (G) Zeta potential, (H) fluorescence and (I) UV-Vis spectra of CV and CV@CaP.

Figure 2

Oxygeneration performance of CV@CaP to enhance RT efficiency in vitro. (A) Oxygen production curves of water, CV and CV@CaP over time (5 hours). (B) Cytotoxicity of CV and CV@CaP to breast cancer cell line 4T1. (C) The process (top) of CV@CaP modulating cell hypoxia by photosynthesis, and fluorescence images (bottom) showing the alleviation of hypoxia in the 4T1 tumor cells treated with CV@CaP. Cells and hypoxia signals were stained with DAPI (blue) and hypoxyprobe (red), respectively. Scale Bar = 100 µm. (D) Colony formation assay and (E) Survival curves of 4T1 cells treated with or without CV@CaP under X-ray irradiation (0 to 8 Gy). (F) Cell migration assay and (G) corresponding quantitative analysis of 4T1 cells treated with or without CV@CaP under X-ray irradiation (0 and 6 Gy). **p < 0.01, ***p < 0.001.

Figure 3

Chlorophyll-induced ROS generation combined with thermal effects for phototherapy in vitro. (A) Chlorophyll released from CV@CaP could be used as photosensitizer to generate ROS under 650 nm laser irradiation (1.0 W/cm, 3 min) for photodynamic therapy. (B) Fluorescence images of 4T1 cells stained with DCFH-DA after different treatments. CV@CaP were pre-treated with X-ray. Scale Bar = 200 µm. (C) Flow cytometry analysis showing the expression of ROS in 4T1 cells after various treatments. CV@CaP were pre-treated with X-ray. (D) IVIS images and (E) quantification of the total radiant efficiency of ROS signals in the dissected tumors after various treatments. (F) Fluorescence images of live/dead staining of 4T1 cells in different treated groups. The cells were co-stained with Calcein-AM (green, living cells) and PI (red, dead cells). Scale Bar = 100 µm. ***p < 0.001.

Figure 4

In vivo fluorescence imaging and biodistribution of CV@CaP. (A) Time-dependent in vivo FL images of 4T1 tumor-bearing mice (red circles: the tumor sites) after intravenous injection of CV and CV@CaP. (B) Ex vivo FL images of major organs and tissues from 4T1 tumor-bearing mice after 4 h and 24 h intravenous injection of CV@CaP. (C) Biodistribution of CV@CaP in major organs and tissues quantitated from ex vivo FLI analysis. (D) SEM (left) and pseudo-color (right) images showing the accumulation of CV@CaP (green) in the tumor tissues (red). Scale Bar = 5 µm.

Figure 5

In vivo photoacoustic imaging and tumor oxygenation of CV@CaP. (A) Time-dependent in vivo PA images of 4T1 tumor-bearing mice (white dashed circles: the tumor sites) after intravenous injection CV@CaP. (B) PA images of Hb (750 nm), HbO₂ (850 nm) and CV@CaP (680 nm) of 4T1 tumor-bearing mice (white dashed circles: the tumor sites), indicating that CV@CaP effectively relieved tumor hypoxia by photosynthesis. Scale Bar = 5 mm. (C) Quantitative analysis of the PA intensity of Hb and HbO₂ at the tumor sites. (D) Quantitative analysis of the PA intensity of CV@CaP at the tumor sites.

Figure 6

In vivo antitumor efficacy of CV@CaP in combination with radiotherapy and phototherapy. (A) Experimental schematic of CV@CaP-mediated enhanced radio-phototherapy to inhibit tumor growth in the orthotopic 4T1 breast tumor model. (B) Representative photographs of the 4T1 tumor-bearing mice (black dashed circles: the tumor sites) at 18 days after different treatments (n =5). Treatments: 1) Control; 2) CV@CaP alone; 3) Laser alone (1.0 W/cm, 3 min); 4) RT alone (6 Gy); 5) RT + Laser; 6) CV@CaP + Laser; 7) CV@CaP + RT; and 8) CV@CaP + RT + Laser. (C) Tumor growth curves of mice in different groups (D) Photograph of the dissected tumors at 18 days in different groups. ***p < 0.001.

Figure 7

Histological analysis of the tumor tissues. (A) Representative H&E, CD31, Ki-67, and HIF-1α staining images of tumors (n = 5) at 18 days in different groups. Scale bar = 50 µm. Quantitative analysis of (B) necrosis, (C) CD31-, (D) Ki-67- and (E) HIF-1α-positive intensity at 16 days in different groups. ***p < 0.001.

Figure 8

The potency of CV@CaP combined radio-phototherapy in inhibiting distant metastasis of 4T1 cells. (A) Representative photograph of lung pathological sections stained with H&E from each group of mice (n = 5) at 18 days (black dotted circles: metastatic foci). Scale bar = 200 µm. (B) Representative photograph of liver pathological sections (black arrows: micrometastases) stained with H&E; upper: scale bar = 200 µm; lower: scale bar = 50 µm.