Application of Mitochondrially Targeted Nanoconstructs to Neoadjuvant X-ray-Induced Photodynamic Therapy for Rectal Cancer

Abstract

In this work, we brought together two existing clinical techniques used in cancer treatment-X-ray radiation and photodynamic therapy (PDT), whose combination termed X-PDT uniquely allows PDT to be therapeutically effective in deep tissue. To this end, we developed mitochondrially targeted biodegradable polymer poly(lactic-co-glycolic acid) nanocarriers incorporating a photosensitizer verteporfin, ultrasmall (2-5 nm) gold nanoparticles as radiation enhancers, and triphenylphosphonium acting as the mitochondrial targeting moiety. The average size of the nanocarriers was about 160 nm. Upon X-ray radiation our nanocarriers generated cytotoxic amounts of singlet oxygen within the mitochondria, triggering the loss of membrane potential and mitochondria-related apoptosis of cancer cells. Our X-PDT strategy effectively controlled tumor growth with only a fraction of radiotherapy dose (4 Gy) and improved the survival rate of a mouse model bearing colorectal cancer cells. In vivo data indicate that our X-PDT treatment is cytoreductive, antiproliferative, and profibrotic. The nanocarriers induce radiosensitization effectively, which makes it possible to amplify the effects of radiation. A radiation dose of 4 Gy combined with our nanocarriers allows equivalent control of tumor growth as 12 Gy of radiation, but with greatly reduced radiation side effects (significant weight loss and resultant death).

Figure 1

(A) Schematic illustration of X-ray-induced PDT via PLGA nanocarriers incorporating verteporfin (VP) and gold nanoparticles. (B) SEM image of PLGA nanocarriers; inset is a TEM image of the same sample under high magnification. The gold nanoparticles were clearly observed under TEM with high magnification. (C) ζ potential of PLGA–VP and PLGA–TPP. (D) Absorption spectra of PLGA samples with and without TPP conjugation. (E) Percentage increase of SOSG fluorescence intensities in PLGA samples with different molar ratios of gold and VP under X-ray radiation at different doses. ** p < 0.01, *** p < 0.001 determined by Student t-test, n = 3.

Figure 2

Intracellular ¹O₂ generation indicated by SOSG fluorescence signal in HCT116 cells obtained (A) immediately and (B) 24 h after the treatments indicated in the images. (C) Real-time detection of ΔΨ_m in HCT116 cells after treatment with PLGA–TPP in combination with 4 Gy radiation at the time points indicated in the images. The membrane potential differences are indicated by the ratio of red versus green fluorescence intensities in the cells. Scale bar is 50 μm. *** indicates a significant difference between groups (p < 0.001) as determined by Student t-test, n = 4.

Figure 3

(A–D) Flow cytometry plots showing distribution of annexin-V vs 7-AAD staining after different treatments on HCT 116 cells using the Muse Annexin V and Dead Cell Assay. (E) % of cells and the mean percentages ± SD of apoptosis (early and late apoptosis) of three independent experiments. (F) Western blotting analyses of the expressions of proteins involved in the cell proliferation and apoptosis in HCT 116 cells that received different treatments as indicated. (G–J) Densitometric analysis of the protein bands shown in part F. Values represent the means ± standard deviation of three experiments. * p < 0.05, *** p < 0.001 as determined by Student t-test.

Figure 4

Tumor control of X-PDT in a xenograft model of colorectal cancer. (A) Changes in tumor volume after various treatments as indicated; repeated measures two-way ANOVA statistical analysis with Tukey multiple comparison test was used to determine differences between treatment groups, * p < 0.05 indicates significant difference between the groups (4 Gy vs 4 Gy and PLGA–TPP; PLGA–TPP vs 4 Gy and PLGA–TPP). (B) A Kaplan–Meier curve reveals that treatment with X-PDT extended the survival of mice bearing colorectal cancer. Black arrow indicates the time of treatment administration. (C) Photographs of tumors isolated at the end point. Tumor size and survival data were plotted using GraphPad prism v7.0.

Figure 5

(A) Left image: example of a scanned image of a whole-sample section applied for further color segmentation. The image demonstrates two halves of the same tumor and the areas of different tissue structure. T, live tumor tissue featuring intensive staining with hematoxylin; N, necrotic and paranecrotic areas in the central part of the tumors notable by enhanced eosinophilic and reduced basophilic staining; C, artificial cavities in the tumors reflecting the areas with mucous content; F, peritumoral fibrotic capsule. Right image: example of automated classification of the live tumor tissue in the samples (shown in white color). (B) Average number of Ki-67-positive cells per field of view in the histological specimens of the tumors. Error bars represent lower and upper boundaries of CI 95% for the mean. (C) Relative amount of live tumor tissue on the histological sections in the studied groups. (D) Calculated volume of live tumor tissue in the tumor samples from studied groups, n = 5 in each group.

Figure 6

(A) Top image: peritumoral capsule and periphery of the tumor with visible accumulation of aligned collagen fibers (blue staining). Bottom image: accumulation of nonorganized collagen in the central necrotic area of the tumor. The histograms for fibrotic feature distributions based on (B) fibrotic capsule volume, (C) fibrotic capsule collagen staining intensity, and (D) collagen staining intensity in the central necrotic area. “0” indicates the absence of fibrotic signs, and “3” indicates obvious fibrotic transformation.