Photodynamic Therapy Activity of New Porphyrin-Xylan-Coated Silica Nanoparticles in Human Colorectal Cancer

Abstract

Photodynamic therapy (PDT) using porphyrins has been approved for treatment of several solid tumors due to the generation of cytotoxic reactive oxygen species (ROS). However, low physiological solubility and lack of selectivity towards tumor sites are the main limitations of their clinical use. Nanoparticles are able to spontaneously accumulate in solid tumors through an enhanced permeability and retention (EPR) effect due to leaky vasculature, poor lymphatic drainage, and increased vessel permeability. Herein, we proved the added value of nanoparticle vectorization on anticancer efficacy and tumor-targeting by 5-(4-hydroxyphenyl)-10,15,20-triphenylporphyrin (TPPOH). Using 80 nm silica nanoparticles (SNPs) coated with xylan-TPPOH conjugate (TPPOH-X), we first showed very significant phototoxic effects of TPPOH-X SNPs mediated by post-PDT ROS generation and stronger cell uptake in human colorectal cancer cell lines compared to free TPPOH. Additionally, we demonstrated apoptotic cell death induced by TPPOH-X SNPs-PDT and the interest of autophagy inhibition to increase anticancer efficacy. Finally, we highlighted in vivo, without toxicity, elevated anticancer efficacy of TPPOH-X SNPs through improvement of tumor-targeting compared to a free TPPOH protocol. Our work demonstrated for the first time the strong anticancer efficacy of TPPOH in vitro and in vivo and the merit of SNPs vectorization.

Keywords: anticancer drug; drug delivery; photodynamic therapy; porphyrin; silica nanoparticles.

Figure 1

In vitro phototoxic effects of 5-(4-hydroxyphenyl)-10,15,20-triphenylporphyrin (TPPOH)-X silica nanoparticles (SNPs)- Photodynamic therapy (PDT) and reactive oxygen species (ROS) production. (A) HT-29 cells were treated or not treated with free TPPOH and TPPOH-X SNPs based on TPPOH concentration. Then, cells were exposed or were not exposed to PDT. Phototoxic effects were determined 48 h post-PDT using the MTT assay. Cell viability, expressed as a percentage of each condition, was compared to controls. IC₅₀ values were calculated using 550.2 nM for TPPOH-X SNPs-PDT and around 6 μM for free TPPOH-PDT. (B) HT-29 cells were treated or not treated with TPPOH-X SNPs and SNPs based on nanoparticles concentration. Then, cells were exposed or not exposed to PDT. Phototoxic effects were determined 48 h post-PDT using the MTT assay. Cell viability, expressed as a percentage of each condition, was compared to controls. (C) HT-29 cells were treated or not treated with free TPPOH or (D) TPPOH-X SNPs with or without NAC co-treatment and then photoactivated or not photoactivated. (E) Comparison of free TPPOH and TPPOH-X SNPs on ROS generation in HT-29 cells. Intracellular ROS levels using DCFDA staining were measured 4 h post-PDT by flow cytometry. A greater right shift implied higher fluorescence intensity resulting from higher amounts of 2’,7’-dichlorofluorescein (DCF) formation and thus greater ROS generation. Data are shown as mean ± SEM (n = 3). ** p < 0.01 and *** p < 0.001.

Figure 2

Cell uptake of TPPOH-X SNPs by HT-29 cells. (A) HT-29 cells were treated with free TPPOH and TPPOH-X SNPs at 1 μM and cell uptake of these compounds was studied 24 h post-treatment by AMNIS^® imaging flow cytometry. The first graph highlights the size/structure of HT-29 cells. After selection of the cell population, TPPOH intensity in HT-29 cells was shown in the second graph and in representative images. The table summarizes the amount of positive TPPOH cells relative to all cells compared to free TPPOH and TPPOH-X SNPs treatments. White scale bar = 10 μm. (B) Representative TEM images of HT-29 cells treated or not treated with TPPOH-X SNPs 24 h post-treatment are shown. Red arrows indicate intracellular nanoparticles. Black scale bar = 1 μm. (C) HT-29 cells were co-treated with TPPOH-X SNPs and LysoTracker or MitoTracker and co-localization was studied 24 h post-treatment by using AMNIS^® imaging flow cytometry analysis. The first graph shows TPPOH intensity in HT-29 cells and the second graph shows similarity of TPPOH positive cells compared to LysoTracker or MitoTracker. The table summarizes the amount of TPPOH positive cells co-localized with LysoTracker or MitoTracker. Representative images of co-localization of TPPOH-X SNPs and LysoTracker in HT-29 cells are shown. White scale bar = 10 μm. Data are shown as three independent experiments.

Figure 3

Effects of TPPOH-X SNPs-PDT on HT-29 cell line apoptosis. (A) HT-29 cells were treated or not treated with TPPOH-X SNPs and then photoactivated or not photoactivated. The mitochondrial membrane potential was analyzed by flow cytometry with JC-1 at 48 h post-PDT. R2 represents the aggregate ratio and R3 represents the monomer ratio. (B) HT-29 cells were also stained, 48 h post-PDT, with Annexin V-FITC and PI, and apoptosis was analyzed by flow cytometry. The upper right quadrant represents the percentage of late apoptosis, and the lower right quadrant represents early apoptosis. (C) Caspase-3/7 activity, with the same conditions in HT-29 cells, was evaluated every 2 h during 48 h post-PDT by IncuCyte imaging live cell analysis and green count/cell count/well are shown. Representative images at 48 h post-PDT are shown. Yellow scale bar = 400 μm. (D) DNA fragmentation in HT-29 cells 48 h post-PDT was quantified from cytosol extracts by ELISA. Results are reported as n-fold compared to light control. (E) Representative TEM images of HT-29 cells treated or not treated with TPPOH-X SNPs and photoactivated or not 48 h post-PDT were shown. Red arrows indicate intracellular nanoparticles. Black scale bar = 1 μm. Data are shown as mean ± SEM (n = 3). *** p < 0.001.

Figure 4

Effects of autophagy inhibition on HT-29 apoptosis. (A) HT-29 cells were treated or not with TPPOH-X SNPs in the presence or absence of 3-MA for 24 h. Expression of autophagy-related proteins was analyzed by Western blotting 48 h post-PDT. β-actin was used as a loading control. Representative images were shown. (B) Representative TEM images of HT-29 cells treated or not with TPPOH-X SNPs 48 h post-PDT protocol are shown. Red arrowheads indicate autophagosomes in the treated cells. Scale bar = 1 μm. (C) HT-29 cells were treated or not with TPPOH-X SNPs with or without 3-MA co-treatment and then were photoactivated or not photoactivated. At 48 h post-PDT, cells were stained with Annexin V-FITC and PI, and apoptosis was analyzed by flow cytometry. Upper right quadrant represents the percentage of late apoptosis, and the lower right quadrant represents early apoptosis. (D) With the same conditions of treatment, caspase-3/7 activity was evaluated each 2 h during 48 h post-PDT protocol by IncuCyte imaging live cell analysis and green count/cell count/well were shown. Representative images at 48 h post-PDT protocol were shown. Yellow scale bar = 400 μm. (E) With the same conditions of treatment, DNA fragmentation was quantified from cytosol extracts with ELISA. Results were reported as n-fold compared to light control. Data are shown as mean ± SEM (n = 3). *** p < 0.001.

Figure 5

In vivo phototoxic effects on tumor growth. (A) Tumor growth curves of different groups over the treatment period until mouse sacrifice. (B) Representative images of HT-29 tumor-bearing nude mice and ex-vivo tumors after the mice being sacrificed on day 20. Data are shown as mean ± SEM (n = 5). * p < 0.05 and ** p < 0.01.

Figure 6

In vivo antitumor efficacy. (A) Tumors sections from treatment groups at 24 h post-PDT were stained with hematoxylin/eosin/saffron (HES), terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) or LC3 staining. (B) Tumors sections from treatment groups after sacrifice were stained with HES and Ki-67. Representative images of each condition are shown. Black scale bar = 100 μm.

Figure 7

In vivo and ex vivo fluorescence imaging for biodistribution and safety evaluation. (A) In vivo fluorescence imaging of HT-29 tumor-bearing mice at 24 h post-intravenous injection of Cy5.5-labeled free TPPOH and TPPOH-X SNPs, at 1/100^e LD₅₀ for each group. The red circles indicate tumor sites. (B) Ex-vivo fluorescence imaging of tumors and organs at 24 h post-injection. (C) ROI analysis of fluorescence intensity of tumors and organs at 24 h post-injection. (D) Representative images of histological analyses of major organ (kidney, liver, lung, heart and spleen) sections by HES staining. Black scale bar = 100 μm. Data are shown as mean ± SEM (n = 3). * p < 0.05 and NS: not significant.