Combining photodynamic therapy and cascade chemotherapy for enhanced tumor cytotoxicity: the role of CTT2P@B nanoparticles

Abstract

The mitochondria act as the main producers of reactive oxygen species (ROS) within cells. Elevated levels of ROS can activate the mitochondrial apoptotic pathway, leading to cell apoptosis. In this study, we devised a molecular prodrug named CTT₂P, demonstrating notable efficacy in facilitating mitochondrial apoptosis. To develop nanomedicine, we enveloped CTT₂P within bovine serum albumin (BSA), resulting in the formulation known as CTT₂P@B. The molecular prodrug CTT₂P is achieved by covalently conjugating mitochondrial targeting triphenylphosphine (PPh₃), photosensitizer TPPOH₂, ROS-sensitive thioketal (TK), and chemotherapeutic drug camptothecin (CPT). The prodrug, which is chemically bonded, prevents the escape of drugs while they circulate throughout the body, guaranteeing the coordinated dispersion of both medications inside the organism. Additionally, the concurrent integration of targeted photodynamic therapy and cascade chemotherapy synergistically enhances the therapeutic efficacy of pharmaceutical agents. Experimental results indicated that the covalently attached prodrug significantly mitigated CPT cytotoxicity under dark conditions. In contrast, TPPOH₂, CTT₂, CTT₂P, and CTT₂P@B nanoparticles exhibited increasing tumor cell-killing effects and suppressed tumor growth when exposed to light at 660 nm with an intensity of 280 mW cm^-2. Consequently, this laser-triggered, mitochondria-targeted, combined photodynamic therapy and chemotherapy nano drug delivery system, adept at efficiently promoting mitochondrial apoptosis, presents a promising and innovative approach to cancer treatment.

Keywords: ROS-responsive; bovine serum albumin; camptothecin; mitochondria-targeting; photodynamic therapy.

SCHEME 1

Design of mitochondria-targeted ROS-responsive nanomedicine. (A) Schematic of each part of the CTT₂P prodrug-linked compound and its encapsulation into CTT₂P@B NPs by BSA. (B) Schematic diagram of CTT₂P NPs release and treatment at local tumor sites.

FIGURE 1

Analysis of CTT₂P@B nanoparticles. (A) Particle size distribution of blank BSA NPs and CTT₂P@B NPs. (B) Zeta potential of blank BSA NPs and CTT₂P@B NPs. (C) Stability of CTT₂P@B NPs in deionized aqueous solution. (D) TEM images of blank BSA NPs and CTT₂P@B NPs. Scale bar = 200 nm. (E) The release of CTT₂P from CTT₂P@B NPs in a PBS solution at 37°C was examined using in vitro testing.

FIGURE 2

ROS generation detection in vitro. (A) The release of singlet oxygen from each drug was detected in vitro under dark conditions. Data were presented as the mean ± SD (n = 3). (B) Detection of singlet oxygen release of each drug under 660 nm laser irradiation at a power density of 280 mW cm^-2 was performed in vitro. Data were presented as the mean ± SD (n = 3). (C) ROS generation was observed in 4T1 cells treated with each medication while being exposed to laser irradiation at a wavelength of 660 nm and intensity of 280 mW cm⁻². Bright: Bright field. DCF: Green fluorescence represents intracellular ROS. Merge: Superimpose the image. Scale Bar = 100 μm.

FIGURE 3

Evaluation of mitochondrial targeting function. (A) Confocal images of the mitochondrial sites of TPPOH₂, CTT₂, CTT₂P, and CTT₂P@B NPs in 4T1 cells. Mito-Tracker Green was used to stain the mitochondria in the green channel. The red channel was derived from the emission of the photosensitizer fraction (PS) itself. Merge stands for superimposed image. The Green and red curves in the Plot Profile represent the gray value of Mito-Tracker Green and PS, respectively. Scale bar = 20 μm. (B) Flow cytometry JC-1 method was used to analyze the mitochondrial function of cells treated with different drugs. Red fluorescence: normal mitochondria (J-aggregate); Green fluorescence: depolarized mitochondria (J-monomer).

FIGURE 4

Evaluation of cell death in vitro through cytotoxicity assessment and examination of apoptosis and necrosis. (A) The in vitro cytotoxicity of 4T1 cells treated with CPT, TPPOH₂, CTT₂, CTT₂P, and CTT₂P@B NPs in the dark was assessed using the CCK-8 assay. Data were presented as the mean ± SD (n = 5). **p < 0.01, ****p < 0.0001. (B) The in vitro cytotoxicity of 4T1 cells treated with TPPOH₂, CTT₂, CTT₂P and CTT₂P@B NPs under laser irradiation (660 nm, 280 mW cm⁻²) was determined using the CCK-8 assay. Data were presented as the mean ± SD (n = 5). **p < 0.01, ****p < 0.0001. (C) Cell apoptosis and necrosis were analyzed using flow cytometry with Annexin V-FITC/PI double staining following treatment with various drugs at a concentration of 5 μM.

FIGURE 5

Biodistribution in vivo. (A) Blood compatibility test of CPT, TPPOH₂, CTT₂, CTT₂P, CTT₂P@B NPs. Data were presented as the mean ± SD (n = 3). ***p < 0.001, ****p < 0.0001. (B) Time-lapse live fluorescence imaging of mice with 4T1 tumors following the administration of free CTT₂P and CTT₂P@B NPs via intravenous injection. (C) Fluorescent images of major organs and tumors were obtained 24 h after injection, using ex vivo methods. (D) The mean fluorescence intensity of each organ and tumor was used to determine the biodistribution of free CTT₂P and CTT₂P@B NPs in mice. Data were presented as the mean ± SD (n = 3). *p < 0.05.

FIGURE 6

In vivo anti-tumor study of each drug in 4T1 tumor-bearing mice. (A) Tumor images of different drug administration treatments after the antitumor study. (B) During the administration, the growth of tumors in mice was observed in each group receiving treatment. Data were presented as the mean ± SD (n = 5), **p < 0.01. (C) The weight of mice in each treatment group was monitored throughout the administration period. Data were presented as the mean ± SD (n = 5).

FIGURE 7

Investigation of the effects of each medication on mice with 4T1 tumors through pathological examination. (A) After administering various medications, the major organs (including the heart, liver, spleen, lung, and kidney) were subjected to H&E staining. Scale bar = 50 μm. (B) After administering various medications, tumors were subjected to H&E staining and TUNEL staining. Scale bar = 50 μm.