Mitochondrial-Targeted Triphenylphosphonium-Hydroxycamptothecin Conjugate and Its Nano-Formulations for Breast Cancer Therapy: In Vitro and In Vivo Investigation

Abstract

Mitochondria are involved in various stages of cancer cell diffusion and metastasis. Therefore, targeting tumor mitochondria with antineoplastic medicines to cause mitochondria to initiate apoptosis may be an effective strategy for cancer therapy. Here, in order to enhance the anti-tumor efficacy of the antineoplastic agent hydroxycamptothecin (HCPT), the mitochondrial targeting ligand 4-(carboxybutyl) triphenylphosphine bromide (TPP) was attached to HCPT by an ester linkage. The resultant TPP-HCPT (TH) conjugate could self-assemble into nano-aggregates, with a mean particle size of 203.2 nm and a polydispersity index (PDI) value of 0.312. The TH conjugate could also co-assembly with mPEG₃₀₀₀-PLGA₅₀₀₀ into nanoparticles (TH-NPs), with a mean diameter of 86.41 nm, a PDI value of 0.256 and a zeta potential of -0.125 mV. In contrast to HCPT injections, TH aggregates displayed enhanced cellular uptake, mitochondria-targetability and cytotoxicity against 4T1 cells, while TH-NPs showed even better improvement than TH aggregates. In the in vivo study, TH aggregates displayed higher anti-tumor efficacy in 4T1 tumor-bearing mice than HCPT injections (tumor inhibition rate, 55.71% vs. 69.17%), and TH-NPs displayed more superior anti-tumor effects (tumor inhibition rate, 80.02%). In conclusion, our research demonstrated that the TPP-HCPT conjugate and its nano-formulations, including TH aggregates and TH-NPs, may be a promising mitochondria-targeting anticancer medicine for cancer therapy. As far as we know, this is the first report in which TPP and HCPT have been conjugated directly for this aim.

Keywords: hydroxycamptothecin; mitochondrial targeting; nanoparticles; triphenylphosphine.

Figure 1

The schematic diagram showing the synthesis of TH, the preparation of TH-NPs by the anti-solvent precipitation method, how TH-NPs were in vivo delivered into tumor cells.

Figure 2

Synthesis scheme of TPP-HCPT conjugate.

Figure 3

Determination of critical aggregation concentration (CAC) of TH using pyrene fluorescence method.

Figure 4

The actual photo and the particle size distribution of TH aggregates (a). The particle size change of TH aggregates in various physiological media (b). The particle size and PDI change of TH aggregates during the 7 days shelf storage (c). Transmission electron microscopy image of TH aggregates (d).

Figure 5

The actual photo and the particle size distribution of TH-NPs (a). The particle size and PDI change of TH-NPs in various physiological media (b). The particle size and PDI change of TH-NPs during the seven days shelf storage (c). Transmission electron microscopy image of TH-NPs (d).

Figure 6

The cumulative in vitro drug release of HCPT injection, TH aggregates and TH-NPs in PBS (pH 7.4) (n = 3, mean ± SD).

Figure 7

Cytotoxicity of HCPT (DMSO solution, (a)) and TPP-HCPT (DMSO solution, (b)), TH aggregates (c) and TH-NPs (d) against 4T1 cells after incubation for 48 h (n = 3).

Figure 8

Fluorescent microscopy images for cellular uptake of HCPT injection (a), TH aggregates (b), TH-NPs (c) by 4T1 cells. blue: 40,6-diamidino-2-phenylindole (DAPI); green: Coumarin-6 labeled nanoparticles. The semi-quantitative analysis of cellular uptake of HCPT injection, TH aggregates and TH-NPs by 4T1 cells (d).

Figure 9

Co-localization analysis. The red fluorescence is the staining of the mitochondria in 4T1 cells, the green fluorescence is coumarin-labeled HCPT injection, TH aggregates and TH-NPs. The blue fluorescence is DAPI (a) 2D intensity histogram (orange points represent correlated part) (b).

Figure 10

The tumor volume change, * p < 0.05 vs. normal saline. ** p < 0.01 vs. normal saline (a) and body weight change of the 4 groups of mice during the dosing period (b), and the actual photo of the tumors collected for the 4 groups at the end of the experiment (c). Data represent the mean ± SD (n = 8).

Figure 11

Dynamic biodistribution of DIR-labeled HCPT injection (a) and TH-NPs (b) in 4T1 tumor-bearing mice (from left to right: 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 36 h, 48 h). The fluorescence image of dissected tumor and major organs (from left to right: tumor, heart, liver, spleen, lung and kidney) from HCPT injections group (c) and TH-NPs (d) at the end of the 48th hour, and the corresponding fluorescence semi-quantitative analysis ((e) for HCPT injection, (f) for TH-NPs).