A paclitaxel prodrug nanoparticles with glutathion/reactive oxygen species dual-responsive and CD206 targeting to improve the anti-tumour effect

Abstract

As a first-line anticancer drug, paclitaxel has shortcomings, such as poor solubility and lack of tumour cell selectivity, which limit its further applications in clinical practice. Therefore, the authors aimed to utilise the characteristics of prodrug and nanotechnology to prepare a reactive oxygen species (ROS) and GSH dual-responsive targeted tumour prodrug nanoparticle Man-PEG-SS-PLGA/ProPTX to improve the clinical application status of paclitaxel limitation. The characterisation of Man-PEG-SS-PLGA/ProPTX was carried out through preparation. The cytotoxicity of nanoparticles on tumour cells and the effect on apoptosis of tumour cells were investigated by cytotoxicity assay and flow cytometry analysis. The ROS responsiveness of nanoparticles was investigated by detecting the ROS level of tumour cells. The tumour cell selectivity of the nanoparticles was further investigated by receptor affinity assay and cell uptake assay. The particle size of Man-PEG-SS-PLGA/ProPTX was (132.90 ± 1.81) nm, the dispersion coefficient Polymer dispersity index was 0.13 ± 0.03, and the Zeta potential was (-8.65 ± 0.50) mV. The encapsulation rate was 95.46 ± 2.31% and the drug load was 13.65 ± 2.31%. The nanoparticles could significantly inhibit the proliferation and promote apoptosis of MCF-7, HepG2, and MDA-MB-231 tumour cells. It has good ROS response characteristics and targeting. The targeted uptake mechanism is energy-dependent and endocytosis is mediated by non-clathrin, non-caveolin, lipid raft/caveolin, and cyclooxygenase (COX)/caveolin with a certain concentration dependence and time dependence. Man-PEG-SS-PLGA/ProPTX is a tumour microenvironment-responsive nanoparticle that can actively target tumour cells. It restricts the release of PTX in normal tissues, enhances its selectivity to tumour cells, and has significant antitumour activity, which is expected to solve the current limitations of PTX use.

Keywords: cellular effects of radiation; drug delivery systems; drugs; nanoparticle; tumours.

FIGURE 1

The particle size and potential of Blank‐PP‐NPs. (a) ProPTX‐NPs (b) ProPTX‐SS‐NPs (c), and Man‐ProPTX‐SS‐NPs (d) (Mean ± SD, n = 3).

FIGURE 2

Particle size changes of ProPTX‐NPs and ProPTX‐SS‐NPs nanoparticles in PBS containing 10% foetal bovine serum (pH 7.4) (Mean ± SD, n = 3).

FIGURE 3

Cytotoxicity of PTX, ProPTX, ProPTX‐NPs, ProPTX‐SS‐NPs, and Man‐ProPTX‐SS‐NPs after 48 h incubation with MCF‐7 (a), HepG2 (b), and MDA‐MB‐231 (c) cells (mean ± SD, n = 3).

FIGURE 4

Cell morphology of PTX, ProPTX, ProPTX‐NPs, ProPTX‐SS‐NPs, and Man‐proPTX‐SS‐NPs after 48 h incubation with MDA‐MB‐231 cells. The magnification was 60×. Scale bar was 50 μm.

FIGURE 5

Comparison of 48 h apoptosis results of Control (a), PTX (b), ProPTX (c), ProPTX‐NPs (d), ProPTX‐SS‐NPs (e), and Man‐ProPTX‐SS‐NPs (f) on MDA‐MB‐231 cells (a). Histogram analysis of the apoptosis rate (b) (Mean ± SD, n = 3) (**P < 0.01, ***P < 0.001, vs. PTX, ^### P < 0 0.001 vs. ProPTX).

FIGURE 6

Blank (PBS Sterile) (a), PTX (b), Blank PP‐NPs and PTX physical mixture (c), PTX‐NPs (d), ProPTX (e), Blank PP‐NPs and ProPTX physical mixture (f), ProPTX‐NPs (g), ProPTX‐SS‐NPs (h), and Man‐ProPTX‐SS‐NPs (i) in MDA‐MB‐21 reactive oxygen species (ROS) determination (a). Statistical analysis of test results (b) (Mean ± SD, n = 3) (*P < 0.05, **P < 0.01, ***P < 0.001, vs. PTX).

FIGURE 7

The uptake of C6‐Sol and C6 nanoparticles on MDA‐MB‐231 cells at 0.5 h (a) and 1.5 h (b) (Mean ± SD, n = 3) was observed by the confocal laser microscopy. The magnification was 60×. Scale bar was 50 μm.

FIGURE 8

The consumption of paclitaxel (paclitaxel prodrug + paclitaxel) (a) and the drug alone (paclitaxel) (b) in MDA‐MB‐231 cells as a function of time (mean ± SD, n = 3).

FIGURE 9

Total drug (paclitaxel prodrug + paclitaxel) (a) and drug alone (paclitaxel) (b) intake curves of paclitaxel and each nanoparticle on MDA‐MB‐231 cells as a function of concentration (mean ± SD, n = 3).

FIGURE 10

(a and b) The effect of various endocytic inhibitors on the uptake of Man‐C6‐SS‐NPs in MDA‐MB‐231 cells was quantified by flow cytometry (mean ± SD, n = 3) (*P < 0.05, vs. Man‐C6‐SS‐NPs).

FIGURE 11

Laser confocal images of C6‐Sol and C6 nanoparticles incubated with MDA‐MB‐231 cells for 1.5 h (Mean ± SD, n = 3) magnification: 60×. Scale bar: 50 μm.

FIGURE 12

The uptake of C6‐Sol in MDA‐MB‐231 cells after 2 h incubation was quantified by flow cytometry (mean ± SD, n = 3) (***P < 0.001 vs. C6‐Sol, ^# P <   0.05 vs. Man‐C6‐SS‐NPs).