Glutathione-Responsive Prodrug Nanoparticles for Effective Drug Delivery and Cancer Therapy

Abstract

Spurred by recent progress in medicinal chemistry, numerous lead compounds have sprung up in the past few years, although the majority are hindered by hydrophobicity, which greatly challenges druggability. In an effort to assess the potential of platinum (Pt) candidates, the nanosizing approach to alter the pharmacology of hydrophobic Pt(IV) prodrugs in discovery and development settings is described. The construction of a self-assembled nanoparticle (NP) platform, composed of amphiphilic lipid-polyethylene glycol (PEG) for effective delivery of Pt(IV) prodrugs capable of resisting thiol-mediated detoxification through a glutathione (GSH)-exhausting effect, offers a promising route to synergistically improving safety and efficacy. After a systematic screening, the optimized NPs (referred to as P6 NPs) exhibited small particle size (99.3 nm), high Pt loading (11.24%), reliable dynamic stability (∼7 days), and rapid redox-triggered release (∼80% in 3 days). Subsequent experiments on cells support the emergence of P6 NPs as a highly effective means of transporting a lethal dose of cargo across cytomembranes through macropinocytosis. Upon reduction by cytoplasmic reductants, particularly GSH, P6 NPs under disintegration released sufficient active Pt(II) metabolites, which covalently bound to target DNA and induced significant apoptosis. The PEGylation endowed P6 NPs with in vivo longevity and tumor specificity, which were essential to successfully inhibiting the growth of cisplatin-sensitive and -resistant xenograft tumors, while effectively alleviating toxic side-effects associated with cisplatin. P6 NPs are, therefore, promising for overcoming the bottleneck in the development of Pt drugs for oncotherapy.

Keywords: glutathione; nanoparticle; pharmacodynamics; pharmacokinetics; platinum(IV).

Figure 1.

Schematic illustration of self-assembled Pt(IV) NPs for specific delivery of Pt drugs and effective suppression of cisplatin-resistant tumors. Redox-responsive P6 NPs were self-assembled with superhydrophobic Pt(IV) 6 and coated with amphiphilic lipid-PEG via nanoprecipitation. Benefiting from the extended blood circulation and selective tumor accumulation, P6 NPs could be endocytosed into tumor cells through macropinocytosis and then disintegrated by consumption of cytoplasmic thiol-containing species, especially GSH. The redox-triggered process contributed to the release of Pt(II) ions and their reduced probability of deactivation, which went on to rapidly diffuse into nuclei and covalently bind large amounts of DNA, ultimately resulting in the mitochondria-controlled apoptosis of cisplatin-resistant tumors.

Figure 2.

PEGylated Pt(IV) 6 delivery platform exhibition of redox-triggered degradation. (a) Histogram of particle-size distribution of P6 NPs obtained by DLS. (b) Particle-size and ζ-potential changes of P6 NPs monitored over the course of 1 week. (c, d) TEM morphologies of P6 NPs stored in (c) water or (d) 10 mM DTT at 25 °C for 24 h.

Figure 3.

Self-assembly of Pt(IV) 6 into NPs quantitatively maximized cellular uptake of Pt, which was easily interfered with by macropinocytosis inhibitors. (a–d) Pt uptake into (a, b) A2780 and (c, d) A2780cis cells after 4 and 18 h of exposure to cisplatin, P6 NPs, or P6 Soln at Pt concentrations of (a, c) 50 and (b, d) 100 μM. (e, f) Prior exposure of A2780 and A2780cis cells to (e) EIPA and (f) cytochalasin D reduced uptake of Dil-P6 NPs in a dose-dependent fashion.

Figure 4.

Confocal laser scanning microscopy utilization to visualize cytoplasmic delivery of P6 NPs. (a) Dil-P6 NPs were incubated with A2780cis cells in the presence of Alexa-Fluor 488-labeled markers of various endocytic pathways. NPs co-localized with dextran, a fluid-phase marker known to enter cells via macropinocytosis (white arrows). (b) Alexa-Fluor 488-labeled actin fibers revealed membrane ruffling and actin rearrangement (white arrows), hallmarks of uptake by macropinocytosis.

Figure 5.

Rapid intracellular disintegration of P6 NPs accompanied by a GSH-exhausting effect. (a) A2780 and A2780cis cells incubated with Coumarin 6 and Nile red-P6 NPs for 4 and 18 h. To investigate the effect of GSH on NP disintegration, cells were also pretreated with NEM to consume cytosolic GSH. Images were taken under a 60× objective. (b, c) GSH and (d, e) GSSG percent of (b, d) A2780 and (c, e) A2780cis cells treated with cisplatin, P6 NPs, or P6 Soln at Pt concentrations of 0, 1.56, 6.25, or 25 μM.

Figure 6.

P6 NPs enhancement of toxicity to various tumor types through the augmentation of apoptosis. (a, b) In vitro cytotoxicity of cells treated with cisplatin, P6 NPs, or P6 Soln for (a) 48 or (b) 72 h. (c, d) In vitro apoptosis of (c) A2780 and (d) A2780cis cells treated with cisplatin, P6 NPs, or P6 Soln for 24 h.

Figure 7.

Pharmacokinetics and biodistribution of P6 NPs. (a) Pharmacokinetics of cisplatin, P6 NPs, or P6 Soln in BALB/c mice (n = 3). (b–d) Biodistribution of (b) cisplatin, (c) P6 NPs, or (d) P6 Soln in A2780cis tumor-bearing athymic nude mice (n = 3). All data were normalized by weight or volume, i.e., microgram per gram or milligram per liter.

Figure 8.

NIR images of dye or dye-loaded NPs in single or multiple tumor-bearing athymic nude mice. In vivo fluorescence images of mice captured using the Maestro 2 in vivo imaging system (n = 3).

Figure 9.

P6 NPs mediation of an antitumor effect in a A2780 xenograft model. (a) Tumor growth curve of each mouse from different groups of A2780 tumor-bearing athymic nude mice during chemotherapy (n = 5). (b) The harvested A2780 xenograft tumors after systemic treatment captured using the Maestro 2 in vivo imaging system. (c) Western blot quantification of p53, Caspase 3, PARP, and cleaved PARP for A2780 xenograft tumors following treatment with PBS, cisplatin, P6 NPs, P6 Soln, or DP 3000.

Figure 10.

P6 NPs mediation of an antitumor effect in a A2780cis xenograft model. (a) Tumor growth curve of each mouse from different groups of A2780cis tumor-bearing athymic nude mice during chemotherapy (n = 5). (b) The harvested A2780cis xenograft tumors after systemic treatment captured using the Maestro 2 in vivo imaging system. (c) Western blot quantification of p53, Caspase 3, PARP, and cleaved PARP for A2780cis xenograft tumors following treatment with PBS, cisplatin, P6 NPs, P6 Soln, or DP 3000.