Cholesterol-tethered platinum II-based supramolecular nanoparticle increases antitumor efficacy and reduces nephrotoxicity

Abstract

Nanoscale drug delivery vehicles have been harnessed extensively as carriers for cancer chemotherapeutics. However, traditional pharmaceutical approaches for nanoformulation have been a challenge with molecules that exhibit incompatible physicochemical properties, such as platinum-based chemotherapeutics. Here we propose a paradigm based on rational design of active molecules that facilitate supramolecular assembly in the nanoscale dimension. Using cisplatin as a template, we describe the synthesis of a unique platinum (II) tethered to a cholesterol backbone via a unique monocarboxylato and O→Pt coordination environment that facilitates nanoparticle assembly with a fixed ratio of phosphatidylcholine and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000]. The nanoparticles formed exhibit lower IC(50) values compared with carboplatin or cisplatin in vitro, and are active in cisplatin-resistant conditions. Additionally, the nanoparticles exhibit significantly enhanced in vivo antitumor efficacy in murine 4T1 breast cancer and in K-Ras(LSL/+)/Pten(fl/fl) ovarian cancer models with decreased systemic- and nephro-toxicity. Our results indicate that integrating rational drug design and supramolecular nanochemistry can emerge as a powerful strategy for drug development. Furthermore, given that platinum-based chemotherapeutics form the frontline therapy for a broad range of cancers, the increased efficacy and toxicity profile indicate the constructed nanostructure could translate into a next-generation platinum-based agent in the clinics.

Fig. 1.

Synthesis and characterization of SACNs. (A) Scheme for synthesis of cholesterol-cisplatin conjugate from cholesteryl chloroformate. Schematic representation shows synthesis of SACNs by self-assembly from PC, cholesterol-cisplatin conjugate, and DSPE-PEG. (B) High-resolution cryo-TEM image of SACNs at lower magnification (Upper) and magnified image (Lower). (Scale bar, Upper, 500 nm). (C) Distribution of hydrodynamic diameter of SACNs measured using dynamic light scattering. (D) Graph shows the pH-dependent release of platinum from SACNs as quantified over a 120-h period.

Fig. 2.

In vitro characterization of SACNs. (A–C) Graphs show cell viability of (A) LLC, (B) 4T1, and (C) 7404-CP20 cell lines, respectively, after 48-h incubation with increasing concentrations of cisplatin, carboplatin, and SACNs. (D) Treatment with SACNs induces cell death by apoptosis. Representative FACS distribution of 4T1 cells treated with carboplatin, cisplatin, and SACNs at 1 μM Pt concentration. The cells were incubated for 24 h, following which they were labeled with Annexin-V FITC and counter-stained with propidium iodide. Each quadrant represents the percentage of cells in early apoptosis (Lower Right), late apoptosis (Upper Right), necrosis (Upper Left), and healthy cells (Lower Left). Data shown are mean ± SE from n = 3 independent experiments. (E) Representative epifluorescence imaging of 4T1 tumor cells for monitoring internalization of SACNs. SACNs were labeled with FITC (green), the endolysosomal compartment was labeled by LysoTracker red, and the nucleus was labeled with DAPI blue. Colocalization of the signals in the merged images reveals internalization of FITC-SACNs in the endolysosomal compartments within 4 h. (F). Epifluorescence imaging of 4T1 and CP20 tumor cells to monitor the internalization of FITC tagged SACN at 37 °C and 4 °C. Images were captured using a Nikon Ti epifluorescence microscope at 40× magnification. 4T1 images were captured at 1,000 × 700 pixel resolution and the 7404-CP20 images are 900 × 600 pixels. (G) Graph shows Pt levels in 7404-CP20 cells treated with cisplatin or SACNs (20 μM Pt concentration). Cells incubated with similar concentration of SACNs at 4 °C to inhibit energy-dependent endocytosis exhibit lower intracellular Pt concentrations (*P < 0.05, **P < 0.01, ANOVA, Newman–Keuls post hoc test).

Fig. 3.

In vivo antitumor activity of SACNs in 4T1 breast cancer model. (A) Graph shows body weight loss of animals with increasing doses of cisplatin or SACNs (Cisplatin NP). Maximum tolerated dose is calculated at 20% body weight loss. (B) Graph shows change in tumor volume in different treatment groups in 4T1 murine breast cancer model following a single dose of platinum chemotherapy at the MTD platinum dose of cisplatin. (C) Kaplan–Meier curve shows effect of different treatments on survival at MTD platinum dose of cisplatin (P = 0.0189 Logrank test for trend). (D) Multiple-dose effects of treatment on 4T1 breast cancer growth. Cells were implanted subcutaneously on day 0. Mice were treated with PBS, carboplatin (3 mg/kg), cisplatin (3 mg/kg and 1 mg/kg), and SACNs (3 mg/kg and 1 mg/kg) (n = 4, doses are Pt equivalent) on days 9, 11, and 13 posttumor implantation. Upper row shows representative images of excised tumors, and Lower row shows tumor cross sections processed for TUNEL as marker for apoptosis. Images were captured using a Nikon Ti epifluorescence microscope at 20× magnification to capture a large view field. (E) Growth curves show the effect of the different multiple-dose treatments on tumor volume. (F) Graph shows change in body weight of animals in different treatment groups. (G) Kaplan–Meier curve shows effect of different treatments on survival (P = 0.0022, Logrank Mantel–Cox test).

Fig. 4.

SACNs preferentially accumulate in the tumor bypassing the kidney, and exert reduced nephrotoxicity. Mice were treated with PBS, carboplatin (3 mg/kg), cisplatin (3 mg/kg and 1 mg/kg), and SACNs (cisplatin NP, 3 mg/kg and 1 mg/kg) (n = 4, doses are Pt equivalent) on days 9, 11, and 13 posttumor implantation. (A) Bar graph shows weight of excised kidney in different treatment groups. (B) Representative images of cross sections of kidney stained for KIM1 expression. Images were captured using a Nikon Eclipse 90i microscope (Left). (C) Representative images of kidney cross-sections processed for TUNEL as marker apoptosis (Right). Images were captured using a Nikon Ti epifluorescence microscope at 20× magnification to capture a large view field; 1,000 × 700 pixels. (D) Tissue distribution of platinum in different treatment groups as determined by inductively coupled plasma-MS. *P < 0.05, **P < 0.01 vs. cisplatin (3 mg/kg)-treated group (ANOVA followed by Newman–Keuls post hoc test).