Rationally designed oxaliplatin-nanoparticle for enhanced antitumor efficacy

Abstract

Nanoscale drug delivery vehicles have been extensively studied as carriers for cancer chemotherapeutics. However, the formulation of platinum chemotherapeutics in nanoparticles has been a challenge arising from their physicochemical properties. There are only a few reports describing oxaliplatin nanoparticles. In this study, we derivatized the monomeric units of a polyisobutylene maleic acid copolymer with glucosamine, which chelates trans-1,2-diaminocyclohexane (DACH) platinum (II) through a novel monocarboxylato and O --> Pt coordination linkage. At a specific polymer to platinum ratio, the complex self-assembled into a nanoparticle, where the polymeric units act as the leaving group, releasing DACH-platinum in a sustained pH-dependent manner. Sizing was done using dynamic light scatter and electron microscopy. The nanoparticles were evaluated for efficacy in vitro and in vivo. Biodistribution was quantified using inductively coupled plasma atomic absorption spectroscopy (ICP-AAS). The PIMA-GA-DACH-platinum nanoparticle was found to be more active than free oxaliplatin in vitro. In vivo, the nanoparticles resulted in greater tumor inhibition than oxaliplatin (equivalent to 5 mg kg⁻¹ platinum dose) with minimal nephrotoxicity or body weight loss. ICP-AAS revealed significant preferential tumor accumulation of platinum with reduced biodistribution to the kidney or liver following PIMA-GA-DACH-platinum nanoparticle administration as compared with free oxaliplatin. These results indicate that the rational engineering of a novel polymeric nanoparticle inspired by the bioactivation of oxaliplatin results in increased antitumor potency with reduced systemic toxicity compared with the parent cytotoxic. Rational design can emerge as an exciting strategy in the synthesis of nanomedicines for cancer chemotherapy.

Fig 1. Structure activity-inspired engineering of oxaliplatin nanoparticle

(a) Chemical structure of oxaliplatin. The broken lines demarcate the leaving group. (b) Scheme shows the chemical synthesis of PIMA-GA-DACH-platinum complex. Conjugation of PIMA with glucosamine, followed by complexation introduces a monocarboxylato and O→Pt coordinate linkage between the copolymer and Pt of DACH-platinum. (c) High-resolution transmission electron micrographs show the morphology of PIMA-GA-oxaliplatin nanoparticles formed. Bar= 500 nm. (d) Plot shows the size distribution of nanoparticle measured using a dynamic laser light scatter. (e) Graph shows zeta potential of a representative sample of nanoparticles suspended in double distilled water.

Fig 2. Release kinetics profile of aquated DACH-Pt from the nanoparticles with time under different pH conditions

Free oxaliplatin or PIMA-GA-DACH-platinum conjugates (containing equivalent amounts of Pt) were dialyzed at an acidic pH (5.5) mimicking the endosomal fraction as compared with a reference alkaline pH of 8.5 or at the physiological pH (7.4). The release of active cyclohexanediamine platinum (II) molecule from the polymer complex was found to be temporally sustained and pH-dependent, with faster release observed in acidic conditions as measured using inductive-coupled plasma atomic absorption spectrometry.

Fig 3. Cellular uptake and cytotoxity assay

(a) Murine breast cancer cells 4T1, human hepatocellular carcinoma cells CP20, and human metastatic breast cancer MDA-MB-231 cells, were treated with fluorescein isothiocyanate (FITC)-conjugated PIMA-GA-DACH-platinum nanoparticles for different durations (4 h, 6 h, 12 h, and 24 h). The cells were labeled with lysotracker red at different time-points following incubation with the nanoparticles. The merge images show co-labeling, indicating that the nanoparticles are internalized into the endolysosomes over time. (b) Graphs show the concentration-effect of different treatments on cellular viability as measured using MTS assay. Breast cancer cells, 4T1 and MDA-MB-231, hepatocellular carcinoma CP20 cells, and ovarian cancer cells SKOV3, were used for this study. X-axis shows the equivalent concentrations of platinum. Where blank polymeric controls were used, dose of polymer used was equivalent to that used to deliver that specific dose of DACH-platinum in the complexed form. PIMA-GA-Ox refers to the isomer [PIMA-GA-DACH-platinum].

Fig 4. PIMA-GA-DACH-platinum nanoparticle exerts superior anti-tumor effect with reduced systemic toxicity compared to free oxaliplatin in a 4T1 breast cancer model

(a) Graphs show the effect of treatments on (i) tumor volume and (ii) body weight over the treatment period. The formulations were prepared and validated such that 100 μL of phosphate buffer saline contained 5 mg/kg and 15 mg/kg of DACH-Pt either as free oxaliplatin or nanoparticle (i.e. all animals received an equivalent dose of platinum (administered via tail vein injections). The animals were dosed i.v. on Day 9, 11 and 13. Data shown are mean ± SE, n=4–8. (b) Images of representative tumors from different treatment groups.

Fig 5. Biodistribution of PIMA-GA-DACH-platinum nanoparticles in vivo

Graph shows the levels of Pt in the organs after 4T1 tumor-bearing animals were dosed thrice every alternate day starting Day 9 over duration of the experiment. The formulations were prepared and validated such that 100 μL of phosphate buffer saline contained 5 mg/kg and 15 mg/kg of DACH-Pt either as free oxaliplatin or nanoparticle (i.e. all animals received an equivalent dose of platinum (administered via tail vein injections). The Pt concentration was measured using ICP-MS, and normalized to the total weight of the tissue. Data shown are mean ± SEM (n=3–5), *P<0.05 vs free oxaliplatin-treated group (at equivalent platinum dose).

Fig 6. PIMA-GA-DACH-platinum nanoparticle exhibits lesser renal toxicity but increased tumor apoptosis compared with free oxaliplatin in vivo

The formulations were prepared and validated such that 100 μL of phosphate buffer saline contained 5 mg/kg and 15 mg/kg of DACH-Pt either as free oxaliplatin or nanoparticle (i.e. all animals received an equivalent dose of platinum (administered via tail vein injections). Representative epifluorescence images of kidney and tumor sections following TUNEL staining for apoptosis. (d) Graphs show the effect of treatment on the weight of kidney as a marker for nephrotoxicity Data shown are mean ± SEM [n=4–6]. *P<0.05 vs free oxaliplatin-treated group (at equivalent platinum dose).