Cisplatin Prodrug-Loaded Nanoparticles Based on Physalis Mottle Virus for Cancer Therapy

Abstract

Nanoparticle-based prodrugs offer an effective strategy to improve the safety and delivery of small-molecule therapeutics while reducing the risk of drug resistance. Here, we conjugated a maleimide-functionalized cisplatin prodrug containing Pt(IV) to the internal and/or external surface of virus-like particles (VLPs) derived from Physalis mottle virus (PhMV) to develop a pH-sensitive drug delivery system. The internally loaded and PEGylated VLPs (Pt-PhMVCy5.5-PEG) were taken up efficiently by cancer cells where they released platinum, presumably as a reduced, DNA-reactive Pt(II) complex, rapidly under acidic conditions in vitro (>80% in 30 h). The efficacy of the VLP-based drug delivery system was demonstrated against a panel of cancer cell lines, including cell lines resistant to platinum therapy. Furthermore, Pt-PhMVCy5.5-PEG successfully inhibited the growth of xenograft MDA-MB-231 breast tumors in vivo and significantly prolonged the survival of mice compared to free cisplatin and cisplatin-maleimide. Pt-PhMVCy5.5-PEG therefore appears promising as a prodrug to overcome the limitations of conventional platinum-based drugs for cancer therapy.

Keywords: cisplatin resistance; nanomedicine; pH-responsive drug delivery; plant virus-like nanoparticles; prodrug.

Figure 1.

Physicochemical characterization of PhMV-based nanoparticles. (A) Cartoon, TEM image (scale bars: 100 nm), and size exclusion chromatography profile of native PhMV and the three VLPs: PhMVCy5.5-PEG, Pt-PhMVCy5.5-PEG, and Pt-PhMVCy5.5-Pt. (B) SDS-PAGE analysis of the four nanoparticles followed by staining with Coomassie Brilliant Blue (left) or fluorescence imaging (right). (C) Agarose gel electrophoresis of the four nanoparticles followed by staining with Coomassie Brilliant Blue (left) or fluorescence imaging (right).

Figure 2.

Platinum release from nanoparticles and the comparative toxicity of nanoparticles and free drugs. Data from triplet experiments and standard deviations are shown. (A) Cisplatin release profiles from nanoparticles in pH-adjusted solutions. (B) Cytotoxicity of the control particle PhMVCy5.5-PEG determined by the MTT assay in MDA-MB-231 cells. (C) IC₅₀ values (mean values ± standard deviations) of free cisplatin, Pt-Mal, Pt-PhMVCy5.5-PEG, and Pt-PhMVCy5.5-Pt particles against a panel of cancer cells determined by the MTT assay. All concentrations were normalized to Pt. Asterisk: ref.

Figure 3.

Analysis of VLP uptake and distribution within MDA-MB-231 cells. (A) Quantitative analysis by flow cytometry and (B) mean fluorescence intensities (MFIs) of cells in each sample (n = 3 ± standard deviations, ***p < 0.001 per Student’s t-test). Data were analyzed using FlowJo v10. (C, D) Intracellular distribution and nuclear content of Pt following the incubation of MDA-MB-231 cells with free Pt-Mal or Pt-PhMVCy5.5-PEG for 24 h (**p < 0.01).

Figure 4.

Inhibition of tumor growth in an athymic mouse model (n = 10) with MDA-MB-231 xenografts after treatment with different formulations. The treatment began when tumors reached a volume of ~150 mm³ and involved a twice-weekly intravenous bolus of 1.0 mg Pt/kg in the form of cisplatin (cis-Pt), Pt-Mal, or Pt-PhMVCy5.5-PEG (Pt-PhMV). PBS or PhMVCy5.5-PEG (PhMV) particles were administered as controls, with PhMVCy5.5-PEG particle dosage normalized to Pt-PhMVCy5.5-PEG. Tumor volumes and body weight were measured before each injection. Statistical analysis was carried out by two-way ANOVA. Mean tumor volumes and standard errors of the mean are shown. (A) Pt-Mal vs cis-Pt, (B) Pt-Mal vs Pt-PhMV, and (C) cis-Pt vs Pt-PhMV. (D) Statistical analysis for survival curves: log-rank (Mantel–Cox) test. ns = no statistical significance, ***: p < 0.001, ****: p < 0.0001.

Figure 5.

(A) Biodistribution of Pt in organs/tissues as determined by ICP-MS after three injections of PBS, Pt-Mal, cisplatin (cis-Pt), or Pt-PhMVCy5.5-PEG (Pt-PhMV). (B) Body weight of tumor bearing mice over the course of the study. For (A) and (B), mean values ± standard deviations are shown.

Scheme 1.

Strategies for the internal (A) and external (B) surface functionalization of VLPs based on PhMV