Targeted delivery of cisplatin to prostate cancer cells by aptamer functionalized Pt(IV) prodrug-PLGA-PEG nanoparticles

Abstract

Cisplatin is used to treat a variety of tumors, but dose limiting toxicities or intrinsic and acquired resistance limit its application in many types of cancer including prostate. We report a unique strategy to deliver cisplatin to prostate cancer cells by constructing Pt(IV)-encapsulated prostate-specific membrane antigen (PSMA) targeted nanoparticles (NPs) of poly(D,L-lactic-co-glycolic acid) (PLGA)-poly(ethylene glycol) (PEG)-functionalized controlled release polymers. By using PLGA-b-PEG nanoparticles with PSMA targeting aptamers (Apt) on the surface as a vehicle for the platinum(IV) compound c,t,c-[Pt(NH(3))(2)(O(2)CCH(2)CH(2)CH(2)CH(2)CH(3))(2)Cl(2)] (1), a lethal dose of cisplatin was delivered specifically to prostate cancer cells. PSMA aptamer targeted delivery of Pt(IV) cargos to PSMA(+) LNCaP prostate cancer cells by endocytosis of the nanoparticle vehicles was demonstrated using fluorescence microscopy by colocalization of green fluorescent labeled cholesterol-encapsulated NPs and early endosome marker EEA-1. The choice of linear hexyl chains in 1 was the result of a systematic study to optimize encapsulation and controlled release from the polymer without compromising either feature. Release of cisplatin from the polymeric nanoparticles after reduction of 1 and formation of cisplatin 1,2-intrastrand d(GpG) cross-links on nuclear DNA was confirmed by using a monoclonal antibody for the adduct. A comparison between the cytotoxic activities of Pt(IV)-encapsulated PLGA-b-PEG NPs with the PSMA aptamer on the surface (Pt-NP-Apt), cisplatin, and the nontargeted Pt(IV)-encapsulated NPs (Pt-NP) against human prostate PSMA-overexpressing LNCaP and PSMA(-) PC3 cancer cells revealed significant differences. The effectiveness of PSMA targeted Pt-NP-Apt nanoparticles against the PSMA(+) LNCaP cells is approximately an order of magnitude greater than that of free cisplatin.

Scheme 1.

Chemical structure of the hydrophobic platinum(IV) compound 1 and the chemistry by which the active drug, cisplatin is released, after reduction in the cell.

Fig. 1.

Construction and properties of aptamer-functionalized Pt(IV) nanoparticles. (A) Synthesis of Pt(IV)-encapsulated PLGA-b-PEG-COOH nanoparticles by nanoprecipitation and conjugation of PSMA aptamer to NP. (B) Loading of 1 in the PLGA-b-PEG-COOH nanoparticles. (C) Size of the Pt(IV)-encapsulated nanoparticles.

Fig. 2.

In vitro release kinetics of encapsulated Pt(IV) compound 1 from PLGA-b-PEG nanoparticles in PBS (pH 7.4) at 37 °C.

Fig. 3.

Detection of endosome formation and cellular uptake of Pt-NP-Apt in LNCaP cells by fluorescence microscopy. Green fluorescent 22-NBD-cholesterol and 1 were encapsulated in the PLGA-b-PEG nanoparticles and PSMA aptamers were conjugated to the surface of the particles. The early endosomes were visualized in red by using the early endosome marker EEA-1.

Fig. 4.

Cytotoxicity profiles of PSMA aptamer-targeted Pt(IV)-encapsulated PLGA-b-PEG nanoparticles (Pt-NP-Apt) (red circles), nontargeted nanoparticles (Pt-NP) (black squares), and compound 1 (blue triangles) with (A) PSMA⁺ LNCaP cells and (B) PSMA⁻ PC3 cells after 72 h as determined by the MTT assay.