Pt(II)-PLGA Hybrid in a pH-Responsive Nanoparticle System Targeting Ovarian Cancer

Abstract

Platinum-based agents are the main treatment option in ovarian cancer (OC). Herein, we report a poly(lactic-co-glycolic acid) (PLGA) nanoparticle (NP) encapsulating platinum (II), which is targeted to a cell-spanning protein overexpressed in above 90% of late-stage OC, mucin 1 (MUC1). The NP is coated with phospholipid-DNA aptamers against MUC1 and a pH-sensitive PEG derivative containing an acid-labile hydrazone linkage. The pH-sensitive PEG serves as an off-on switch that provides shielding effects at the physiological pH and is shed at lower pH, thus exposing the MUC1 ligands. The pH-MUC1-Pt NPs are stable in the serum and display pH-dependent PEG cleavage and drug release. Moreover, the NPs effectively internalize in OC cells with higher accumulation at lower pH. The Pt (II) loading into the NP was accomplished via PLGA-Pt (II) coordination chemistry and was found to be 1.62 wt.%. In vitro screening using a panel of OC cell lines revealed that pH-MUC1-Pt NP has a greater effect in reducing cellular viability than carboplatin, a clinically relevant drug analogue. Biodistribution studies have demonstrated NP accumulation at tumor sites with effective Pt (II) delivery. Together, these results demonstrate a potential for pH-MUC1-Pt NP for the enhanced Pt (II) therapy of OC and other solid tumors currently treated with platinum agents.

Keywords: in vivo imaging; nanoparticles; ovarian cancer; pH-sensitive; platinum therapy.

Figure 1

A schematic of an active targeting module (A), the proposed pH-MUC1-Pt NP platform (B), and activation of Pt complex from PLGA-Pt (II) hybrid (C).

Figure 2

(A) TEM image of negatively stained pH-MUC1-Pt NPs; (B) stability of pH-MUC1-Pt NPs in 10% FBS at pH 7.4, 6.8 and 5.5; (C) Measurement of the hydrazone bond cleavage of the pH-MUC1 NP via fluorescamine test after incubation of the NP at different pHs; (D) Time dependent hydrazone bond cleavage of pH-MUC1 NP at pH 6.8. Data in (B–D) presented as mean ± st. dev (n = 3).

Figure 3

In vitro Pt (II) release from pH-MUC1-Pt NP in PBS at 37 °C. Data presented as mean ± st. dev. (n = 3).

Figure 4

(A) ELISA test results for MUC 1 expression in OC cells. Data in presented as mean ± st. dev (n = 3). (B) Confocal microscope images of OC cell lines after 15 min incubation with pH-MUC1-Cy5.5 NPs. The NPs are shown in red, MUC1in green, and nuclei are in blue. Scale bars are 5 µm in all images.

Figure 5

Flow cytometry analysis of A2780 and CP70 cells after 15 min incubation with pH-MUC1-FITC NP.

Figure 6

Cellular viability of seven different OC cell lines, following 72 h incubation with pH-MUC1-Pt NP and controls. Data presented as mean ± st. dev (n = 3) and p < 0.01 all cell lines, p < 0.2 in TOV-21G. Asterisks denote a statistically significant difference. The concentrations of Pt(II) are constant in each cell line and are as follows: SKOV-3 3.44 × 10⁻⁵ M, CP70 5.28 × 10⁻⁵ M, OV-90 2.95 × 10⁻⁵ M, TOV-21G 2.95 × 10⁻⁵ M, A2780 3.08 × 10⁻⁵ M, OVCAR-3 3.08 × 10⁻⁵ M, ES-2 3.44 × 10⁻⁵ M.

Figure 7

(A) Pt:DNA molar ratio in A2780 cells after incubation with pH-MUC1-Pt NP. (B) The ratio of Pt (II) to DNA base pairs in all OC cell lines following 72 h incubation with pH-MUC1-Pt NP. Data in (A,B) presented as mean ± st. dev (n = 3). The concentrations of Pt (II) in all cell lines are the same as used for in vitro studies.

Figure 8

The images of NPs biodistribution in vivo (A,B) in an ovarian cancer mouse model and excised tumors (C) from an NP-injected mouse (A) and a control mouse (B). White arrows indicate the tumor site. The data were collected with the help of the “Small Animal Imaging Center in the Translational and Molecular Imaging Institute.”

Figure 9

(A) Ex-vivo study of Apt-PLGA-Pt NPs biodistribution in organs at four days after the NPs injection (left) and control untreated organs (right). The data were collected with the help of the “Small Animal Imaging Center in the Translational and Molecular Imaging Institute.” (B) AAS analysis of a platinum (II) biodistribution in organs at four days after the NPs injection.