Nanoscale metal-organic frameworks for the co-delivery of cisplatin and pooled siRNAs to enhance therapeutic efficacy in drug-resistant ovarian cancer cells

Abstract

Ovarian cancer is the leading cause of death among women with gynecological malignancies. Acquired resistance to chemotherapy is a major limitation for ovarian cancer treatment. We report here the first use of nanoscale metal-organic frameworks (NMOFs) for the co-delivery of cisplatin and pooled small interfering RNAs (siRNAs) to enhance therapeutic efficacy by silencing multiple drug resistance (MDR) genes and resensitizing resistant ovarian cancer cells to cisplatin treatment. UiO NMOFs with hexagonal-plate morphologies were loaded with a cisplatin prodrug and MDR gene-silencing siRNAs (Bcl-2, P-glycoprotein [P-gp], and survivin) via encapsulation and surface coordination, respectively. NMOFs protect siRNAs from nuclease degradation, enhance siRNA cellular uptake, and promote siRNA escape from endosomes to silence MDR genes in cisplatin-resistant ovarian cancer cells. Co-delivery of cisplatin and siRNAs with NMOFs led to an order of magnitude enhancement in chemotherapeutic efficacy in vitro, as indicated by cell viability assay, DNA laddering, and Annexin V staining. This work shows that NMOFs hold great promise in the co-delivery of multiple therapeutics for effective treatment of drug-resistant cancers.

Figure 1

Preparation and characterization of siRNA/UiO-Cis. (a) Schematic presentation of siRNA/UiO-Cis synthesis and drug loading. TEM images of UiO (b), UiO-Cis (c), and siRNA/UiO-Cis (d). Bars: 200 nm. (e) Electron diffraction pattern of UiO by FFT. (f) High-resolution TEM image of UiO. Bar: 20 nm. (g) PXRD patterns of UiO-68 (black), UiO (red), and UiO-Cis (blue). (h) Serum stability of pooled siRNAs in siRNA/UiO-Cis as evaluated by electrophoresis. (i) BSA binding to UiO-Cis with and without siRNA loading. The results were expressed as the amount of BSA (μg) adsorbed to per μg of UiO-Cis.

Figure 2

Cellular uptake and endosomal escape of siRNA/UiO-Cis in SKOV-3 cells. (a) siRNA/UiO-Cis significantly increase (by >11-fold) the siRNA uptake amount compared to naked siRNA (n = 3). (b) CLSM image showing the internalization of siRNA (TAMRA-labeled) into the cytoplasm. Nuclei were stained with DAPI. Bar represents 20 μm. (c) siRNA release from NMOFs was dramatically promoted in 2 mM PBS compared to water. (d) siRNA (TAMRA-labeled, red) successfully escaped from endosomes as evidenced by the separation of green and red fluorescence (white arrows). Endosome/lysosome and nuclei were stained with Lysotracker Green and DAPI, respectively. Bar represents 5 μm.

Figure 3

In vitro gene silencing efficiency and anticancer efficacy. (a) siRNA/UiO-Cis-mediated efficient gene silencing in SKOV-3 cells at a 30 nM siRNA dose. Silencing efficiency was expressed as percentage values of control group treated with PBS. (b) SKOV-3 cells were incubated with free cisplatin, UiO-Cis, pooled siRNAs/UiO-Cis, free cisplatin plus free pooled siRNAs, and free cisplatin plus pooled siRNAs/UiO at different concentrations for 72 h, and then the cytotoxicity was determined by MTS assay. (c) Analysis of DNA ladder on 2% (w/v) agarose gel at 35 V for 5 h after DNA extraction from SKOV-3 cells treated with siRNA/UiO-Cis at an equivalent cisplatin concentration of 10 μM. Lanes 1–5: DNA marker, control, UiO-Cis, siRNA/UiO-Cis, and free cisplatin. (d–f) CLSM images showing cell apoptosis and siRNA (TAMRA-labeled, red) internalization in SKOV-3 cells after incubation with UiO-Cis (d), siRNA/UiO (e), and siRNA/UiO-Cis (f) for 24 h. The apoptotic cells were stained with Alexa Fluor 488 Annexin V conjugate, and the nuclei were stained with DAPI. Bar represented 10 μm.