Nanoenabled intracellular zinc bursting for efficacious reversal of gefitinib resistance in lung cancer

Abstract

Following the identification of specific epidermal growth factor receptor (EGFR)-activating mutations, gefitinib, one of the first-generation tyrosine kinase inhibitors (TKIs), has proven efficacious in targeting NSCLC that is driven by specific EGFR-activating mutations. However, most patients who initially respond to gefitinib, develop acquired resistance. In the current study, we devised a novel strategy to enhance the efficacy of gefitinib. We developed a simple and effective, nano-interrupter termed zeolitic imidazolate framework-8@Gefitinib@hyaluraonic nanoparticle (ZIF-8@G@HA NP). This nanoparticle was prepared by loading gefitinib onto a ZIF-8 nanoplatform followed by coating with hyaluronic acid (HA). The burst of Zn²⁺ release triggered by pH-sensitive degradation of ZIF-8@G@HA NPs was shown to enhance the efficacy of gefitinib in parental lung carcinoma HCC827 cells and overcame acquired gefitinib resistance in gefitinib drug resistant (GDR) HCC827 cells. We found that when treated with ZIF-8@G@HA NPs, Zn²⁺ acts synergistically with gefitinib via increased apoptosis in both parental and GDR HCC827 cells. Consistently, this in vitro activity was correlated with in vivo tumor growth inhibition. Interestingly, GDR cells were more sensitive to Zn²⁺ when compared with parental cells. We further found that ZIF-8 NPs overcame gefitinib resistance by triggering reactive oxygen species (ROS) generation and consequent cell cycle arrest at the G2/M phase, resulting in cancer cell apoptosis. Zn²⁺ was also found to block P-gp activity, facilitating the accumulation of gefitinib in GDR cells, thus enhancing the anti-tumor efficacy of gefitinib resulting in reversal of gefitinib resistance. Thus, this study offers a novel and promising strategy to surmount acquired gefitinib resistance via cell cycle arrest at the G2/M phase by facilitating gefitinib accumulation in GDR cells.

Keywords: Cancer therapy; Drug resistance; Lung cancer; Reactive oxygen species (ROS); Zeolitic imidazolate framework-8 (ZIF-8); Zn2+.

Schematic 1

a) Preparation procedure of zeolitic imdazolate framework-8@Gefitinib@hyaluraonic nanoparticle (ZIF-8@G@HA nano-interrupters). b) Schematic illustration for tumor killing and overcoming gefitinib resistance by ZIF-8@G@HA nano-interrupters.

Figure 1

Preparation and characterization of ZIF-8@G@HA. (A) Transmission electron microscopy (TEM) image of ZIF-8, ZIF-8@HA, ZIF-8@G, and ZIF-8@G@HA NPs. (B) Average particle sizes of ZIF-8, ZIF-8@HA, ZIF-8@G, and ZIF-8@G@HA. (C) Zeta potential profiles of ZIF-8, ZIF-8@G, and ZIF-8@G@HA (D) The pH-responsive release of Zn²⁺ from ZIF-8@G@HA in pH 5.4, and pH 7.2. (E) The pH-responsive release of gefitinib from ZIF-8@G@HA in pH 5.4, and pH 7.2. (F) Representative TEM images of ZIF-8@G@HA at pH 7.4 and pH 5.4. (G) Representative size distribution of ZIF-8@G@HA in water and physiological buffer (phosphate-buffered saline [PBS]) (scale = 100 nm).

Figure 2

Efficacy of ZIF-8@G@HA NPs in parental HCC827 cells. (A) Fluorescence microscopy images of Zn²⁺ release in HCC827 cells after treatment with ZIF-8@G@HA NPs for 1,6,12 and 24 h (scale bar = 50 μm). (B) The corresponding flow cytometric analysis of Zn²⁺ release in HCC827 cells after treatment with ZIF-8@G@HA NPs for 1,6,12 and 24 h. (C) Detection of HCC827 cell viability upon gefitinib treatment for 24h using the tetrazolium MTT assay. (D) Detection of HCC827 cell viability upon treatment with ZIF-8@HA/ZIF-8@G@HA NPs and the corresponding gefitinib within ZIF-8@G@HA NPs for 24h using the MTT assay. (E) HCC827 cell apoptosis analysis after treatments of gefitinib/ZIF-8@HA/ZIF-8@G@HA for 24h. (F) Live and dead HCC827 cell staining images after treatments of gefitinib/ZIF-8@HA/ZIF-8@G@HA for 24h (scale bar = 200 μm).

Figure 3

Efficacy of the ZIF-8@G@HA NPs in HCC827 xenograft mice. (A-C) Analysis of tumor progression in terms of tumor size, tumor volume, and tumor weight after tumor excision from HCC827 xenograft mice that received different treatments for three weeks: (1) PBS (via oral administration), (2) Gefitinib (25 mg/kg, via oral administration), (3) ZIF-8@HA (20mg/kg, via intravenous injection), (4) Gefitinib (25 mg/kg, via oral administration) + ZIF-8@HA (20mg/kg, via intravenous injection) and (5) ZIF-8@G@HA (20mg/kg, via intravenous injection). (D) Tumor cell apoptosis in the excised HCC827 tumors. Scale bar= 200 μm. (E) H&E staining of the paraffin-embedded sections of the HCC827 tumor tissues (scale bar = 250 μm).

Figure 4

Efficacy of the ZIF-8@G@HA NPs in GDR cells. (A) Determination of cell viability after gefitinib treatment of both parental HCC827 cells and GDR cells using the tetrazolium MTT assay. (B) Fluorescence microscopy images of Zn²⁺ release in GDR cells after treatment with ZIF-8@G@HA NPs for 1,6,12 and 24 h (scale bar = 50μm). (C) The corresponding flow cytometric analysis of Zn²⁺ release in GDR cells after treatment with ZIF-8@G@HA NPs for 1,6,12 and 24 h. (D) Determination of cell viability after treatment with ZIF-8@HA/ZIF-8@G@HA NPs and the corresponding gefitinib within ZIF-8@G@HA NPs for 24h in GDR cells using the MTT assay. (E) Comparison of the viability of parental HCC827 cells and GDR cells after different treatments. (F) Cell apoptosis of GDR cells after different treatments. (G) Live and dead staining images of GDR cells with different treatments after culturing for 24 h (scale bar = 200 μm).

Figure 5

Efficacy of the ZIF-8@G@HA NPs in mice bearing GDR xenografts. (A-C) Analysis of tumor progression in terms of the tumor size, tumor volume, and tumor weight of the tumors excised from the GDR xenograft mice that received different treatments for three weeks: (1) PBS (via oral administration), (2) Gefitinib (25 mg/kg, via oral administration), (3) ZIF-8@HA (20mg/kg, via intravenous injection), (4) Gefitinib (25 mg/kg, via oral administration) + ZIF-8@HA (20mg/kg, via intravenous injection) and (5) ZIF-8@G@HA (20mg/kg, via intravenous injection). (D) Analysis of cell apoptosis in excised GDR tumors. Scale bar = 200 μm. (E) H&E staining of the paraffin-embedded sections of the GDR tumor tissues (scale bar = 250 μm).

Figure 6

Release of Zn²⁺ and generation of ROS mediating tumor cell killing. (A) Detecting ROS production in HCC827 cells after treatments of gefitinib/ZIF-8@HA/ZIF-8@G@HA for 24h via DCFH-DA staining. Scale bar = 150 μm. (B) Determination of HCC827 cell viability after NPs treatments and EDTA rescue. (C) Cell apoptosis of HCC827 cells after NPs treatments and EDTA rescue. (D) Fluorescence microscopy images of ROS generation in HCC827 cells after NPs treatments and EDTA rescue. Scale bar = 150 μm. (E) Determination of HCC827 cell viability after NPs treatments and NAC rescue. (F) Cell apoptosis of HCC827 cells after NPs treatments and NAC rescue. (G) Fluorescence microscopy images of ROS generation in HCC827 cells after NPs treatments and NAC rescue (scale bar = 150 μm).

Figure 7

Release of Zn²⁺ and generation of ROS medaiting the reversal of gefitinib resistance and tumor cell killing. (A) Detection of ROS production in GDR cells after treatments of gefitinib/ZIF-8@HA/ZIF-8@G@HA for 24h and staining with DCFH-DA. Scale bar = 150 μm. (B) Determination of HCC827 cell viability after NPs treatments and EDTA rescue. (C) Determination of GDR cell viability after NPs treatments and NAC rescue. (D) Cell apoptosis of GDR cells after NPs treatments and EDTA rescue. (E) Cell apoptosis of GDR cells after NPs treatments and NAC rescue. (F) Fluorescence microscopy images of ROS generation in GDR cells after NPs treatments and EDTA rescue. Scale bar = 150 μm. (G) Microscope images of ROS generation in GDR cells after NPs treatments and NAC rescue (scale bar= 150 μm).

Figure 8

The difference between parental HCC827 and GDR cells and the mechanism underlying the reversal of gefitinib resistance. (A) The flow chart of RNA-sequencing analysis among parental HCC827 and GDR cells. (B) Volcano plot of differentially expressed genes between HCC827 and GDR cells (C) Gene ontology (GO) analysis among differentially expressed genes. (D) Heat map among cell cycle related genes in HCC827 and GDR cells. (E) Protein-protein interaction (PPI) analysis among cell cycle related genes. (F,H) Cell cycle phase analysis in GDR cells after treatments of gefitinib/ZIF-8@HA/ZIF-8@G@HA for 24h. (G,I) Cell cycle phase analysis in HCC827 cells after treatments of gefitinib/ZIF-8@HA/ZIF-8@G@HA for 24h. (J-L) Expression levels of cell cycle related markers in GDR cells after treatments of gefitinib/ZIF-8@HA/ZIF-8@G@HA for 24h. (M,O) Expression levels of P-gp among HCC827 cells and GDR cells. (N,P) Expression of P-gp in GDR cells after treatments of gefitinib/ZIF-8@HA/ZIF-8@G@HA for 24h.