A Zeolitic Imidazolate Framework-Based Antimicrobial Peptide Delivery System with Enhanced Anticancer Activity and Low Systemic Toxicity

Abstract

Background: The clinical efficacies of anticancer drugs are limited by non-selective toxic effects on healthy tissues and low bioavailability in tumor tissue. Therefore, the development of vehicles that can selectively deliver and release drugs at the tumor site is critical for further improvements in patient survival.

Methods: We prepared a CEC nano-drug delivery system, CEC@ZIF-8, with a zeolite imidazole framework-8 (ZIF-8) as a carrier, which can achieve the response of folate receptor (FR). We characterized this system in terms of morphology, particle size, zeta potential, infrared (IR), x-ray diffraction (XRD), and transcriptome analysis, and examined the in vitro cytotoxicity and cellular uptake properties of CEC@ZIF-8 using cervical cancer cells. Lastly, we established a TC-1 tumor-bearing mouse model and evaluated its in vivo anti-cervical cancer activity.

Results: The CEC@ZIF-8 nano-delivery system had favorable biocompatibility, heat stability, and pH responsiveness, with a CEC loading efficiency of 12%, a hydrated particle size of 174 ± 5.8 nm, a zeta potential of 20.57 mV, and slow and massive drug release in an acidic environment (pH 5.5), whereas release was 6% in a neutral environment (pH 7.4). At the same time, confocal imaging and cell viability assays demonstrated greater intracellular accumulation and more potent cytotoxicity against cancer cells compared to free CEC. The mechanism was analyzed by a series of transcriptome analyses, which revealed that CEC@ZIF-8 NPs differentially regulate the expression levels of 1057 genes in cancer cells, and indicated that the enriched pathways were mainly cell cycle and apoptosis-related pathways via the enrichment analysis of the differential genes. Flow cytometry showed that CEC@ZIF-8 NPs inhibited the growth of HeLa cells by arresting the cell cycle at the G0/G1 phase. Flow cytometry also revealed that CEC@ZIF-8 NPs induced greater apoptosis rates than CEC, while unloaded ZIF-8 had little inherent pro-apoptotic activity. Furthermore, the levels of reactive oxygen species (ROS) were also upregulated by CEC@ZIF-8 NPs while ROS inhibitors and caspase inhibitors reversed CEC@ZIF-8 NPs-induced apoptosis. Finally, CEC@ZIF-8 NPs also reduced the growth rate of xenograft tumors in mice without the systemic toxicity observed with cisplatin treatment.

Conclusions: The CEC@ZIF-8 nano-drug delivery system significantly enhanced the anti-cervical cancer effect of CEC both in vivo and in vitro, providing a more promising drug delivery system for clinical applications and tumor management. At the same time, this work demonstrates the clinical potential of CEC-loaded ZIF-8 nanoparticles for the selective destruction of tumor tissues.

Keywords: ZIF-8; antibacterial peptide; anticancer; drug delivery; pH response.

Scheme 1

Synthesis of CEC@ZIF-8.

Figure 1

Characterization of the CEC@ZIF-8 nanoparticles and ZIF-8 nanoparticles. (a) TEM images of CEC@ZIF-8 and ZIF-8 nanoparticles. Scale bar, 200 nm. (b) SEM images of CEC@ZIF-8 and ZIF-8 nanoparticles. (c) DLS profile of ZIF-8, CEC@ZIF-8. (d) Zeta potentials of ZIF-8 NPs and CEC@ZIF-8 NPs (The red line represents ZIF-8, and the black line represents CEC@ZIF-8).

Figure 2

Basic properties of ZIF-8 and CEC@ZIF-8. (a) XRD patterns of CEC@ZIF-8, ZIF-8, and simulated ZIF-8. (b) FTIR spectrum of ZIF-8 and CEC@ZIF-8. (c) TGA curves of ZIF-8 and CEC@ZIF-8.

Figure 3

In vitro therapy effect of CEC@ZIF-8 against cervical cancer cells. (a) CLSM images of the distribution of drugs in HeLa cells incubated with CEC@ZIF-8 (scale bar = 25 µm). (b) Cell viability of HeLa cells after treatment with (1) free CEC, (2) CEC@ZIF-8, and (3) ZIF-8 for 24 h. (c) Differences in the number of HeLa cell clones formed by different concentrations of CEC@ZIF-8; (d) Statistics of the number of HeLa cell clones formed by different concentrations of CEC@ZIF-8; Compared to the control group, *** p < 0.001, ** p < 0.01.

Figure 4

(a) Principal component analysis of HeLa cells based on (control) untreated control (Triangle representation) and (CEC@ZIF-8) CEC@ZIF-8 treatment groups (Circular representation). (b) Volcano plots to determine the DEGs of the control vs. CEC@ZIF-8 groups. (c) GO pathway annotation analysis of differential genes.

Figure 5

Anticancer action mechanism of CEC@ZIF-8 on HeLa cells. (a,b) Flow cytometric analysis of HeLa cell cell cycle arrest induced by CEC@ZIF-8 and free CEC. (c,d) Flow cytometric analysis of HeLa cell apoptosis induced by CEC@ZIF-8 and free CEC. Compared to the control group, *** p < 0.001, ** p < 0.01, * p < 0.05.

Figure 6

Anticancer action mechanism of CEC@ZIF-8 on HeLa cells. (a) ROS production in HeLa cells after CEC@ZIF-8 NPs treatment for 3, 6, and 24 h, the cells were stained with the fluorescent probe DCFH-DA and analyzed using flow cytometry. (b) The statistical figure. (c) HeLa cells were treated with 30 and 35 μg/mL CEC@ZIF-8NPs and free CEC (35 μg/mL) as the control. After 24 h, cells were treated with JC-1 dye and analyzed by inverted fluorescence microscope. (d) Flow cytometry was used to analyze the changes in JC-1 fluorescence. (e) HeLa cells were pretreated with 10 mM NAC for 1 h, and then treated with CEC@ZIF-8 NPs and CEC for 24 h. The cells were stained with the fluorescent probe DCFH-DA and analyzed using flow cytometry, and the statistical figure. Compared to the control group, *** p < 0.001. At the same concentration, the group treated with inhibitor was compared with the group without inhibitor treatment, # p < 0.05, ### p < 0.001.

Figure 7

Effect of different concentrations of CEC@ZIF-8 nanoparticles on the migration of tumor cells. (a) HeLa cells were treated with different concentrations of CEC@ZIF-8 nanoparticles for 0 h and 24 h. (b) Statistical chart of the migration ability of HeLa cells. (c) Tumor cell invasion and statistics of HeLa cells treated with different concentrations of CEC@ZIF-8 nanoparticles for 24 h. (d) Statistical plot of the invasion ability of HeLa cells. Compared to the control group, *** p < 0.001.

Figure 8

1 × 10⁵ TC-1 cells were injected into the right back of mice, and when the tumor was palpable, the mice were randomly divided into 5 groups. (a) The tumor volume and (b) body weight of the mice were measured every two days during drug treatment. Compared to the control group, * p < 0.05. (c,d) On day 25, the mice were sacrificed and weighed for tumor photography and weight. (e) Tissue sections of TC-1 tumor-bearing mice after CEC@ZIF-8NPs treatment: (1) a section of tumor tissue, (2) liver tissue section, (3) kidney tissue section. Compared to the control group, ** p < 0.01, *** p < 0.001. ZIF-8 group compared with CEC@ZIF-8 group, # p < 0.05.