Lipid-Polymer Hybrid Nanoparticle-Based Combination Treatment with Cisplatin and EGFR/HER2 Receptor-Targeting Afatinib to Enhance the Treatment of Nasopharyngeal Carcinoma

Abstract

Purpose: Nasopharyngeal carcinoma (NPC) is one of the most prevalent carcinomas among the Cantonese population of South China and Southeast Asia (responsible for 8% of all cancers in China alone). Although concurrent platinum-based chemotherapy and radiotherapy have been successful, metastatic NPC remains difficult to treat, and the failure rate is high.

Methods: Thus, we developed stable lipid-polymer hybrid nanoparticles (NPs) containing cisplatin (CDDP) and afatinib (AFT); these drugs act synergistically to counter NPC. The formulated nanoparticles were subjected to detailed in vitro and in vivo analysis.

Results: We found that CDDP and AFT exhibited synergistic anticancer efficacy at a specific molar ratio. NPs were more effective than a free drug cocktail (a combination) in reducing cell viability, enhancing apoptosis, inhibiting cell migration, and blocking cell cycling. Cell viability after CDDP monotherapy was as high as 85.1%, but CDDP+AFT (1/1 w/w) significantly reduced viability to 39.5%. At 1 µg/mL, AFT/CDDP-loaded lipid-polymer hybrid NPs (ACD-LP) were significantly more cytotoxic than the CDDP+AFT cocktail, indicating the superiority of the NP system.

Conclusion: The NPs significantly delayed tumor growth compared with either CDDP or AFT monotherapy and were not obviously toxic. Overall, the results suggest that AFT/CDDP-loaded lipid-polymer hybrid NPs exhibit great potential as a treatment for NPC.

Keywords: afatinib; antitumor; apoptosis; cisplatin; nanoparticles; nasopharyngeal carcinoma.

Figure 1

(A) A schematic of cisplatin- and afatinib-loaded lipid–polymer hybrid nanoparticles prepared via emulsification. (B) The particle size distribution as revealed by dynamic light scattering. (C) Nanoparticle morphology as revealed by transmission electron microscopy.

Figure 2

The release of cisplatin and afatinib from lipid–polymer hybrid nanoparticles over 72 h at pH 7.4 and pH 5.0. Afatinib was assayed via high-performance liquid chromatography and cisplatin via mass spectrometry.

Figure 3

(A) Cellular uptake of cisplatin- and afatinib-loaded lipid–polymer hybrid nanoparticles by HONE1 cells as revealed by flow cytometry. (B) The viability of HONE1 cells. Concentration-dependent cytotoxicity was assayed using the MTT assay. (C) The effects of the drugs at different molar ratios on cell viability. (D) The effects of different formulations on cell viability. *p<0.05 and ***p<0.0001 is the statistical difference.

Figure 4

(A) Apoptosis of HONE1 cells as revealed by flow cytometry after staining with Annexin V-FITC and propidium iodide. (B) Qualitative analysis of apoptosis employing Hoechst 33258 staining. The free drugs (alone or in combination) and cisplatin- and afatinib-loaded lipid–polymer hybrid nanoparticles were incubated with cancer cells for 24 h.

Figure 5

Cell cycle analysis of HONE1 cells using flow cytometry after staining with PI. The proportions of cells in the G0/G1, S, G2/M, and sub-G0 cell cycle phases were analyzed.

Figure 6

Migration of HONE1 cells after treatment with different formulations. Images were captured at 0 and 24 h using the Axiovert 200M microscope (Zeiss) equipped with the CoolSNAP ES camera.

Figure 7

(A) Inhibition of tumor growth by different formulations in a nasopharyngeal carcinoma-bearing xenograft model. The groups included an untreated control group and groups given empty nanoparticles, the free drugs (alone or in combination), and cisplatin- and afatinib-loaded lipid–polymer hybrid nanoparticles. All formulations were given intravenously every third day (three injections). The data are means ± standard deviation. (B) The tumor weights. The data are means ± standard deviations. *p<0.05, **p<0.01 and ***p<0.0001 is the statistical difference. (C and D) Histochemical analysis of tumor tissues treated with different formulations.

Figure 8

Body weights of tumor-bearing mice treated with different formulations. Mice were weighed daily until day 18 to monitor drug-induced toxicities. *p<0.05 is the statisitcal difference between AFT+CDDP vs ACD-LP. **p<0.05.