Construction of a programmed activation nanosystem based on intracellular hypoxia in cisplatin-resistant tumor cells for reversing cisplatin resistance

Abstract

Cancer poses a significant threat to human life and health. Cancers treated with cisplatin invariably develop drug resistance. This challenge can be overcome by identifying and exploiting the vulnerabilities acquired by drug-resistant cancer cells, paving the way for finding effective novel treatment options for cisplatin-resistant cancers. Our previous study revealed that cisplatin resistance in cancer cells comes at the cost of increased intracellular hypoxia. In this study, we used 2-nitroimidazole modified hyaluronic acid (HA-NI) as the carrier. The cisplatin-resistant tumor cell specific intracellular hypoxia programmed activation nanomedicine (T/C@HN NPs) was constructed by the hypoxic toxic drug tirapazamine (TPZ) and encapsulating chlorin e6 (Ce6) into HA-NI using polymer assembly technology. The amphiphilic carrier could release free Ce6 molecules under the stimulation of intracellular hypoxic environment, and exhibit specific "activated state" photodynamic properties in cisplatin-resistant tumor cells. Upon irradiation, Ce6-mediated photodynamic therapy further intensifies hypoxia, amplifying its cytotoxicity. This project systematically evaluated the effects of T/C@HN NPs on the identification and recognition of cisplatin-resistant tumors using drug-resistant patient-derived xenograft (PDX) models. This study provides a promising avenue for the development of novel treatment of cisplatin-resistant tumors.

Keywords: Cisplatin-resistant; Hypoxia activation; Intracellular hypoxia; Nanomedicine; Program activation.

Graphical abstract

Scheme 1

Schematic diagram of specific recognition of nanomaterials for cisplatin-resistant tumors. Nanomaterials release drugs by specific structural disintegration induced by intracellular hypoxia in cisplatin-resistant tumors. Free Ce6 can aggravate the cytotoxicity of TPZ by photodynamic therapy.

Fig. 1

Preparation and characterization of HA-NI. A) Synthetic scheme of HA-NI conjugates. B) The solid and solution morphology of HA-NI. C) The ¹H NMR spectra of HA and HA-NI. D) Ultraviolet visible absorption spectra of HA, NI, HA-NI. E) Plot of I₃₇₃/I₃₈₄ intensity versus concentrations of HA-NI. F) The characteristic UV absorption values of NI and HA-NI at 327 nm under hypoxic conditions.

Fig. 2

Preparation and characterization of T/C@HN NPs. A) Representative DLS images showcase T/C@HN NPs under normal conditions and after incubation for 15, 30 min, and 1.5h in hypoxic setting. B) Representative TEM images showcase T/C@HN NPs under normal conditions and after incubation for 15, 30 min, and 1.5h in hypoxic setting. Scale bar, 100 nm. C) The UV absorption curve of TPZ, Ce6 and T/C@HN NPs. D) Release curve of TPZ in T/C@HN NPs in normal setting and in hypoxia setting. E) Release curve of Ce6 in T/C@HN NPs in normal setting and in hypoxia setting. F) The trend of changes in the UV absorption values of DPBF at 410 nm in different groups. G) The oxygen change curves in different groups detected by a portable dissolved oxygen analyzer. H) Hemolysis rate and corresponding images of distilled water, saline and T/C@HN NPs with different concentrations. ∗∗∗P < 0.001.

Fig. 3

The inhibitory effect and mechanism of T/C@HN NPs on three types of cells. A) Viability of BEAS-2B, A549 and A549 DDP cells treated with T/C@HN NPs+L for 6 h. B) One-week analysis of colony formation by nanoparticles on BEAS-2B, A549, and A549 DDP cell proliferation post-treatment with control, C@HN NPs, T@HN NPs, T/C@HN NPs, C@HN NPs+L and T/C@HN NPs+L. Scale bar, 500 μm. C) The fluorescence signals of Ce6 in BEAS-2B, A549 and A549 DDP cells co-cultured with different formulations for 6 h. D) Quantitative analysis of the red fluorescence signal of Ce6 in C. E) The fluorescence signals of Ce6 in BEAS-2B, A549 and A549 DDP cells co-cultured with T/C@HN NPs for 6 h under normoxic and hypoxic conditions. F) Quantitative analysis of the red fluorescence signal of Ce6 in E. Scale bar, 40 μm. Error bars represent ± s.d. Statistical significance was determined by two-way ANOVA (B and D). ∗P < 0.1, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001.

Fig. 4

In vitro inhibition evaluation of T/C@HN NPs on A549 DDP cells. A) The viability of A549 DDP cells determined by CCK-8 after 24 h incubation with different concentrations and formulations. B) CLSM images of A549 DDP cells co-stained with calcein AM and propidium iodide (PI) after treatment with different formulations at a Ce6 equivalent concentration of 0.54 μΜ. Scale bars, 200 μm. C) A549 DDP cell apoptosis detected by Annexin V-FITC and PI double staining after treatment with different formulations at a Ce6 equivalent concentration of 0.54 μΜ. D) Quantification of early and late apoptosis of A549 DDP cell in C. E) Detection of ROS production by DCFH-DA in A549 DDP cells after different treatments at a Ce6 equivalent concentration of 0.54 μΜ. F) Quantification of the green fluorescence intensity of DCFH-DA staining in E. Scale bars, 100 μm. Error bars represent ± s.d. Statistical significance was determined by one-way ANOVA (A) or Student's t-test (F). ∗∗∗∗P < 0.0001, ns P > 0.05.

Fig. 5

Specific inhibition efficacy of T/C@HN NPs in nude mouse bearing PDX model. A) Schematic diagram of in vivo anti-tumor experiments. B) PDX tumor growth kinetics of each BALB/c nude mice in different treatment groups. C) Statistical analysis of tumor growth curves in mice. D) Tumor weight collected from mice in different treatment groups. E) Tumor images collected from mice. F) Tumor inhibition rates in different treatment groups. G) Body weight changes in mice of the six treatment groups. H) Staining of tumor tissue sections obtained from mice after treatment, including H&E, Ki67 and Tunel. Scale bar, 100 μm, 50 μm, 100 μm respectively. Error bars represent ± s.d. Statistical significance was determined by Student's t-test (C, D and F). ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001, ns P > 0.05.