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. 2021 Aug 16;19(1):246.
doi: 10.1186/s12951-021-00989-z.

Nitric oxide-releasing micelles with intelligent targeting for enhanced anti-tumor effect of cisplatin in hypoxia

Yan Chen  1 , Lei Fang  1 , Weixin Zhou  1 , Jinghan Chang  1 , Xiaojuan Zhang  1 , Chuanchuan He  1 , Chen Chen  1 , Ruicong Yan  1 , Yakai Yan  1 , Yao Lu  1 , Chuanrui Xu  2 , Guangya Xiang  3
Affiliations

Nitric oxide-releasing micelles with intelligent targeting for enhanced anti-tumor effect of cisplatin in hypoxia

Yan Chen et al. J Nanobiotechnology. .

Abstract

Background: Hypoxic tumor microenvironment (TME) promotes tumor metastasis and drug resistance, leading to low efficiency of cancer chemotherapy. The development of targeted agents or multi-target therapies regulating hypoxic microenvironment is an important approach to overcome drug resistance and metastasis.

Methods: In this study, chitosan oligosaccharide (COS)-coated and sialic acid (SA) receptor-targeted nano-micelles were prepared using film dispersion method to co-deliver cisplatin (CDDP) and nitric oxide (NO) (denoted as CTP/CDDP). In addition, we explored the mechanisms by which NO reversed CDDP resistance as well as enhanced anti-metastatic efficacy in hypoxic cancer cells.

Results: Because of the different affinities of COS and SA to phenylboronic acid (PBA) under different pH regimes, CTP/CDDP micelles with intelligent targeting property increased cellular uptake of CDDP and enhanced cytotoxicity to tumors, but reduced systemic toxicity to normal organs or tissues. In addition, CTP/CDDP showed stimulus-responsive release in TME. In terms of anti-tumor mechanism, CTP/CDDP reduced CDDP efflux and inhibited epithelial-mesenchymal transition (EMT) process of tumor by down-regulating hypoxia-inducible factor-1α (HIF-1α), glutathione (GSH), multidrug resistance-associated protein 2 (MRP2) and matrix metalloproteinase 9 (MMP9) expression, thus reversing drug resistance and metastasis of hypoxic tumor cells.

Conclusions: The designed micelles significantly enhanced anti-tumor effects both in vitro and in vivo. These results suggested that CTP/CDDP represented a promising strategy to treat resistance and metastatic tumors.

Keywords: Chitosan oligosaccharide; Cisplatin; Drug resistance; HIF-1α; Hypoxia; Metastasis; Nitric oxide.

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Conflict of interest statement

The authors have declared that no competing interest exists.

Figures

Fig. 1
Fig. 1
Schematic diagram of CTP/CDDP targeting SA residues, and reversing drug resistance and metastasis of hypoxic cancer cells
Fig. 2
Fig. 2
Characterization and drug release properties of micelles. a Schematic representation and b TEM image of CTP/CDDP. c Particle size and d zeta potential of TD, TD/CDDP, TP/CDDP and CTP/CDDP. e In vitro release of NO from CTP/CDDP incubated with or without 10 mmol/L GSH at 37 ℃. f In vitro release of CDDP from CTP/CDDP at pH 5.5, 6.5 and 7.4. Data are expressed as mean ± SD (n = 3)
Fig. 3
Fig. 3
Cellular uptake of micelles in 4T1 cells. The affinity of free PBA to a SA or b COS in pH 7.4 or 6.5 PBS at 37 ℃ for 1 h. c Cellular uptake of 4T1 cells after incubation with different drug formulations at pH 6.5 or 7.4 for 3 h. Data are expressed as mean ± SD (n = 3)
Fig. 4
Fig. 4
In vitro anti-tumor effects of different drug formulations in 4T1 cells. IC50 was calculated to evaluate the cytotoxicity of various drug formulations after incubation with 4T1 cells for 24 h under a normoxia and b hypoxia. c Apoptosis of 4T1 cells treated with different drug formulations for 24 h was detected by Annexin V-FITC/PI double staining. Cells treated with PBS were used as control. Data are expressed as mean ± SD (n = 3), *p < 0.05, ***p < 0.001
Fig. 5
Fig. 5
Mechanism of reversing drug resistance in vitro. a CDDP retention after incubation with free CDDP, TD/CDDP, TP/CDDP and CTP/CDDP for 20 h in hypoxic 4T1 cells. b Intracellular GSH levels of hypoxic 4T1 cells treated with various drug formulations for 24 h. c Western blot of indicated proteins expression in hypoxic 4T1 cells after 24 h incubation with different drugs. d The expression of relative proteins was calculated by the signal intensity of protein bands. Cells treated with PBS were used as control. Data are expressed as mean ± SD (n = 3), **p < 0.01, ***p < 0.001
Fig. 6
Fig. 6
Synergetic anti-metastasis effect in vitro. a Images of 4T1 cells treated with PBS, free CDDP, TD, TD/CDDP, TP/CDDP and CTP/CDDP in wound healing assay at 0 and 24 h. b The quantification of wound healing assay was calculated as: percent closure (%) = length of cell migration/width of wounds × 100. Percent closure of control group was standardized as 100%. c Migration and invasion activities of 4T1 cells treated with various drug formulations for 24 h. The quantification of d migration and e invasion activities. Cells treated with PBS were used as control. Data are expressed as mean ± SD (n = 3), **p < 0.01
Fig. 7
Fig. 7
Mechanism of CTP/CDDP inhibiting tumor metastasis. a Immuno-fluorescence of EMT markers in 4T1 cells after administration with PBS, free CDDP, TD, TD/CDDP, TP/CDDP or CTP/CDDP for 24 h. b Western blot of indicated proteins expression in hypoxic 4T1 cells after 24 h incubation with different drugs. c The expression of relative proteins was calculated by the signal intensity of protein bands. Cells treated with PBS were used as control. Data are expressed as mean ± SD (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 8
Fig. 8
The in vivo imaging and tissue distribution of tumor-bearing mice. a In vivo images of 4T1 tumor-bearing mice administrated of free DiR or CTP/CDDP/DiR for 3 h, 6 h, 12 h and 24 h via tail vein injection. b Ex vivo images of organs and tumors excised from 4T1 xenograft tumor-bearing nude mice at 12 h after intravenous injection of TD/CDDP/DiR, TP/CDDP/DiR and CTP/CDDP/DiR. c Semiquantitative mean fluorescence intensity results of organs and tumors. Data are expressed as mean ± SD (n = 3)
Fig. 9
Fig. 9
In vivo anti-tumor efficiency of different drug formulations in mice bearing 4T1 xenograft tumors. a Tumor growth curves and b relative body weight during treatment. Mice were injected via tail vein with PBS, free CDDP, TD, TD/CDDP, TP/CDDP or CTP/CDDP on days 6, 8, 10 and 12 after tumor inoculation. c Weight and d image of excised tumors at the end of the experiment (day 24 after tumor inoculation). e Immunohistochemical analysis including HIF-1α, MRP2, E-cadherin and N-cadherin of 4T1 xenograft tumors. Data are expressed as mean ± SD (n = 5), *p < 0.05, ***p < 0.001
Fig. 10
Fig. 10
In vivo anti-metastasis effect. a Digital photographs of excised lung tissues at the end of the experiment (day 21 after tumor inoculation). Mice were injected with 4T1 cells through tail vein and administrated of PBS, free CDDP, TD, TD/CDDP, TP/CDDP or CTP/CDDP on days 2, 4, 6 and 8 after tumor inoculation. b Quantitative analysis of the metastatic lung nodules. c H&E staining of excised lung tissues. Data are expressed as mean ± SD (n = 5), ***p < 0.001
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