Controlled gene and drug release from a liposomal delivery platform triggered by X-ray radiation

Abstract

Liposomes have been well established as an effective drug delivery system, due to simplicity of their preparation and unique characteristics. However conventional liposomes are unsuitable for the on-demand content release, which limits their therapeutic utility. Here we report X-ray-triggerable liposomes incorporating gold nanoparticles and photosensitizer verteporfin. The 6 MeV X-ray radiation induces verteporfin to produce singlet oxygen, which destabilises the liposomal membrane and causes the release of cargos from the liposomal cavity. This triggering strategy is demonstrated by the efficiency of gene silencing in vitro and increased effectiveness of chemotherapy in vivo. Our work indicates the feasibility of a combinatorial treatment and possible synergistic effects in the course of standard radiotherapy combined with chemotherapy delivered via X-ray-triggered liposomes. Importantly, our X-ray-mediated liposome release strategy offers prospects for deep tissue photodynamic therapy, by removing its depth limitation.

Fig. 1

The schematic illustration of gene silencing and cancer cell-killing by X-ray-triggered liposomes. This liposomal delivery platform incorporates verteporfin (VP) and gold nanoparticles. Two types of cargos, antisense oligonucleotide and Doxorubicin, are respectively entrapped inside a liposomal middle cavity for demonstration of in vitro gene release and in vivo drug delivery

Fig. 2

Singlet oxygen generation and calcein release from liposomes under light and X-ray triggering. a, b Percentage increase of SOSG fluorescence intensities from different liposome samples under (a) 360 nm irradiation at different time points and (b) X-ray radiation with different doses. c, d Calcein release profiles from liposomes under (c) 360 nm irradiation and (d) X-ray radiation. Error bars show standard deviation from four measurements

Fig. 3

Cellular uptake activity of liposomes in rat PC12 cells. a–c Representative confocal laser scanning microscopy images of PC12 cells incubated with liposome nanoparticles (25 µM) for 1, 4 and 10 h, respectively. Scale bar is 20 µm

Fig. 4

Cellular uptake of folate-conjugated liposomes in HCT 116 cells and CCD 841 CoN cells. a, b Representative confocal laser scanning microscopy images of incubated (a) HCT 116 cells and (b) CCD 841 CoN cells with folate-conjugate liposomes (25 µM) for 1 h. Scale bar is 75 µm

Fig. 5

In vitro gene silencing by X-ray triggered liposomes loaded with antisense oligonucleotide. a, b Representative confocal images of indirect immunofluorescence staining of PAC1R at different time points after cells were treated with (a) X-ray-triggered liposomes and (b) liposomes alone. The concentration of liposomes incubated with cells was 25 µM. Scale bar was 75 µm. Boxplots in c, d show quantitative assessment of PAC1R gene silencing induced by antisense oligonucleotide released from liposomes at different time points (c) with and (d) without X-ray radiation. Decreased PAC1R fluorescence intensity was expressed as percentage of the control. The box is bounded by the first and third quartile with a horizontal line at the median and whiskers extend to 1.5 times the interquartile range. The mean value was analysed using the t test (n = 5). *** P < 0.001, compared with the control group

Fig. 6

Antitumour activity of X-ray triggered LipoDox in a xenograft model of colorectal cancer. a,b Changes of (a) tumours’ volume and (b) mouse body weight after various treatments as indicated. A black arrow indicates the time of treatment administration. Error bars show standard deviation from four experiments. The mean tumour volumes were analysed using the t test (n = 4). * P < 0.05, ** P < 0.01, *** P < 0.001. c The structural components of treated tumour (H&E staining). Viable tumour tissues (1) were composed of uniform cells with basophilic (blue) cytoplasm and large roundish hyperchromatic nuclei. The areas of cellular paranecrosis and necrosis (2) were recognised by disorganised groups of tumour cells with eosinophilic (pink) cytoplasm, with and without nuclei, respectively. Arrows indicate congested blood vessels. Note the spatial association between the viable tumour tissue and blood vessels. Scale bar is 50 µm. d Boxplot shows morphometric analysis of the effect of the experimental treatment regimens on the structural composition of the xenograft tumours. The relative areas of the viable and non-viable (paranecrotic and necrotic) tumour tissues were measured using ImageJ open source software. The box is bounded by the first and third quartile with a horizontal line at the median and whiskers extend to 1.5 times the interquartile range. The mean tumour necrosis percentage was analysed using the t test (n = 5). * P < 0.05, ** P < 0.01, *** P < 0.001, compared with X-ray-triggered LipoDox-treated group