Polysialic acid-functionalized liposomes for efficient honokiol delivery to inhibit breast cancer growth and metastasis

Abstract

To improve the anti-metastasis effects of honokiol (HNK) on breast cancer, we designed cationic liposomes (Lip) in which HNK was encapsulated into Lip, and its surface was modified with negatively charged polysialic acid (PSA-Lip-HNK) for efficient treatment of breast cancer. PSA-Lip-HNK possessed a homogeneous spherical shape and high encapsulation efficiency. In vitro 4T1 cell experiments indicated that PSA-Lip-HNK increased cellular uptake and cytotoxicity via the endocytosis pathway mediated by PSA and selectin receptors. Furthermore, the significant antitumor metastasis impact of PSA-Lip-HNK was confirmed by wound healing and cell migration and invasion. Enhanced in vivo tumor accumulation of the PSA-Lip-HNK was observed in 4T1 tumor-bearing mice by living fluorescence imaging. For in vivo antitumor experiments using 4T1 tumor-bearing mice, PSA-Lip-HNK exhibited a higher tumor growth and metastasis inhibition compared with unmodified liposomes. Therefore, we believe that PSA-Lip-HNK well combined biocompatible PSA nano-delivery and chemotherapy, providing a promising drug delivery approach for metastatic breast cancer therapy.

Keywords: Breast cancer; honokiol; liposomes; metastasis; polysialic acid.

Scheme 1.

Scheme of in vivo tumor targeting and antitumor effect verification of HNK delivered by PSA-modified liposomes.

Figure 1.

The characterization of the PSA-Lip-HNK. A: The appearance of PSA-Lip-HNK at different PSA modified density. B and C: The impact of PSA density on particle size and zeta potential of liposomes. D: Typical TEM image of PSA-Lip-HNK. E: In vitro release profiles of Lip-HNK and PSA-Lip-HNK in PBS at 37 °C. Data are presented as mean ± SD (n = 3).

Figure 2.

Intracellular uptake study of Lip-Cou6, PSA + PSA-Lip-Cou6, PSA-Lip-Cou6, and free Cou6 on 4T1 cells after incubation for 1 h at 37 °C. (A) Flow cytometric quantitative determination of Cou6 uptake. B: Quantitative analysis of Cou6 uptake based on flow cytometric plots. Each bar represents the mean ± SD (n = 3), **p < .01. C: Laser confocal scanning microscopy images of 4T1 cells. Green and blue indicate the fluorescence of Cou6 and Hoechst 33258, respectively.

Figure 3.

In vitro cytotoxicity against 4T1 cells of various HNK formulations after 48-h incubation by SRB assay. Each bar represents mean ± SD (n = 5). ** indicate statistically different p < .01.

Figure 4.

In vitro inhibitory effects of PSA-Lip-HNK on cell migration and invasion of 4T1 breast cancer cells. A: Typical images of wound healing, migration and invasion assessments of free HNK, Lip-HNK and PSA-Lip-HNK (5.0 µg/mL) on 4T1 cells. B–D: Quantified inhibitory effect of free HNK, Lip-HNK, and PSA-Lip-HNK (5.0 µg/mL) on 4T1 cells in comparison with negative control. Data are shown as mean ± SD (n = 3), **p < .01.

Figure 5.

Effect of various HNK formulations (HNK = 10 μg/mL) on the growth of 4T1 tumor spheroids. A: Typical images of tumor spheroids under inverted microscope. B: The quantified size of tumor spheroids in different time points. Data are shown as mean ± SD (n = 3), **p < .01.

Figure 6.

Biodistribution of the Lip-DiR and PSA-Lip-DiR in 4T1 tumor-bearing mice. A: In vivo real-time imaging of 4T1 tumor-bearing mice over 24 h after tail vein injection. B: Ex vivo fluorescence images of isolated tumors and organs. C: Semi-quantitative analysis of fluorescence intensity in the tumor site at the indicated time points. D: Semi-quantitative analysis of the ex vivo fluorescent intensity at the tumors and organs. Data are shown as mean ± SD (n = 3), *p < .05. The tumors were indicated by red ellipses.

Figure 7.

Antitumor efficacy of various HNK formulations (15 mg/kg) in 4T1 tumor-bearing mice models. A: Tumor volume change curves. B: The excised tumor weight. C: Body weight change curves. D: Images of excised tumors. E: Image of excised lungs (The pulmonary metastatic nodules are denoted by blue arrows). F: TUNEL fluorescent staining of apoptotic cells in excised tumor tissue. The various HNK formulations were injected every two days for the total of four times, data are given as mean ± SD (n = 6), *p < .05, **p < .01.

Figure 8.

H&E staining photographs of tumors and main organs from different HNK treatment groups. The necrosis areas in tumor sections and metastasis lesions in liver and lung sections were circled by blue lines.