SREBP1 siRNA enhance the docetaxel effect based on a bone-cancer dual-targeting biomimetic nanosystem against bone metastatic castration-resistant prostate cancer

Abstract

Until recently, there have been limited options for patients with bone metastatic castration-resistant prostate cancer (BmCRPC) following the failure of or development of resistance to docetaxel (DTX), which is one of the frontline treatments. Sterol regulatory element-binding protein 1 (SREBP1) is reported to regulate abnormal lipid metabolism and to promote the progression and metastasis of prostate cancer (PCa). The siRNA interferes SREBP1 may provide an efficient treatment when combined with DTX. Methods: In this study, lipoic acid (LA) and cross-linked peptide-lipoic acid micelles were cross-linked (LC) for DTX and siSREBP1 delivery (LC/D/siR). Then, cell membrane of PCa cells (Pm) and bone marrow mesenchymal stem cells (Bm) were fused for cloaking LC/D/siR (PB@LC/D/siR). Finally, the synthesized PB@LC/D/siR was evaluated in vitro and in vivo. Results: PB@LC/D/siR is internalized in PCa cells by a mechanism of lysosome escape. Tumor targeting and bone homing studies are evaluated using bone metastatic CRPC (BmCRPC) models, both in vitro and in vivo. Moreover, the enhanced anti-proliferation, anti-migration and anti-invasion capacities of DTX- and siSREBP1- loaded PB@LC (PB@LC/D/siR) were observed in vitro. Furthermore, PB@LC/D/siR was able to suppress the growth of the tumor effectively with deep tumor penetration, high safety and good protection of the bone at the tumor site. Additionally, the mRNA levels and protein levels of SREBP1 and SCD1 were able to be significantly downregulated by PB@LC/D/siR. Conclusion: This study presented a bone-cancer dual-targeting biomimetic nanodelivery system for bone metastatic CRPC.

Keywords: SREBP1 siRNA; bone marrow mesenchymal stem cells; bone metastatic prostate cancer; docetaxel; fused cell membrane.

Scheme 1

The designation and mechanism of PB@LC/D/siR. A) The preparation of co-loading nanoparticles LC/D/siR. B) The fusion and coating of PBm. C) The schematic illustration of PB@LC/D/siR targeting the microenvironment of BmCRPC based on the fundamental of bone homing and homotypic targeting ability of PBm. D) The effect mechanism of PB@LC/D/siR.

Figure 1

The characterization of gene compression and gene transfection abilities of LC. A) The agarose gel electrophoresis results of different N/P ratios of LC/pEGFP. B-D) The transfection ability of PEI or LC gene carriers in HeK-293T cells at different N/P ratios. B) The statistical results of green fluorescent protein (GFP) positive cells of LC and PEI (n=3, mean ± SD). C-D) The flow cytometry results of C) PEI and D) LC.

Figure 2

The characterization of PBm and PB@LC. A-B) The fluorescence spectrophotometer results of different membrane protein ratios of 5:1, 4:1, 3:1, 2:1, 1:1, and 0:1 of Bm to Pm. C) The representative TEM images of LC and PB@LC (scale bars = 50 nm). D-E) The representative CLSM images of D) Pm, Bm, Mixture of P Bm, and Fused PBm (scale bars = 20 μm) and E) PB@LC (scale bars = 20 μm). F) The SDS-PAGE gel electrophoresis results of Pm, Bm, PBm, and PB@LC. G) The immunogold TEM images of -11 (green arrows, small gold = 10 nm) and STRO-1 (blue arrows, large gold = 20 nm) probed LC, P@LC, B@LC and PB@LC, followed by negative staining with phosphotungstic acid (scale bars = 50 nm).

Figure 3

The targeting abilities of PB@LC. CLSM images of A) intracellular colocalization of each group (scale bars = 20 μm). B) The CLSM results of lysosome escape of PB@LC (scale bars = 20 μm). C) Statistic results of flow cytometry, LC was coated with different cell membranes Rm (RWPE-1), Km (KETR-3), Um (U251), Pm, and PBm (n=3, mean ± SD), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA. D-E) The statistic results of bone targeting ability evaluation, PC-3 cells were co-incubated with P@LC and PB@LC in normal media or in conditioned media of D) MG-CM or E) MC-CM (n=3, mean ± SD), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA.

Figure 4

In vitro antiproliferation ability of PB@LC/D/siR. A-B) The cytotoxicity results of each group in A)PC-3 cells and B) C4-2B cells, the concentration gradients of DTX or siSREBP1 were 1 to 32 nM (n=3, mean ± SD). *p < 0.05, one-way ANOVA. C) The representative results of apoptosis of co-incubating PC-3 cells with each group for 24 h, DTX: 20 nM, siSREBP1: 5 nM, PBS was as control.

Figure 5

The anti-migration, anti-invasion and anti-lipogenesis assays. A) and C) PC-3 cells were incubated with each group, and 1, 2, 3, 4, 5, 6, 7, 8, 9 represented PBS (as Control), DTX, siSREBP1, LC/D, LC/siR, PB@LC/D, PB@LC/siR, LC/D/siR, and PB@LC/D/siR for 24 h or 48 h, DTX: 20 nM, siSREBP1: 5 nM, PBS was as control (scale bars = 200 μm). B) and D) The statistical analysis of cells in nine fields of view, respectively (n=9, mean ± SD). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA. E) The RTFQ PCR results of the expression of SREBP1 and SCD1 in PC-3 cells (n = 3, mean ± SD). *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA. F) The Western blotting results of protein levels of SREBP1 and SCD1 in each group.

Figure 6

In vivo study of PB@LC. A) The representative small animal living images of each group of the BmCRPC-bearing mice at 0-24 h, post-injection (yellow circle: tumor area). B) The qualified distribution in major organs of each group (n=3, mean ± SD). *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA. C) The representative photoacoustic images of each group, saline as control. D) The CLSM images of fluorescence distribution in the tumor mass of each group, siFAM was as model drug (scale bars = 500 μm). E) The in vivo study protocol of PB@LC/D/siR, the concentration of siRNA or DTX was 1 mg/kg or 0.25 mg/kg, respectively. F) The tumor volume curves or G) body weight growth curves of the 9 groups (n = 5, mean ± SD). *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA. H) The BMD values of the BmCRPC-bearing tibias of each group, tumor-free normal tibias were as control (n = 5, mean ± SD). ***p < 0.001, ****p < 0.0001, n.s. no significance, one-way ANOVA. I) The micro-CT images of each group (n = 5, red arrows: bone damaged sites).

Figure 7

The safety and mechanism of PB@LC/D/siR. A) The HE images (200 ×) of the heart, liver, spleen, lung, kidney, tumor, bone (right hind limb tibia) of each group. B) The RTFQ PCR results of the transcription of SREBP1 and SCD1 in each group (n = 3, mean ± SD). *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA. C- D) The expression of SREBP1 and SCD1 in the tumor site of each group, C) Western blotting results. D) IHC images (400 ×).