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

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.

Preparation and characterization of LC and LC/D/siR. LA-NP, LA-NP/DTX, LACL micelles and LACL/siRNA micelles were prepared as previous report. 1,2 Briefly, 100 mg and 17 mg cysteine (cys) were immersed in 1 mL methanol and stirred for 8 h at room temperature (RT) to get cross-linked lipoic acid (LA). 100 mg cross-linked LA and 5 mg DTX were immersed in 1 mL chloroform and 4 mL sodium cholate (1%), sonicated at 400 W for 15 s, replicated 3 times. The obtained emulsion were added to 10 mL double-distilled (DD) water and stirred for overnight. LA-NP/DTX were purified by ultrafiltration at 3500 rpm for 10 min with 100 k MWD ultrafiltration tube. Meanwhile, a N/P ratio of 50:1 of LACL to siSREBP1 was co-incubated for 30 min at RT and purified by dialysis (MW3500). LA-NP/DTX and LACL/siSREBP1 micelles were co-incubated at RT for 30 min to obtain LC/D/siR, purified by ultrafiltration (100 k MWD).
Different weight ratios of LA-NP to LACL micelles were tested to confirm the optimal ratio. LA-NP, LA micelles and LC were prepared using the same method. Moreover, the size, PDI and zeta potentials were detected by DLS, and the stability of LC was evaluated in 30 days at 4℃. The morphology of nanoparticles was observed by TEM (FEI TECNAI G 2 S-TWIN, USA). Further, the gene compression capacity of LC was estimated by agarose gel electrophoresis (DYY-6C, China), using pEGFP as a model drug.

Gene transfection assay.
To evaluate the gene transfection efficiency, 2 × 10 5 HEK-293T cells were seeded in each well of 12-well plates for overnight. HEK-293 cells were treated with different N/P ratios of LC/pEGFP for 24 h, followed by flow cytometry detection (FACS Calibur, BD Biosciences, USA). Traditional polycationic material PEI was as a control for gene delivery vector.
Vehicle and material safety test. CCK-8 kit was used to investigate the cytotoxicity of PB@LC. PCa cells were seeded in 96-well plates at a concentration of 10 4 cells/well. PCa cells were co-incubated with a concentration gradients of vehicles of 0-400 μg/mL for 24 h. Microplate reader (Thermo, USA) was used for detection at 450 nm.
Isolation and purity detection of rat BMSC cells. 3 For extraction of BMSC cells, 4-week old SD male rats were sacrificed and immersed in 75% alcohol for 10 s. The tibias and femurs were dissected, and washed with HBSS. Bones were cut into small pieces and bone marrow was blown out with 1-mL syringes until the bones became whitened. Then, the suspensions were gathered and centrifuged  China) for 5 min. Cellular content was removed by centrifugation at 2000 g for 10 min at 4℃ (Eppendorf, Centrifuge 5804R, Germany). The supernatants were collected and centrifuged at 10,000 g for 30 min at 4℃. Cell membrane precipitation was washed and resuspended with TM-Buffer containing 0.25 M sucrose (SinoPharm, China) for centrifugation at 10,000 g for 30 min at 4℃. The zeta potential was evaluated by DLS system, and the total membrane proteins were detected by BCA method. Cell membranes were stored at -80℃ for further use.

Preparation of PCa-BMSC fused membrane (PBm). Pm and Bm were
obtained through methods of Zhang et al. 6 To obtain the optimal ratio of the Pm to Bm, we prepared a pair of Förster resonance energy transfer (FRET) dyes C6-NBD /DOPE-RhB. Different membrane protein weight ratios (Pm : Bm) of 0:1, 1:1, 2:1, 3:1, 4:1, and 5:1 were tested by fluorescence spectrophotometry (Hitachi, F-7000, Japan). Furthermore, the colocalization of Pm and Bm was evaluated by CLSM. One hundred microliters of Pm or Bm was dyed with 5 μL of 10 μg/mL green fluorescent dye DiO or red fluorescent dye DiR. The two stained cell membranes were mixed to obtain the mixed membrane or fused (ultrasound for 5 min) to obtain the fused membrane. These cell membrane materials were analyzed by CLSM.
Preparation and characterization of PB@LC. One milligram (membrane protein) of PBm was added to 1mL LC (1 mg/mL) to obtain PB@LC by ultrasound for 5 min. Then, PB@LC were resuspended in PBS at a membrane protein concentration of 0.2 mg/mL. To assess the best weight ratio of LC to PBm, different ratios (1:0, 1:10, 1:5, 1:2, 1:1 2:1, 4:1, 5:1, 6:1, 10:1, 0:1) were examined by DLS system for optimal size and zeta potential. PB@LC was purified as reported. 1 The coverage rate of PB@LC were evaluated through BCA method. Moreover, the morphology of PB@LC was detected through TEM imaging. To assay the fusion by CLSM, LC was coated with DiO/DiR stained PBm, while the nuclei were stained with DAPI. For more accurate measurement, cell membrane-specific markers of BMSCs (STRO-1) and PC-3 (CDH11) were selected for immunogold TEM study. 6  Cells were co-incubated with each group for 1-4 h, simultaneously the medium of each group was changed with serum-free medium. The fluorescence intensity of each group was detected by flow cytometry.
Lysosome escape assay. 7 Cells were seeded in 24-well plates at a concentration of 10 5 cells/ well for overnight. PB@LC was loaded with Coumarin-6 (20 ng/mL) and lysosome was stained with Lysotracker Red (50 ng/mL) for 30 min.
8 CLSM images of 1 and 4 h were obtained to reveal the transport mechanism of PB@LC.
Homologous targeting assay. 8   In vivo biosafety evaluation. The heart, liver, spleen, lung, kidney, tumor and right hind limb tibia of each mouse were harvested for H&E staining to investigate the biosafety of PB@LC/D/siR.

Statistics analysis.
All numerical data were analyzed with Prism 7.0 software, mean ± SD, and at least tropical samples. The comparison of each group was performed by one-way ANOVA, while a P value < 0.05 was considered to be statistically significant.