Exosome-mediated SiRNA Delivery To Suppress Postoperative Breast Cancer Metastasis

Objective We developed a biomimetic nanoscale drug delivery platform(CBSA/si S100A4@Exosome)that consist of S100A4 si RNA(si S100A4)and autologous breast cancer cells derived-exosome membrane.CBSA/si S100A4@Exosome nanoparticle was used to target and regulate the lung pre-metastatic niche(PMN),achieving the postoperative lung metastasis of triple-negative breast cancer(TNBC)prevention and therapy.Methods In this study,the optimal cationization degree of cationic bovine serum albumin(CBSA)was obtained by the surface modification of bovine serum albumin(BSA)with ethylamine through an amide linkage.Mouse breast cancer cells(4T1)were inoculated into the mammary fat pad of each female BALB/c mouse to establish the in situ breast cancer model.When the tumor size reached 100 mm~3,autologous tumors were surgically excised.Exosomes were isolated and purified from autologous breast cancer cells using a differential centrifugation protocol.Exosomes membrane was isolated by the combination of freeze-thaw cycle and sucrose gradient centrifugation.CBSA/si S100A4@Exosome was fabricated by coating CBSA/si S100A4with autologous breast cancer cells derived-exosome membranes by incubation and extrusion method.The molecular weight of CBSA was detected by MALDI-TOF/TOF and the Biuret method performed to determine the isoelectric point(p I)of CBSA.The agarose gels electrophoresis was performed to detect the binding ability of CBSA and si S100A4.The expressions of the exosome-specific proteins were examined by western blot analysis.The optimal weight ratio of CBSA/si S100A4 and exosome membranes was assessed by agarose gels electrophoresis.The particle size,zeta potential and morphological of this nanoparticle were characterized.The hydrodynamic size of CBSA/si S100A4@Exosome was monitored in physiological condition for two weeks to test the stability of this nanoparticle over time.To assess the ability of CBSA/si S100A4@Exosome to protect the si S100A4 from degradation,the stability of si S100A4 in fetal bovine serum(FBS)or ribonuclease(RNase)was examined by agarose gels electrophoresis.To confirm the successful exosome membranes encapsulation around CBSA/si S100A4,the sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)analysis was performed.The viability assessment was performed by MTT assay using human umbilical vein endothelial cells(HUVEC).Confocal laser scanning microscope(CLSM)was used to monitor the internalization of CBSA/si S100A4@Exosome in mouse embryonic lung fibroblast.We established an in vitro metastasis model with co-cultured breast cancer cells and mouse embryonic lung fibroblast.Western blot was used to evaluate the gene slicing efficiency at the protein level of CBSA/si S100A4@Exosome under an in vitro simulation of pulmonary metastasis environment.Wound healing assay was performed to determine the effect of CBSA/si S100A4@Exosome on inhibiting metastatic potential of breast cancer under an in vitro simulation of pulmonary metastasis environment.Healthy female BALB/c mice were injected with CBSA/si S100A4@Exosome via the tail vein to evaluate the lung targeting capability of this nanoparticle and the sliced tissues were imaged using a CLSM at the designated time point.To further investigate the lung PMN targeting ability of this nanoparticle,we established the breast cancer postoperative pulmonary metastasis model.Female BALB/c mice with established postoperative lung metastasis were injected with CBSA/si S100A4@Exosome via the tail vein to evaluate the lung PMN targeting by IVIS Lumina II imaging.Female BALB/c mice with established postoperative lung metastasis were randomly divided into 6 groups,each intravenous injection was administered every other day(S100A4 used here at dose of 1 mg/kg,total for 4 doses)through the tail vein.The body weight of each mouse was measured every other day and monitored daily for physical activity.Ten days after the last injection,animals were sacrificed to harvest lungs,the number of macroscopic metastatic nodules in each lung was recorded,the weight of the lungs was recorded and the index of lung weight was calculated to evaluate the therapeutic effects.Histological examinations of the lung slices were performed with hemetoxylin and eosin(H&E)staining for visualization of metastatic foci.Furthermore,to determine the therapeutic effects of gene silencing at the protein level in lung tissues,the expression of S100A4 in post-operative lung metastasis mice were investigated by western blot and immunofluorescence.Results1.The CBSA was achieved through the modification of BSA with moderate ethylamine,which had an average molecular weight(M_w)of67,583.27 Da analyzed by MALDI-TOF/TOF,which revealed that an average of 43 ethylamines were linked to one BSA molecule.The p I of the CBSA was determined as 8.5 by the Biuret method.The above results indicate that the optimal cationization degree of CBSA was successfully obtained.The results of agarose gels electrophoresis assays indicated the optimum combination ratio of CBSA/si S100A4(si S100A4/CBSA=1/30,w/w).Western blot shows that the expression of the exosome markers TSG101 and CD9 were enriched in the isolated breast cancer cells-derived exosomes.Furthermore,our data also shows that GRP94,which is a protein that indicates cellular contamination of exosomes,was not detected in exosomes,confirming the successful isolation and purity of the exosomes.Transmission electron microscope(TEM)data confirmed the round-shaped nanovesicles surrounded by membrane and narrow size distribution of exosomes.We found that the construction of CBSA/si S100A4@Exosome can be formed and optimized at a weight ratio of 30 as revealed by the agarose gels electrophoresis assay(si S100A4/CBSA/Exosome membrane=1/30/30,w/w/w),indicating an excellent encapsulation capability of exosome membrane around the CBSA/si S100A4.The encapsulation efficiency(EE)of the si S100A4 into the exosome membranes was determined at86.70±1.22%.Our data shows that the resulted core-shell CBSA/si S100A4@Exosome exhibited a hydrodynamic diameter of～200 nm and a zeta potential of-28.63±0.33 m V,which possessed a similar charge to that of exosome membrane shell(-29.49±0.24 m V).A spherical core-shell structure of the CBSA/si S100A4@Exosome was observed by TEM.The above results confirmed that the CBSA/si S100A4@Exosome nanoparticles were successfully constructed.The SDS-PAGE image about the protein profile of CBSA/si S100A4@Exosome reveled that the protein composition in the CBSA and exosome membrane was mostly retained in the CBSA/si S100A4@Exosome,which further indicated that the CBSA/si S100A4@Exosome nanoparticles were successfully constructed.CBSA/si S100A4@Exosome nanoparticles were stable in physiological conditions without developing aggregates for at least 2 weeks and no detectable degradation of si S100A4 in CBSA/si S100A4@Exosome was noticed even after 24 h of incubation with RNase and serum,indicating that the nanoscale drug delivery platform showed great stability and is good for the subsequent in vivo applications.MTT assays verified the autologous breast cancer cells derived-exosome membranes as gene vectors with high biocompatibility.2.The data of in vitro cellular uptake assay shows that CBSA/si S100A4@Exosome was extensively internalized in mouse embryonic lung fibroblast with high affinity.The western blotting analysis shows that the endogenous breast cancer-derived exosomes can effectively deliver anti-metastasis si S100A4 into recipient cells and exhibit a perfect gene silencing efficiency under an in vitro simulation of pulmonary metastasis environment.Wound healing assay revealed that CBSA/si S100A4@Exosome significantly decreased the migration capability of breast cancer cells compared to the controlled cells.The above results revealed that the CBSA/si S100A4@Exosome deadened the effect of S100A4 by downregulating the expression of S100A4,and then inhibited the formation of a favorable microenvironment for malignant breast cancer cells to develop metastasis,leading an efficient anti-metastatic efficacy.3.CBSA/si S100A4@Exosome showed their excellent target ability and great lung adhesive capacity in normal mice or postoperative lung metastasis mice.The results about the images of the lung tissue and the index of lung weight all indicated that the number of macroscopic metastatic nodules of the CBSA/si S100A4@Exosome treated group was dramatically decreased in postoperative lung metastasis mice.Besides,from the H&E staining of the lung metastatic lesions,metastatic foci and inflammation could be clearly detected in the negative control group,but were barely visible in the CBSA/si S100A4@Exosome group,indicating a higher anti-metastatic efficacy of CBSA/si S100A4@Exosome.The expression of S100A4 in the lung tissues of postoperative lung metastasis mice were investigated by western blot and confirmed by immunofluorescence.CBSA/si S100A4@Exosome shows the downregulation of S100A4,indicating outstanding gene silencing effects in the lung PMN.The therapy effect of CBSA/si S100A4@Exosome was further demonstrated to downregulate the expression of S100A4 and regulate the lung PMN,inducing significantly suppression of postoperative metastasis in TNBC.Conclusion1.The biomimetic nanoscale drug delivery platform(CBSA/si S100A4@Exosome)based on the autologous breast cancer cells-derived exosomes that consists of a CBSA/si S100A4 core and an exosome membrane shell was successfully engineered.The nanoparticles formed homogeneous sizes and without developing aggregates.Besides,the nanoscale drug delivery platform not only protected si S100A4 from degradation but also had excellent physical stability and biocompatibility.2.Based on the effective lung targeting ability of autologous breast cancer cells-derived exosome membranes,CBSA/si S100A4@Exosome revealed great lung adhesive and preferable target uptake to pre-metastatic niche of lung both in vivo an in vitro,which is a promising strategy for targeting delivery of nucleic acid drug.3.CBSA/si S100A4@Exosome-mediated RNAi was further demonstrated to downregulate the expression of S100A4(metastasis-related protein),regulate the lung PMN and then induced significantly suppression of postoperative metastasis in triple negative breast cancer.The nanoscale drug delivery platform is expected to achieve the individualized prevention and treatment of postoperative pulmonary metastasis of TNBC.