Cell Penetrating Peptide Modified SN38 For Triple Negative Breast Cancer Antimetastasis Study

Triple negative breast cancer(TNBC)is the most aggressive and the worst prognosis subtype of breast cancer and is mainly characterized by a high probability of distant metastasis,with 20% of TNBC patients dying of metastatic disease progression within five years.Studies have shown that SN38 can significantly kill TNBC cells,and a previous study found that SN38 also has a certain inhibitory effect on the metastasis of TNBC.But the solubility of SN38 is extremely poor,and there are barriers for efficient delivery and penetration into tumor tissue in vivo,which cannot be directly applied in the clinic.Amphiphilic micelles modified by cell penetrating peptides can effectively improve the above problems,but need its certain selective accumulation ability to tumor cells.In this study,we synthesized a PEG-SN38 prodrug by conjugating hydrophilic polyethylene glycol(PEG)to the hydrophobic end of SN38 and selected the cell penetrating peptide TAT to modify PEG-SN38,which self-assembled in aqueous solution to form micelles.TAT-PEG-SN38 micelles greatly improved the water solubility of SN38,and the introduction of TAT also enhanced the selective uptake ability of micelles into tumor cells and the penetration ability into tumor tissue,effectively inhibiting the tumor activity and distal metastasis of TNBC.As novel,efficient and safe anti TNBC agents,they have potential clinical applications and deserve further in-depth study.This study mainly includes the following:1.Synthesis and characterization of TAT-PEG-SN38TAT and SN38 were linked on both ends using PEG as a linker to prepare TATPEG-SN38.The amphiphilic TAT-PEG-SN38 can self-assemble to form micelles in aqueous solution with a particle size of approximately 142 nm and a potential of approximately + 8.44 MV;The morphology of micelles was observed to be uniform near sphere by SEM,and the micelles had good solubility,stability,moderate particle size and potential,which can meet the conditions of drug efficacy evaluation in vivo and in vitro.2.In vitro anti-tumor efficacy of TAT-PEG-SN38TAT-PEG-SN38 exhibited significant cytotoxicity against 4T1 cells,a murine triple negative breast cancer cell line,and BT-549,a human triple negative breast cancer cell line: for the 4T1 cell line,after modification with TAT,PEG-SN38 exhibited a 3.61 fold increase in cytotoxicity;However,for the BT-549 cell line,the cell killing effect of TAT-PEG-SN38 was also significantly higher than that of SN38,CPT-11,and PEGSN38.The enhanced toxicity of TAT-PEG-SN38 on tumor cells was due to the cell penetrating peptide modification improving the cellular uptake ability of PEG-SN38,which was enhanced by 4.2 fold and 1.7 fold compared with PEG-SN38 by 4T1 and BT-549 cells,respectively.The HC-11 cell line,a normal mammary epithelial cell line,was introduced as a reference to analyze the selectivity of TAT modification for tumor cells,and it was found that the uptake of TAT-PEG-SN38 was not significantly enhanced compared with PEG-SN38,possibly due to the difference in cell surface charge,and the internalization of TAT-PEG-SN38 into tumor cells was found to be somewhat selective.To mimic the tumor penetration ability of TAT-PEG-SN38 in a 3D tumorsphere model,TAT-PEG-SN38 penetrated into the interior of tumorspheres after24 h of incubation,PEG-SN38 was only present on the surface of tumorspheres,and TAT-PEG-SN38 clearly inhibited the growth of tumorspheres.The above experiments demonstrated that TAT-PEG-SN38 can be selectively taken up by tumor cells and permeate into the interior of the tumor with superior in vitro anti-tumor activity.3.Evaluation of the in vivo anti-tumor activity of TAT-PEG-SN38An orthotopic tumor bearing model of murine breast cancer was established to evaluate the in vivo anti-tumor efficacy of TAT-PEG-SN38: intravital imaging results showed that 8 h after intravenous injection of micelles,TAT-PEG-SN38 efficiently accumulated at the tumor site,further demonstrating that TAT was able to deliver the drug efficiently to the tumor region for enhanced uptake.Compared with saline,TATPEG-SN38 inhibited tumor growth by 52.42%,and the inhibitory effect was 1.57 fold greater than that of PEG-SN38.Through observation of the metastasis situation in lung tissues,it can be found that the TAT-PEG-SN38 group had a significantly higher metastasis inhibitory effect on TNBC in vivo organs than the rest of the administered groups.Pathological section analysis validated the same results,with the exception of the TAT-PEG-SN38 group,in which tumors showed various degrees of metastasis to the liver and lung.It follows that TAT-PEG-SN38 clearly improves organ metastasis in the 4T1 mouse tumor bearing model.It was found by immunohistochemical analysis that the expression of antigen CD31 was the lowest in the TAT-PEG-SN38 group compared with the other groups,indicating that the TAT-PEG-SN38 group could effectively inhibit tumor neovascularization.TAT modifcation signifcantly improved the in vivo TNBC anti-tumor effcacy of PEG-SN38 and inhibited TNBC distal metastasis.4.Anti metastasis studies of TAT-PEG-SN38First,the metastasis inhibitory ability of TAT-PEG-SN38 on two cell lines,4T1 and BT-549,was examined at the cellular level: as can be found from studies of cell scratch healing and Transwell cell migration,the TAT-PEG-SN38 scratch healing rates were only 16.17% and 22.19%.Respectively,whereas in the Transwell cell migration assay,the cells of the TAT PEG SN38 treated group barely underwent migration.CPT-11 did not significantly inhibit cell migration compared with the control,and TAT-PEGSN38 significantly improved the cell metastasis inhibition ability of CPT-11.Differential protein expression analysis of mouse tumor tissues by proteomics revealed that the expression of 15 proteins was upregulated and 29 proteins downregulated in the TAT-PEG-SN38 administration group compared with the control group,including DSG2 and LDLR,JUP proteins in proteins associated with tumor metastasis,and the expression differences were verified by Western blot experiments,Demonstrated that TAT-PEG-SN38 exerted an effect on the distal metastasis of breast cancer tumors at the protein level.TAT-PEG-SN38 was validated against TNBC metastasis at the cellular level according to the results of proteomic analysis.The up-regulated protein DSG2 and down regulated protein LDLR were selected as gene knockdown and overexpression plasmids,respectively,and transfection with the above two plasmids revealed that after the expression of the two proteins was reversed,the migration inhibition ability and tumorsphere killing ability of PEG-SN38 and TAT-PEG-SN38 on 4T1 cells were significantly attenuated,suggesting that the two proteins DSG2 and LDLR might be critical for SN38’s inhibition of the metastatic ability of TNBC.In summary,this study established a cell penetrating peptide TAT modified SN38 drug delivery strategy that improved the water solubility and cellular uptake of the poorly soluble drug SN38,which was successfully demonstrated to inhibit the growth and distal metastasis of triple negative breast cancer in vitro and in vivo,and validated the key proteins affecting the metastasis of triple negative breast cancer by analyzing the differential proteins in the administered groups,Making it an easily translatable drug delivery platform for the treatment of triple negative breast cancer.