Study On Anti-tumor Activity Of Cell-penetrating Peptides Linked SN38 Prodrug And Its SiRNA Co-delivery System

Malignant tumor is one of the deadly disease in the world,which seriously threats human health.Surgery and chemotherapy are the main treatment of malignant tumor clinical practice.However,chemotherapeutic drugs usually have the disadvantages of poor solubility,large toxicity and no targeting,which limits their wide clinical application.In this paper,self-assembled cell-penetrating peptides(CPP)linked SN38prodrugs and its co-delivery system was prepared to deliver insoluble chemotherapy drugs.7-Ethyl-10-hydroxycamptothecin(SN38)is a kind of traditional antitumor drug,has a broad spectrum of anti-tumor activity.However,its physicochemical property,pharmacological characteristics and toxicity limition make SN38 difficult to apply in clinical treatment of anti-tumor.In this paper,SN38 was used as the model drug,PEG and CPP were used to synthesize prodrugs to improve solubility and cellular uptake of SN38.These prodrugs can be self-assembled to form micelles.The charge shielding effect of polyethylene glycol(PEG)on CPP made SN38 prodrugs could be applied in vivo anti-tumor research.CPP-PEG-SN38 micelles showed significant advantages in cellular uptake,in vitro and vivo antitumor activity and tissue distribution compared with positive control of irinotecan(CPT-11).And optimized to obtain the optimal CPP-PEG-SN38(TAT-PEG-SN38).But CPP-PEG-SN38 did not improve the histological toxicity than CPT-11.Survivin siRNA can improve the sensitivity of tumor cells to chemotherapy drugs.In this paper,co-delivery system of TAT-PEG-SN38 and survivin siRNA transferrin targeting liposomes(Tf-L-SN38/P/siRNA)was prepared to improve SN38 cellular uptake,anti-tumor activity and reduce the histological toxicity.Survivin siRNA and protamine formed the core of liposomes by electrostatic adsorption;Phospholipid and TAT-PEG-SN38 composed the middle layer skeleton of delivery system;The outer of Tf provides a targeting capability for liposomes.Tf-L-SN38/P/siRNA increased the anti-tumor activity of survivin siRNA and TAT-PEG-SN38,and significantly reduced the histological toxicity compared with CPT-11.The research content of this paper mainly involves the following parts:1.Synthesis of CPP-PEG-SN38In this paper,a series of CPP-PEG-SN38 were synthesized using PEG as linker to connect CPP and SN38 by three steps:(1)Five kinds of CPPs were synthesized by standard Fmoc-chemistry,C terminal of CPPs were modified by cysteine,these CPPs were R8,P28,PFV,SAP(E)and TAT,respectively.(2)C₁₀ hydroxy of SN38 was reacted with triphosgene by acyl chlorination.Next,OPSS-PEG₂₀₀₀-OH conjugated with SN38 through ester bond.(3)Cysteine modified CPP was added to the reaction product to form disulfide bond,CPP-PEG-SN38 were synthesized.CPP-PEG-SN38were self-assembled to form micelles with a particle size of 124 nm to 249 nm,with relatively low potential.2.Anti-tumor activity evaluation of CPP-PEG-SN38 in vitroCellular uptake of CPP-PEG-SN38 micelles were qualitatively and quantitatively analysed by confocal and flow cytometry.CPP-PEG-SN38 could be integrallty absorbed by cells,cellular uptake of CPP-PEG-SN38 relys on the hydrophobic and positive charge of CPP.TAT-PEG-SN38 had the highest cellular uptake efficiency and had the nuclei targeting property.The uptake mechanism of different CPP-PEG-SN38 were explored by cell uptake inhibitor.It was found that CPP-PEG-SN38 was mainly uptaken into cells via clathrin-mediated endocytosis pathway.In vitro antitumor activity of CPP-PEG-SN38 is closely related to the property of CPP,cytotoxicity of CPP-PEG-SN38 had similar tendency to cellular uptake.In addition,cytotoxicity of CPP-PEG-SN38 were time and concentration dependent.In the A549 and MCF-7 cells,the cytotoxicity of TAT-PEG-SN38 were 4.3 and 3.6times than SN38,respectively.3.Anti-tumor activity and toxicity of CPP-PEG-SN38 in vivoThe blood compatibility of CPP-PEG-SN38 were analyzed by in vitro hemolysis experiment.CPP-PEG-SN38 did not cause hemolysis or coagulation,they could be administered by intravenous administration.A549 cell xenograft tumor-bearing nude mice model was established to evaluate the anti-tumor activity of CPP-PEG-SN38 in vivo.Series of CPP-PEG-SN38 exhibited significant anti-tumor activity,TAT-PEG-SN38 showed the most powerful tumor growth inhibition effect among them.TAT-PEG-SN38 inhibited 73.6%and 61.4%of tumor growth compared with the control and the CPT-11,respectively.In vitro and in vivo anti-tumor experiments proved that TAT-PEG-SN38 had the best anti-tumor activity.The toxicity of CPP-PEG-SN38 was evaluated by changes of weight and pathological section.The toxicity of TAT-PEG-SN38 was similar to CPT-11,which did not significantly reduce the weight of nude mice.However,the pathological section of tissues showed that the liver and small intestine of the nude mice were damaged,the damage extent of TAT-PEG-SN38 was similar to CPT-11.4.Pharmacokinetics and tissue distribution of TAT-PEG-SN38Analysis method of TAT-PEG-SN38,SN38 and CPT-11 in plasma and tissue were builded up to measuer the content of prodrugs and SN38 after TAT-PEG-SN38 or CPT-11 administration.Plasma drug concentration of CPT-11 was significantly higher than TAT-PEG-SN38,TAT-PEG-SN38 did not show anti-tumor advantage than CPT-11.A549 cell xenograft tumor-bearing nude mice model was established to analyze the tissue distribution of TAT-PEG-SN38 in nude mice.The AUC_last of TAT-PEG-SN38in the tumor tissues was 37.5 times than CPT-11,free SN38 released from TAT-PEG-SN38 in the tumor tissues was 8.7 times than CPT-11.The effect of TAT on the tumor accumulation of TAT-PEG-SN38 was further analyzed by IVIS,the results showed that TAT could promote the accumulation of SN38 in tumor tissues and perform better anti-tumor activity.5.Evaluation of anti-tumor activity of Tf-L-SN38/P/siRNASN38 is a topoisomerase I(TOP?)inhibitor with broad spectrum antitumor activity.However,SN38 has the disadvantages of poor solubility and strong side effects.Although TAT-PEG-SN38 could improve the anti-tumor activity,it did not significantly improve the tissue toxicity compared with CPT-11.Survivin is highly expressed in tumor cells,and survivin protein has the effect of inhibiting apoptosis and promoting angiogenesis.Survivin siRNA can improve the sensitivity of tumor cells to SN38 and enhance the anti-tumor activity of SN38.In this paper,we established the targeted liposome system to co-deliver survivin siRNA and TAT-PEG-SN38 for improving the anti-tumor activity and reducing the tissue toxicity.The particle size of Tf-L-SN38/P/siRNA was 148 nm,and?-potential was+7.8 mV.When the protamine and survivin siRNA form the core of liposomes by electrostatic adsorption,liposomes have better binding efficiency of siRNA.Tf-L-SN38/P/siRNA could synchronously induced survivin siRNA and the TAT-PEG-SN38 into cells,Tf and TAT-PEG-SN38 significantly enhanced the uptake of Tf-L-SN38/P/siRNA.Results showed that Tf-L-SN38/P/siRNA mainly entered cells by transferrin receptor and clathrin-mediated endocytosis pathway.In MTT assay,survivin siRNA liposomes had no significant cytotoxicity to HeLa cells.In HeLa cells,the cytotoxicity of the Tf modified liposomes was 1.25 times than SN38 when only loading TAT-PEG-SN38.However,when survivin siRNA was combined with TAT-PEG-SN38,cytotoxicity increased to 14.56 times than SN38.The silencing effect of Tf-L-SN38/P/siRNA on survivin gene was evaluated by Western blot.Tf and TAT-PEG-SN38 could increase the delivery efficiency of survivin siRNA.Tf-L-SN38/P/siRNA could significantly inhibit the expression of survivin protein in HeLa cells compared with survivin siRNA liposomes.HeLa cell xenograft tumor-bearing nude mice model was established to evaluate the anti-tumor effect of Tf-L-SN38/P/siRNA.L-siRNA had a limited anti-tumor effect.Tf-L-SN38/P/siRNA had the most effective anti-tumor activity,the tumor growth inhibition rate was 76.8%compared with the control group;Tf-L-SN38/P/siRNA combined with siRNA inhibited 33.8%of the tumor growth than Tf-L-SN38;Tf-L-SN38/P/siRNA modified with Tf inhibited 39.1%of the tumor growth than L-SN38/P/siRNA;In addition,Tf-L-SN38/P/siRNA did not significantly damage the liver,kidneys and small intestine.IVIS was used to further evaluate the target efficiency of Tf-L-SN38/P/siRNA,L-SN38/P/siRNA was used as control.Tf-modified liposomes have higher targeting in the tumor site.In conclusion,this paper established the self-assembled cell-penetrating peptides linked SN38 prodrugs and its co-delivery system,evaluated its antitumor activity to obtain high anti-tumor activity,low toxicity SN38 delivery system.