Study On The Treatment Of Liver Metastasis Of Intestinal Cancer With Copper Doped Mesoporous Silicon Loaded Disulfiram Combined With Doxycycline

A triple combination of capecitabine-based drugs（capecitabine+oxaliplatin+folinic acid）is the first-line drug for the treatment of colorectal cancer liver metastases.However,it has the disadvantages of high toxic side effects and drug resistance.There is an urgent development of new drugs with high efficiency and low toxicity drug.Because of the antitumor potential of disulfiram combined with copper ions,this thesis discusses the use of bovine serum albumin-encapsulated copper-doped mesoporous silicon as a carrier and loaded DSF as a DDS to investigate its activities against colorectal cancer liver metastases.The following research results are available.1.Preparation of DSF@Cu-MSNs-BSA nanoparticles and its process optimization:Spherical silica was first prepared by the hard template method,and then mesoporous silica was obtained by removing the template.The optimum preparation condition are as follows:CTAB was0.5 g,TEA was 0.06 g,TEOS was 1.5 m L,temperature was 80℃.The final particle size of DSF@Cu-MSNs-BSA nanoparticles,prepared after all steps such as copper doping and attachment of albumin,was 160.8±20.7 nm with a potential of-22.6±4.5 m V.2.The characterization of DSF@Cu-MSNs-BSA nanoparticles:（1）The stretching vibration peaks of Cu-MSNs-NH2 at 3457 cm^-1and two bending vibration peaks of-NH₂at 1653,1541 cm^-1were measured by FIR,which proved that-NH₂was connected to mesoporous silicon.The drug loading and encapsulation rates of DSF@Cu/MSNs-BSA nanoparticles were obtained by HPLC as 12.69%±2.47%and 44.58%±1.32%,respectively.（2）Mesoporous silicon and Cu-doped mesoporous silicon were measured by scanning electron microscopy and transmission electron microscopy,showing a round spherical morphology and a tightly arranged mesoporous structure.（3）The copper content of mesoporous silicon in surface was 4.74%as measured by X-ray electron spectroscopy and energy dispersive spectroscopy.The stability experiments show that DSF@Cu/MSNs-BSA nanoparticles can maintain stability at 24 h.The particle size and potential were in the range of 160-180 nm and 20-30 m V.（4）The specific surface area of Cu-MSNs was 670.28 m²/g and the pore size was 5.77 nm as measured by BET.（5）In vitro experiments showed that the release amount of DSF@Cu/MSNs-BSA nanoparticles was 27.2±4.1%at 48 h and 48.7±5.1%at 72 h,indicating that the nanoparticles could release drugs slowly.The results showed that the DSF@Cu/MSNs-BSA nanoparticles could achieve sustained release of the encapsulated drugs.3.DOX combined with DSF@Cu-MSNs-BSA nanoparticles inhibited CT26.WT cells in vitro:（1）The IC₅₀values for 48 h of DOX,DSF,DSF@Cu/MSNs-BSA,DOX combined with DSF@Cu/MSNs-BSA and 5-fluorouracil were 37.98,30.82,13.05,6.82/17.14（DSF@Cu/MSNs-BSA/DOX）and 25.92μM,respectively,and the IC₅₀values for 72 h were 18.88,8.57,6.02,2.38/5.95（DSF@Cu/MSNs-BSA/DOX）,8.52μM,respectively.The IC₅₀values of DOX,DSF@Cu/MSNs-BSA,DOX combined with DSF@Cu/MSNs-BSA were 45.55,18.28,7.34/18.34μM for 48 h after adding NAC（DSF@Cu/MSNs-BSA/DOX）.The IC₅₀values at 72 h were 21.66,8.18,4.45/11.13μM（DSF@Cu/MSNs-BSA/DOX）,which suggested that DOX combined with DSF@Cu/MSNs-BSA had strong inhibition on CT26.WT cells,and the inhibition was related to the production of reactive oxygen species.（2）The uptake of Cu-MSNs,DSF-Cu-MSNs and DSF@Cu/MSNs-BSA nanoparticles by CT26.WT cells was determined by HCI or flow cytometry.The fluorescence intensity was 1200±102,1454±155 and 5009±214（high-content analysis）,50443.1±1022.1,37295.2±898.2,29082.2±679.3（flow cytometry）,respectively（n=3,P<0.01）,it was concluded that BSA could increase drug intake by CT26.WT cells.The reactive oxygen species produced of CT26.WT cells under the influence of DOX,DSF@Cu/MSNs-BSA nanoparticles and DOX combined with DSF@Cu/MSNs-BSA nanoparticles were determined by high content imaging,and the fluorescence intensity was 1306±182,3090±272,3927±387,respectively.These results indicated that inhibition of CT26.WT cells by the DSF@Cu/MSNs-BSA was related to reactive oxygen species.Flow cytometry showed that DOX and DSF@Cu/MSNs-BSA could decrease and lose mitochondrial membrane potential,and the proportion of abnormal cells was 61.84%and 53.77%,respectively.（3）Pubchem and Swiss Target Prediction were used to screen the targets of active components of DOX and DSF.The results showed that the active component DDC of disulfide can bind to the carbonic anhydrase family,and DOX may bind to MCL1 and PIK3CA.（4）Pubchem,PDB protein database and Autoduck Vina were used for molecular docking.The results showed that the binding energy of DDC to CA9 and CA12 was less than 0.The binding energy of DOX for docking with MCL1 and PIK3CA was all less than 0,indicating that active ingredients of drugs can bind to screened targets.4.In vitro activity of DOX combined with DSF@Cu-MSNs-BSA nanoparticles in inhibiting liver metastasis of colorectal cancer:（1）The body weight of mice in DOX combined with DSF@Cu-MSNs-BSA nanoparticle group began to increase on the 5th day,while that of mice in DSF@Cu-MSNs-BSA nanoparticle group did not increase until the 8th day,suggesting that DOX combined with DSF@Cu-MSNs-BSA nanoparticle can reduce toxicity.（2）The mean survival of negative control group,DOX group,DSF@Cu/MSNs-BSA nanoparticle group,DOX combined DSF@Cu/MSNs-BSA nanoparticle group and capecitabine group were（18.33±1.37 d）,（22.50±1.87 d）,（24.17±1.17 d）,（27.67±1.75 d）,（28.00±1.78 d）,respectively.The results showed that DOX combined with DSF@Cu/MSNs-BSA nanoparticles group could prolong the survival compared with single administration（n=6,P<0.01）.（3）The tumor inhibition rates in DOX group,DSF@Cu/MSNs-BSA nanoparticle group,DOX combined DSF@Cu/MSNs-BSA nanoparticle group and capecitabine group were 15.02%,21.38%,39.30%and 36.99%,respectively.The inhibitory rate of DOX combined with DSF@Cu/MSNs-BSA nanoparticles group was significantly different from that of single administration group（DOX group,DSF@Cu/MSNs-BSA nanoparticle group）（n=6,P<0.01）,q=1.18（King’s formula）,which proves that the two medicine have synergistic effect.（4）Liver samples of in each group showed that DOX combined with DSF@Cu/MSNs-BSA nanoparticles had the fewest tumor nodules,indicating that DOX combined with DSF@Cu/MSNs-BSA nanoparticles had a strong anti-liver metastasis effect on colorectal cancer（n=6,P<0.01）.Pathological sections showed that DOX combined with DSF@Cu/MSNs-BSA nanoparticles could reduce the toxicity of nanoparticles and enhance their inhibitory effect on tumor cells.In summary,DOX combined with DSF@Cu-MSNs-BSA nanoparticles have the potential to become second-line drugs against liver metastasis of colorectal cancer.