The Construction Of Cisplatin Prodrug And Wortmannin/etoposide Co-encapsulated Nanoparticles And Study On Their Anti-cancer Effect

Objective:At present,the treatment of cancer is still a worldwide medical problem.Among them,ovarian cancer is the highest fatality rate in gynecological cancer diseases.The incidence and mortality of lung cancer account for the first and second position of all malignant tumors in both men and women respectively.Non-small cell lung cancer（NSCLC）,whose long-term survival rate is very low,accounts for 85%of all lung cancers.Cisplatin is a commonly used broad-spectrum anti-tumor drug in clinical practice,which is also the first choice in combination therapy for ovarian cancer and lung cancer.For the treatment of ovarian cancer,the effect of cisplatin in the initial application is remarkable.Yet cured patients is susceptible to recurrence,and often accompany cisplatin resistance.Based on evidences from references,resistance of ovarian cancer to cisplatin may be related to DNA repair.Wortmannin is a drug can block DNA repair by inhibiting PI3K signaling pathway,which is accordingly expected to bring about synergy effect with cisplatin against cisplatin resistance in ovarian cancer.The combination of cisplatin and etoposide is the first-line treatment agents for NSCLC in clinical chemoradiotherapy.However,the therapeutic dose is large and the course of treatment is long.Patients are prone to serious hematopoietic function and liver and kidney damage during the treatment.Nanoparticles are particle delivery vehicles with functions of improveing drug targeting,reducing drug toxicity,and improving efficacy,which can also achieve delivery of two or more drugs in precise ratio for better synergistic effects.In addition to direct killing effect on tumors,cisplatin,wortmannin and etoposide were also known with their radiosensitization effects.Due to poor hydrophobicity,cisplatin is difficult to be encapsulated by common biocompatible carrier materials.In this study,cisplatin octane-derived prodrugs were used to replace cisplatin.Cisplatin prodrug--wortmannin and cisplatin prodrug--etoposide co-encapsulated nanoparticles were prepared,characterized and optimized.Loading ratio of the drug combos were optimized by means of platinum sensitive and resistant ovarian cancer cell models as well as NSCLC models in vitro.The radiosensitization effects of the preparations were evaluated and the tumor-bearing animal model was used to investigate the therapeutic effects of these two nanoparticle encapsulated drug combinations on ovarian cancer and NSCLC chemoradiotherapy.Methods:Cisplatin prodrug—wortmannin and cisplatin prodrug--etoposide nanoparticles were prepared using biodegradable materials such as PLGA and PLGA-PEG.The morphology,particle size,particle size distribution,and zeta potential of the nanoparticle preparations were measured and characterized by TEM and DLS.The drug loading（DL%）,encapsulation rate（EE%）,and drug release of the nanoparticle preparations was determined and optimized by HPLC.Nanoparticles with different drug loading ratios were prepared.Human ovarian cancer cells including A2780 and A2780cis,human lung cancer cells including H460 and A549,and mouse lung cancer cells 344SQ were used to evaluate the concentration-cell survival rate data by MTS method.The IC₅₀ values were fitted by Origin 9 software using a four-parameter model and the best ratio of the drugs were accordingly screened.The sensitization effect of the free drug combination and its nanoparticle form on radiotherapy was examined by colony formation test of drugs and X-ray treated cells.A tumor-bearing animal model was prepared by subcutaneous injection of the corresponding tumor cells.The tumor growth curve was measured to examine the anti-tumor effect of cisplatin prodrug--wortmannin and cisplatin prodrug--etoposide co-delivery nanoparticles.The toxicity of the nanoparticles to blood cell,liver,and kidney were assessed.The cisplatin-intracellular DNA conjugates and Caspase3 fragments in tumor tissues were detected by immunohistochemistry and immunofluorescence methods to evaluate the killing effect of drug combo nanoparticles and X-rays irradiation on tumor tissues.Results:Both cisplatin prodrug--wortmannin and cisplatin prodrug--etoposide nanoparticles were successfully prepared using PLGA and PLGA-PEG as carriers.The TEM results showed that the nanoparticles were spherical or ellipsoidal in appearance.DLS combined with TEM results showed that the nanoparticle size ranged from 60 to120 nm and the zeta potential was-20 to-30 mV.The HPLC results showed that the nanoparticle preparations drug loadings were 1%to 10%for different drugs and the encapsulation efficiency was about 15%to 60%（w/w）.Drug release results showed the release half-lives of wortmannin and cisplatin prodrug in the corresponding co-encapsulated nanoparticles were 2.4 h and 3.9 h respectively,the release half-lives of etoposide and cisplatin prodrug in the corresponding co-encapsulated nanoparticles were 3.1 h and 11.1 h,respectively.In vitro cytotoxicity experiments showed that the IC₅₀ values of the selected tumor cells after treated with nanoparticles were significantly decreased compared with free drugs（p<0.05 or p<0.01）.Analyzed results of IC₅₀ values of showed that cisplatin prodrug--wortmannin made synergistic effects（CI<1）on cisplatin-resistant A2780cis ovarian cancer cells when using co-encapsulated nanoparticles.The optimal drug loading ratios was found 1:1.4（n/n）for wortmannin and cisplatin prodrug.Cisplatin prodrug--etoposide produced an additive effect on A549,H460 grade 344SQ lung cancer cells（CI=1）when using co-encapsulated nanoparticles.The optimal drug-loading ratio was found 1:1.8（n/n）for etoposide and cisplatin.Colony formation experiments showed that the radiosensitization ratio（SER）of drug combo co-encapsulated nanoparticles were significantly higher than that of the free drug combination（p<0.05）.Tumor growth curves showed that co-encapsulated nanoparticles plus radiotherapy（DNP+XRT）made better tumor inhibition effects than single drug-loaded nanoparticle mixtures+radiotherapy（SNP+XRT）as well as free drug+XRT（p<0.05）on tumors of A2780 and A2780cis.For 344SQ and H460 tumor tissues,the inhibitory effect of DNP+XRT on tumor was significantly better than that of Free drug+XRT（p<0.05）,but there was no stastical significance compared with SNP+XRT（p<0.05）.The results of hematological,hepatic,and nephrotoxicity showed that no toxicity was observed when administed with cisplatin prodrug--wortmannin or cisplatin prodrug--etoposide co-encapsulated nanoparticles.Immunofluorescence results showed that DNP+XRT and SNP+XRT played a significant role in promoting the formation of cisplatin-DNA complexes and Cleaved Caspase3 in tumor cells.Conclusion:（1）By using PLGA and PLGA-PEG as a carrier material,cisplatin prodrug--wortmannin and cisplatin prodrug--etoposide nanoparticles were successfully prepared and characterized;（2）By using drug-loaded nanoparticles,antitumor activity and radiotherapy sensitization of cisplatin prodrug--wortmannin and cisplatin prodrug--etoposide drug combinations can be significantly improved;3,Cisplatin prodrug—wortmannin nanoparticles can effectively reverse cisplatin resistance A2780cis ovarian cisplatin resistant cancer cells in vitro and in vivo.4.Cisplatin prodrug--etoposide nanoparticles can increase the killing effect of the drug combo on human non-small cell lung cancer（NSCLC）H460 and mouse metastatic lung cancer 344SQ in vitro and in vivo.