The Construction Of Prodrug-based Nanoparticle Drug Delivery System And The Combination With Dasatinib For Hepatocellular Carcinoma Therapy

Background:Hepatocellular carcinoma(HCC)is one of the most common malignant tumors with high mortality.Because of lacking in early stage specific symptoms,most patients are not diagnosed until they are in the intermediate or advanced stages of HCC,and therefore miss the best opportunity for liver resection or liver transplantation.The therapeutic effect of interventional therapy or systemic chemotherapy is relatively low,resulting the poor prognosis of the patients with unresectable HCC.As a consequence,the research and development of new treatment strategy will be meaningful and significant for HCC patients.Based on different kinds of inorganic materials and organic or polymer materials,we can use nanomaterials to form nanoparticle drug delivery systems.And with the decoration of specific targeting ligands,drug-loaded nanoparticles could improve drug accumulation in tumor site and reduce toxicity.Some specific prodrug could improve compatibility between drugs and nanomaterials,and thus enhance stability of nanoparticles and improve their effects.Besides,the combination of traditional chemotherapy and molecular targeted agents is another important strategy in oncotherapy,which could induce synergistic effect,overcome drug resistance,reduce dosage and lower toxicity.Aim:This research aims to prepare PLA-decorated doxorubicin(DOX)prodrug or SN38 prodrug based on prodrug strategy,and construct drug-loaded nanoparticles by co-assembly of these prodrugs and DSPE-PEG or PEG-PLA.SP94 peptide is used to construct HCC-targeting nanoparticles,and their targeting ability will be studied both in vitro and in vivo.We also expect to study the combination use of chemotherapeutic agent irinotecan/SN38 and tyrosine kinase inhibitor dasatinib in HCC,and explore the possible mechanism of its synergistic effect.The combination effect of SN38 prodrug-loaded nanoparticles and dasatinib will be further validated in vivo.Methods:Section 1:PLA segment was conjugated to DOX through an amide bond to form prodrug PLA-DOX,and an SP94 peptide ligand was attached to maleimide-terminated DSPE-PEG2000 via a thiol-maleimide coupling reaction.Nontargeting or targeting PLA-DOX prodrug-loaded nanoparticles(termed nNP-PLA-DOX and tNP-PLA-DOX)were constructed by co-assembly of PLA and DSPE-PEG2000 or DSPE-PEG2000-SP94 in water.The size,zeta potential,morphology and drug release of these nanoparticles were measured separately.Confocal laser scanning microscopy and flow cytometry were used to evaluate the cell-specific uptake of nanoparticles.CCK-8 was used to assess the cytotoxicity of nNP-PLA-DOX and tNP-PLA-DOX in HCC cells.EdU staining was used to evaluate the anti-proliferation ability of these nanodrugs in HCC cells.An HCC-LM3 cell-derived xenograft model and in vivo and ex vivo imagings were used to x study the in vivo anti-tumor effect of these nanodrugs and their in vivo targeting ability.Section 2:SRB assay was used to evaluate the cytotoxicity of SN38 and dasatinib.Colony formation assay was used to assess the anti-proliferation ability of SN3 8 and dasatinib in different HCC cells.Annexin V-FITC/PI staining and flow cytometry were applied to evaluate dasatinib and SN38-induced apoptosis,and DPAI staining was performed to observe cell nucleus after drug administration.Western Blot(WB)was applied to assess the protein level of c-PARP,c-caspase-3,Bcl-2 and Bax.JC-1 staining and flow cytometry were applied to detect the decrease of mitochondrial membrane potential.qRT-PCR and WB were used to evaluate the mRNA and protein level of PLK1 after the administration of SN38.PLK1 siRNA and PLK1 plasmid were used to study the correlation of PLK1 and SN38 efficacy.Lysosome inhibitor CQ,proteasome inhibitor MG 132 and protein synthesis inhibitor CHX were used to study the mechanism of PLK1 inhibition from combination therapy.An HepG-2 cell-derived xenograft model was used to study the in vivo synergistic effect of irinotecan and dasatinib.Section 3:PLA segment was conjugated to SN38 to form prodrug PLA-SN38 using the method similar to section 1.PLA-SN38 prodrug-loaded nanoparticles(termed NP-PLA-SN38)were constructed by co-assembly of PLA-SN38 and PEG5000-PLA8000 in water.The size,zeta potential,morphology and drug release of these nanoparticles were measured separately.CCK-8 was used to assess the cytotoxicity of NP-PLA-SN38 in HCC cells.A BEL-7402 cell-derived xenograft model was used to compare the in vivo anti-tumor effect of NP-PLA-SN38 and irinotecan.An HCC PDX model was used to study the in vivo synergistic effect of NP-PLA-SN38 and dasatinib.Results:Section 1:Nontargeting or targeting PLA-DOX prodrug-loaded nanoparticles(termed nNP-PLA-DOX and tNP-PLA-DOX)were constructed by co-assembly of PLA and DSPE-PEG2000 or DSPE-PEG2000-SP94 in water.Hydrodynamic diameters of nNP-PLA-DOX and tNP-PLA-DOX were 122.6±3.8nmand 75.3±9.6 nm,respectively.Zeta potentials of nNP-PLA-DOX and tNP-PLA-DOX were-14.83±0.61 mV and-3.10±1.51 mV,respectively.And morphology pictures of nanoparticles were acquired by TEM and SEM.nNP-PLA-DOX and tNP-PLA-DOX possessed a remarkably sustained release property,the amount of the total DOX released from nNP-PLA-DOX and tNP-PLA-DOX were 30.09±2.53%and 32.55±0.48%in PBS buffer(pH 7.4,)at 37 0C for 20 days.CLSM and flow cytometry confirmed that the cell-specific uptake of tNP-PLA-DOX was stronger than nNP-PLA-DOX in HCC cells,while this phenomenon was not observed in liver cells and lung cancer cells.In vitro cytotoxicity assay showed that IC50 of tNP-PLA-DOX were 6.571±0.855 μM and 3.729±0.702μM in HCC-LM3 and BEL-7402 cells,respectively,which were signiifcantly lower than that of nNP-PLA-DOX(9.283±0.422 μM and 6.921±0.841μM in HCC-LM3 and BEL-7402 cells,respectively).But in vitro cytotoxic ability of free DOX was stronger than both nNP-PLA-DOX and tNP-PLA-DOX.EdU staining assay revealed that the anti-proliferation effect of tNP-PLA-SN38 was more potent than nNP-PLA-DOX.In an HCC-LM3 cell-derived xenograft model,in vivo and ex vivo imaging results displayed that tNP-PLA-DOX possessed an enhanced tumor targeting ability than nNP-PLA-DOX,and consequently the improved in vivo anti-tumor efficacy and reduced toxicity(tumor inhibition rate of tNP-PLA-DOX and nNP-PLA-DOX and free DOX,72.83%vs 57.29%31.70%).Section 2:All the combination index(CI)of different concentration of SN38 and dasatinib in BEL-7402 and HepG-2 were below 0.4,indicating the strong synergistic effect of SN3 8 and dastinib in HCC cells.The colony formation assay showed the enhanced anti-proliferation ability of this combination therapy in HCC cells.Annexin V-FITC/PI staining and flow cytometry displayed that dasatinib could improve SN38-induced apoptosis in BEL-7402 and HepG-2 cells.DAPI staining showed the number of typical apoptotic cells was increased in combination therapy group.The protein level of c-PARP,c-caspase-3 and Bax were increased in combination therapy group,while Bcl-2 was decreased.JC-1 staining and flow cytometry displayed that the combination use of SN38 and dasatinib could further promote the decrease of mitochondrial membrane potential.SN38 could inhibit the mRNA and protein level of PLK1 in HepG-2 cells,and the further inhibition of the protein level of PLK1 was observed in combination therapy.Silencing PLK1 could induce the increase of c-PARP,while the overexpression of PLK1 impaired the increase of c-PARP induced by SN38.The pretreatment of CQ or MG 132 were unable to reverse the combination therapy-induced decrease of PLK1.While in the CHX treatment group,the combination therapy could not further inhibit the protein level of PLK1,indicating that dasatinib synergized with SN38 to inhibit PLK1 by the inhibition of its protein synthesis.In an HepG-2 cell-derived xenograft model,we found that the in vivo anti-tumor effect of the combination therapy was more potent than irinotecan or dasatinib alone(tumor inhibition rate of combination therapy,irinotecan or dasatinib alone,49.67%vs 10.70%vs 14.56%).Section 3:PLA-SN38 prodrug-loaded nanoparticles(termed NP-PLA-SN38)were constructed by co-assembly of PLA-SN38 and PEG5000-PLA8000 in water.Hydrodynamic diameter and zeta potential of NP-PLA-SN3 8 were 50.9±0.243 nm and-0.015±0.862 mV,respectively.And morphology pictures of nanoparticles were acquired by TEM.NP-PLA-SN38 possessed a remarkably sustained release property,the amount of the total SN38 released from NP-PLA-SN38 was 51.22±6.56%in PBS buffer(pH 7.4)at 37℃ for 10 days.In vitro cytotoxicity assay showed that IC50 of NP-PLA-SN38 were 20.94±3.171μM and 2.487±0.488μM in HCC-LM3 and BEL-7402 cells,respectively,similar to free SN38 in HCC-LM3 and BEL-7402 cells(15.08±2.396 μM and 1.663±0.219 μM,respectively).In a BEL-7402 cell-derived xenograft model,the in vivo anti-tumor effect was assessed,and we found that NP-PLA-SN38 was more potent than irinotecan(tumor inhibition rate of NP-PLA-SN38 and irinotecan,83.04%vs 50.96%),with lower toxicity.In an HCC PDX model,the in vivo synergistic effect of NP-PLA-SN38 and dasatinib was further validated(tumor inhibition rate 86.86%),which was more efficient than the combination of irinotecan and dasatinib(tumor inhibition rate 54.19%).Conclusion:This research constructed a prodrug-based HCC-targeting nanoparticle drug delivery system.The decoration of SP94 could significantly improve the HCC-targeting ability of PLA-DOX-loaded nanodrugs,thus enhance the accumulation of drugs in tumor site,and greatly improve the anti-tumor efficacy and reduce toxicity.Besides,this research also found that dasatinib synergized with irinotecan/SN38 to suppress HCC via inhibiting the protein synthesis of PLK1.And the synergistic anti-tumor efficacy of PLA-SN38-loaded nanodrugs and dasatinib was further validated.