Experimental Study On Targeting Surface-modified ZrO₂ Nanocomposite In The Interventional Treatment Of Liver Tumor

Objective:Hepatocellular carcinoma（HCC） is one of the most malignant tumors in china.Transarterial Chemotherapy and Embolization（TACE） is recommended as the first-line treatment option for intermediate stage HCC.The antitumor efficacy of TACE was not significantly different compared with that associated with transarterial embolization（TAE）,demonstrated in some clinical studies.Hence,the uneven distribution of chemotherapeutic drug in tumor may account for its unsatisfactory antitumor efficacy in TACE.Previously,transarterial infusion of drug loaded nanoparticles with lipiodol for liver tumors demonstrated effectiveness in tumor treatment.To improve the antitumor efficacy,elevating the passive targeting effect of nanoparticles by surface modification may be a feasible strategy.iRGD contains a disulfide-linked arginine-glycine-aspartic（RGD） tripeptide sequence and a C-terminal end-binding sequence（CendR）.RGD tripeptide sequence can selectively recognize and bind to integrinαvβ3 which overly expresses in the endothelium of tumor vessels or tumor cells.While CendR modification can bind with Nrp1,and the binding can promote vascular and tissue permeability.Pioneering studies demonstrated that iRGD can improve the tumor uptake of nanoparticles and therapeutic drug in liver tumor.ZrO₂nanoparticle is a kind of inorganic nanomaterial which have been applied in the studies of liver tumor therapy and exhibited fine biocompatibility and easily synthesis.iRGD modified ZrO₂ nanoparticle has been demonstrated as a promising drug delivery system in the treatment of liver tumor by our preliminary study.Based on these researches,the doxorubicin（DOX） loaded and iRGD modified ZrO₂ nanoparticles（R-DZCNs） was prepared and we hypothesized that transarterial infusion of R-DZCNs with lipiodol can improved the distribution of DOX and its antitumor efficacy compared with TACE.The passive targeting effect of iRGD modified ZrO₂nanoparticles was evaluated using HepG₂ cells in vitro.In vivo,we conducted an animal experiment to evaluated the anti-tumor effect of transarterial infusion of R-DZCNs with lipiodol in the rabbit VX2 liver tumor model.Methods:Nanosilica spheres were used as templates to synthesize hollow mesoporous nano-sized zirconia by etching.Doxorubicin loaded in the hollow structure of ZrO₂ Nanoparticles.Tumor targeting peptide iRGD were modified on the surface of ZrO₂ by EDC.NHS reaction.The hollow structure,particle size distribution and ZETA potential of the R-DZCNs were characterized by Transmission electron microscopy（TEM）,dynamic light scattering instrument（DLS） and ZETA potentiometers.The release characteristic of DOX in R-DZCNs was determinated.By incubating ZrO₂ nanoparticles at different concentrations with HepG2 cells after,the cytotoxicity of ZrO₂ nanoparticles for HepG2 was evaluated.And the HepG2 cells were incubated with R-DZCNs or DZCNs which contain a serious of DOX concentration,the cytotoxicity of R-DZCNs and DZCNs was compared.DZCNs and R-DZCNs were incubated with HepG2.The cells were stained with DAPI.The target ability of R-DZCNs to HepG2 cells was observed by fluorescence microscopy.The rabbit VX liver tumor was established and observed by the MRI scanning and HE stanning.Under the guidance of DSA,the hepatic proper artery catheterization was performed to infuse lipiodol and R-DZCNs.In the DOX distribution study,24 rabbits were divided into the DOX+Lipiodol group,DZCNs+Lipiodol,and R-DZCNs+Lipiodol group.All rabbits were infused with 0.25 ml of Lipiodol.Besides,DOX,DZCNs and R-DZCNs were infused in the DOX+Lipiodol group,DZCNs+lipiodol group and the R-DZCNs+Lipiodol group respectively.At 10minutes and 4 hours after perfusion,the DOX distribution in tumor was evaluated by calculating the sum of DOX fluorescence and its penetration distance through immunofluorescence.In the antitumor study,the rabbits were divided into the control group,DOX+Lipiodol group,DZCNs+lipiodol group and the R-DZCNs+Lipiodol group respectively.In the control group,the rabbits received transarterial infusion of saline solution.The change of tumor volume was detected by MRI at 7 days and 14days after treatment.The expression of apoptosis-related factors（Caspase-3,Bax,Bcl-2）were analyzed by immunohistochemical（IHC） and western blot（WB）.Results:1.The mean diameter of R-DZCNs was 165.21±8.2 nm.The hydrodynamic diameter of R-DZCNs was 230.1nm with a concentrated size distribution,The encapsulation efficiency of DOX was approximately 50%,while the drug loading efficiency was approximately 20%.The accumulative DOX release ratio was about 37% in 24 hours.2.Cytotoxicity assay and cellular uptake assay in vitro.The viability of HepG2 cells in the R-DZCNs group was lower than that in the DZCNs groups at each drug concentration（P<0.05）,and the red fluorescence intensity in the R-DZCNs group was higher than that in the DZCNs group（118.53±16.85 vs 159.52±23.76,P=0.014）.3.DOX distribution in vivo At 10 minutes after infusion,the sum of fluorescence spots in the DOX+Lipiodol group,DZCNs+Lipiodol group and R-DZCNs+Lipiodol group was（1417.60±353.82,1407.11±520.58,1585.59±484.1,P=0.364）,respectively.4hours after infusion,these values increased to（2449.15±444.14,3464.73±632.75 and 5062.25±585.62,P<0.05）.10 minutes after infusion,the DOX penetration distance in the DOX+Lipiodol group,DZCNs+Lipiodol group and R-DZCNs+Lipiodol group was（33.75±7.3）mm,（27.36±8.13）mm and（34.78±10.04）mm respectively,（P=0.312）.P=0.275).4 hours after infusion,the DOX penetration distance in the R-DZCNs+Lipiodol group was longer than that in the DOX+Lipiodol group DZCNs+Lipiodol group（117.58±19.36）um vs（52.64±8.53）μm or（83.37±13.76）μm,（P=0.005）.4.Anti-tumor assay in vivo.MR imaging:Before transarterial infusion,the tumor volume was no difference in each group（358.32±28.33mm³,368.89±38.85 mm³,416.1±44.29mm,398.97±56.91mm,P=0.526）.At 7 days after infusion,the tumor volume of R-DZCNs+Lipiodol group was smaller tumor compared with the control group,DOX+Lipiodol group or DZCNs+Lipiodol group[（758.70±213.58 vs3349.58±313.15 or 1578.01±299.43 or1151.10±277.65）mm³,P<0.001)].14 days infusion,the tumor volume of R-DZCNs+Lipiodol group was smaller compared with the control group,DOX+Lipiodol or DZCNs+Lipiodol group[（1223.87±223.58 vs8590.69±564.42 or 3695.26±666.25 or 2281.06±457.21）mm³,P=0.012)].Immunohistochemical assay:The percentage of Casepase-3 positively stained cells in the DOX+Lipiodol group,DZCNs+Lipiodol group and R-DZCNs+Lipiodol group was 2.2-fold,3.4-fold and 5.7-fold compared with control group respectively（P=0.01）,and the percentage of Bax positively stained cells of those groups was 2.0-fold,3.8-fold and 5.8-fold compared with control group respectively with an increasing trend（P=0.018）.In contrast,the percentage of Bcl-2 positively stained cells were decreased to0.7-fold,0.5-fold and 0.2-fold respectively compared with the control group with a decreasing trend（P=0.01）Western blot:Western blot assay showed higher gray scale of Caspase-3 and Bax,as well as lower gray scale of Bcl-2 in experimental groups compared with those of control group.The gray scale of Caspase-3 protein bands in the DOX+Lipiodol group,DZCNs+Lipiodol group and R-DZCNs+Lipiodol group was1.7-fold,2.6-fold,4.3-fold as control group respectively with an increasing trend（P<0.001）,and these values for Bax were 1.9-fold,2.5-fold and 3.2-fold respectively（P=0.008）.In contrast,the Bcl-2 expression decreased to 0.8-fold,0.6-fold and 0.3-fold respectively with a decreasing trend（P=0.02）.Conclusion:In this study,we successfully synthesized R-DZCNs,a novel drug delivery system that has the potential of targeting tumor cells.And in vitro experiments have confirmed that R-DZCNs enhanced the cellular uptake and cytotoxicity for hepatocellular carcinoma cells.In vivo experiment,the strategy of infusing R-DZCNs with lipiodol revealed the effect of improving DOX distribution in tumor,and enhanced the antitumor efficacy.