Regorafenib-loaded poly(lactide-co-glycolide) microspheres designed to improve transarterial chemoembolization therapy for hepatocellular carcinoma

Transarterial chemoembolization(TACE)has been widely introduced to treat hepatocellular carcinoma(HCC)especially for unresectable patients for decades.However,TACE evokes an angiogenic response due to the secretion of vascular endothelial growth factor(VEGF),resulting in the formation of new blood vessels and eventually tumor recurrence.Thus,we aimed to develop regorafenib(REGO)-loaded poly(lactide-co-glycolide)(PLGA)microspheres that enabled localized and sustained drug delivery to limit proangiogenic responses following TACE in HCC treatment.REGO-loaded PLGA microspheres were prepared using the emulsion-solvent evaporation/extraction method,in which DMF was selected as an organic phase co-solvent.Accordingly,we optimized the proportion of DMF,which the optimal ratio to DCM was 1:9(v/v).After preparation,the microspheres provided high drug loading capacity of 28.6%,high loading efficiency of 91.5%,and the average particle size of 149μm for TACE.IR spectra and XRD were applied to confirming sufficient REGO entrapment.The in vitro release profiles demonstrated sustained drug release of microspheres for more than 30 d To confirm the role of REGO-loaded microspheres in TACE,the cell cytotoxic activity on HepG2 cells and anti-angiogenic effects in HUVECs Tube-formation assay were studied in combination with miriplatin.Moreover,the microspheres indicated the potential of antagonizing miriplatin resistance of HepG2 cells in vitro.Pharmacokinetics preliminary studies exhibited that REGO could be sustainably released from microspheres for more than 30d after TACE in vivo.In vivo anti-tumor efficacy was further determined in HepG2 xenograft tumor mouse model,demonstrating that REGO microspheres could improve the antitumor efficacy of miriplatin remarkably compared with miriplatin monotherapy.In conclusion,the obtained REGO microspheres demonstrated promising therapeutic effects against HCC when combined with TACE.

Construction and expression of hepatitis B virus vector encoding TC-tagged core protein

Virus tagged with greenfluorescent protein(GFP)contributes to the visualization and study of the virus in living cells.However,the hepatitis B virus(HBV)particle,which is a compact virion with limited internal space,cannot be incorporated with GFP tag as a large fragment.It was recently reported that protein genetically inserted with a smaller size tetracysteine(TC)tag could be specially labeled by a biarsenicalfluorescent dye in living cells.In this study,we constructed a recombinant HBV vector encoding TC-tagged core protein for biarsenical labeling of HBV virion.TC tag was genetically inserted near the immunodominant c/e1 site of HBV core protein by mutagenesis.Western blot and enzyme-linked immu-nosorbent assay(ELISA)analysis showed that the TC-tagged core protein,hepatitis B surface antigen(HBsAg)and hepatitis B e antigen(HBeAg)could be expressed in cells transfected with the recombinant HBV vector,which is similar to the cells transfected with wild-type HBV vector.Reverse transcription-polymerase chain reaction(RT-PCR)and Southern blot analysis showed that HBV virion formation was affected by the genetic insertion of TC tag into core protein in some degree,but cells transfected with the HBV vector could still produce HBV virions incorporated with TC-tagged core proteins.Taken together,the recombinant HBV vector can serve as a useful tool to produce HBV virions incorporated with TC-tagged core proteins to befluorescently labeled by biarsencial dye for visualizing and studying HBV in living cells.