Experimental Study Of Targeted Redox-sensitive Block Polymer Nanomicelle For Hepatocellular Carcinoma

Objective1)Preparation of hepatocellular carcinoma targeted block polymer nanomicelle ?HA-SS-PCL@DOX/SPIO?with reduced sensitivity,containing diagnostic superparamagnetic iron oxide?SPIO?and therapeutic anti-cancer drug DOXorubicin?DOX?,and its physical and chemical properties were characterized in vitro.2)Through in vitro cell experiment,discussed the safety of blank micelle ?HA-SS-PCL?,thecytotoxicityofdrug-loadedmicelle ?HA-SS-PCL@DOX/SPIO?,in vitro release of DOX,and the feasibility of using MRI to monitor the targeted delivery of drug-loaded micellar anticancer drug.3)In vivo animal experiment was conducted to investigate the safety,active target,and redox-susceptibility of drug-loaded micelle?HA-SS-PCL@DOX/SPIO?on human hepatoma HepG₂ cell in nude mice and the feasibility of non-invasive,real-time,dynamic monitoring in MRI.Methods1)Preparationandcharacterizationofblockpolymernanomicelle ?HA-SS-PCL@DOX/SPIO?a)First,hydrophobic polycaprolactone?PCL?was prepared by the ring-opening polymerization of caprolactone??-CL?under the action of a dodecanol initiator,then the PCL was subjected to a two-step chemical reaction.Conversion of hydroxy groups to amino groups containing disulfide linkages?PCL-SS-NH₂?;PCL-SS-NH₂ was then used as a macroinitiator to polymerize glutamate cyclized anhydride?AELG-NCA?monomers.The disulfide-containing block polymer PCL-SS-PAELG was successfully prepared.Then the alkynyl hyaluronic acid ?HA?was coupled to the synthesized copolymer through the"Click"reaction to form HA-SS-PCL.Finally,superparamagnetic iron oxide?SPIO?and the antitumor drug Doxorubicin?DOX?were loaded into the polymer hydrophobic core efficiently by dialysis.b)Characterization of physicochemical properties of polymer micelle:synthesis of HA-SS-PCL by ¹H NMR and FTIR;Using dynamic light scattering?DLS?and transmission electron microscopy?TEM?to measure particle size,shape and distribution;The concentration of critical micelle was detected by pyrene.The loaded rate of SPIO was measured by atomic absorption method,and the loaded rate of DOX was measured by ultraviolet absorption method,and the release of DOX was measured in vitro.2)In vitro cell experimenta)The safety of HA-SS-PCL and the cytotoxicity of HA-SS-PCL@DOX/SPIO were investigated by using methylthiazolyl tetrazolium?MTT?.b)The free DOX,HA-PCL@DOX/SPIO,and HA-SS-PCL@DOX/SPIO nanomicelles were co-incubated with human hepatoma Hep G₂ cells,followed by fluorescence microscopy,flow cytometry,and DAPI staining.The active targeting mechanism of drug-loaded micelle HA-SS-PCL@DOX/SPIO on human hepatoma HepG₂ cells was observed in vitro.c)Through external MRI imaging,the T₂WI signal intensity change was observed and to explore the feasibility of MRI to evaluate the absorption of human hepatoma HepG₂ cells to nanomicelles.3)In vitro animal experimentsa)To establish nude mice subcutaneous transplantation tumor animal model of liver cancer,about 4⁶ weeks when tumor 1² cm in diameter,according to the requirements of group,21 only nude mice were randomly divided into normal saline group,HA-PCL@DOX/SPIO,HA-SS-PCL@DOX/SPIO group,each group of seven,the experimental group by tail vein by Fe 0.5 mg/kg micelles injection,control group by tail vein injection of same amount of saline solution, injection before and after injection of different point?2 h,4 h,8 h?respectively by MRI imaging,measurement of interested area?tumor?T₂WI signal intensity,T₂ and T₁ values.A nude mouse was executed at different scanning time points in each group,and conventional HE staining and prussian blue staining were performed.b)To establish nude mice orthotopic transplantation tumor animal model of liver cancer,about 4⁶ weeks when tumor 0.5¹.5 cm in diameter,according to the requirements of group,12 only nude mice were randomly divided into normal saline group,HA-PCL@DOX/SPIO,HA-SS-PCL@DOX/SPIO group,each group of four,the experimental group by tail vein by DOX 2 mg/kg micelles injection?3 times of continuous injection,one day interval?,control group by tail vein injection of same amount of saline solution.Two hours after the last injection of the drug,the nude mice were sacrificed and routinely stained with HE and Prussian blue.Result1)¹HNMR and FTIR confirmed the successful preparation of block polymer nanomicelle HA-SS-PCL@DOX/SPIO.DLS and TEM show that the prepared micelle was suitable for nanometer size and homogeneity.The ultraviolet absorption method and the atomic absorption method respectively showed the high efficiency of DOX and SPIO.In vitro release experiment confirmed that HA-SS-PCL@DOX/SPIO had a good release for DOX.2)In vitro cell experimentsa)MTT assay showed that HA-SS-PCL had good compatibility with HepG₂ and LO₂ cells under certain concentration.b)MTT assay showed that the polymer micelles HA-SS-PCL@DOX/SPIO had a good release effect on DOX,and with the increase of DOX concentration,the damage to HepG₂ cells increased gradually.c)Fluorescence observation,DAPI dye and flow cell analysis showed significant differences in the fluorescence intensity of Free DOX,HA-PCL@DOX/SPIO and HA-SS-PCL@DOX/SPIO group.The fluorescence of Free DOX group was the strongest,followed by HA-SS-PCL@DOX/SPIO,HA-PCL@DOX/SPIO was the weakest.d)In vitro MRI imaging showed that the T₂WI signal intensity of HA-SS-PCL@DOX/SPIO group and HA-PCL@DOX/SPIO group was significantly lower than that of the blank control group,while the decrease of T₂WI signal intensity was more obvious in the HA-SS-PCL@DOX/SPIO group compared with the HA-PCL@DOX/SPIO group.3)In vitro animal experimentsa)Successfully established nude mice models of hepatocarcinoma subcutaneous heterotopic xenografts and orthotopic liver transplantation.HE staining confirmed that they were consistent with hepatocellular carcinoma model.b)Animal MRI imaging showed that the intensity of T₂WI was significantly lower in the tumor tissue at 2 h and 4 h after injection of nanomicelles HA-PCL@DOX/SPIO and HA-SS-PCL@DOX/SPIO than that of plain scan,while the latter decreased more significantly.The T₂ value was significantly lower in the tumor tissue at 2 h?4 h and 8 h after injection of nanomicelles HA-PCL@DOX/SPIO and HA-SS-PCL@DOX/SPIO than that of plain scan,while the latter decreased more significantly.After injection of nanomicelles HA-PCL@DOX/SPIO and HA-SS-PCL@DOX/SPIO,the T₁ values of tumor tissue at all time points were not significantly lower than those of plain tissue.c)Different time points prussian blue staining confirmed the HA-SS-PCL@ DOX/SPIO group within the tumor tissue see more Fe particles,HA-PCL@ DOX/SPIO group see fewer Fe particles,and normal saline groups did not see the blue dye Fe particles.At the same time,the results further confirmed that HA-SS-PCL@DOX/SPIO micelle was mainly distributed in tumor tissue,and other organs were rarely or undistributed.Conclusion1)This study has successfully prepared a novel hepatocellular carcinoma targeted block polymer nanomicelles with reductive sensitity and diagnostic and therapeutic functions.2)The prepared block polymer nanomicelle HA-SS-PCL@DOX/SPIO has small particle size,uniform size,good water solubility,colloidal stability,biocompatibility,antitumor effect,and active targeting,strong reduction sensitivity and MRI imaging and other functions in one,has a good potential application prospect.