| Objective1.To explore the targeting and cytotoxicity of the nano-drug delivery systems（nano-DDS）RGD-SS-DOX/Ce6@HA-IR-1061（RSSDCHI）with HA/RGD dual-targeting function.2.To explore the second near-infrared window（NIR-II）fluorescence imaging performance of RSSDCHI for tumors in vivo.3.To evaluate the effect of RSSDCHI in combination with targeted combined chemotherapy&photodynamic therapy（PDT）in vivo.Methods1.HA-IR-1061 amphiphilic graft copolymer was synthesized using hyaluronic acid（HA）and NIR-II fluorescence probe IR-1061 at first,and then RGD-DOX/Ce6@HA-IR-1061（RDCHI）was prepared by emulsion-solvent evaporation method,which is modified with disulfide bond（-SS-）to make the nano-theranostics RSSDCHI loaded with hydrophobic chemotherapy drug Doxorubicin（DOX）and photosensitizer Chlorin e6（Ce6）.The physical and chemical properties,spectral analysis,and stability test of RSSDCHI were carried out,and the production of singlet oxygen（1O2）under laser irradiation was detected.Its Hyaluronidase（HAase）and Glutathione（GSH）multi-responsive property was verified through in vitro experiments,its responsive drug release and NIR-II fluorescence recovery ability were explored.The correlation between the responsive release of drugs and the NIR-II fluorescence intensity were also explored.The HA and RGD multi-targeting performance of RSSDCHI was validated through U87 cell.The production of endogenous 1O2 of RSSDCHI was explored.The multi-responsive properties of HAase and GSH for RSSDCHI were verified by U87 cell.U87 cells were inoculated into two groups of 96-well plates,which were respectively set as:（1）no laser irradiation group,（2）laser irradiation group,and their cytotoxicity and phototoxicity were investigated by The Cell Counting Kit-8（CCK-8）assay.2.The human glioma U87 tumor bearing mice model was established,and six U87tumor bearing mice were randomly divided into two groups（n=3）:（1）DOX/Ce6@HA-IR-1061（DCHI）group,（2）RSSDCHI group,to evaluate the ability of RSSDCHI for NIR-II fluorescent imaging in vivo.3.54 U87 tumor-bearing mice were randomly divided into 9 groups（n=6）:（1）Phosphate buffered saline（PBS）group,（2）PBS+laser group,（3）free DOX group,（4）free Ce6+laser group,（5）HA-IR-1061 vesicle（HI）group,（6）DCHI group,（7）DCHI+laser group,（8）RSSDCHI group,（9）RSSDCHI+laser group.The efficacy of tumor-targeted combined PDT&chemotherapy as well as its safety were evaluated.Results1.HA-IR-1061 amphiphilic graft copolymer was successfully synthesized,and the nano-theranostics RSSDCHI was successfully assembled.It showed spherical vesicles with regular shape under the transmission electron microscope（TEM）,with a diameter of about 89.52±6.65 nm.The hydrated particle size of RSSDCHI is about 155.57±1.33 nm,and the Zeta potential is about 7.40±1.10 m V.The fluorescence emission spectrum peak value of RSSDCHI is in the range of 1100-1118 nm when excited by808 nm laser.RSSDCHI has good stability and biocompatibility,and can produce 1O2with 660 nm laser irradiation.It can respond to HAase and GSH and make drug release and fluorescence recovery.Moreover,the NIR-II fluorescence intensity of RSSDCHI is linear with the cumulative drug release（R2>0.97）.RSSDCHI has multiple targeting properties.Blocking group experiment further verified that RSSDCHI can target U87cells through HA and RGD.RSSDCHI can produce a large amount of endogenous 1O2in U87 cells with 660 nm laser irradiation.In the group without laser irradiation,the relative cell activity of U87 cells under different concentrations of HI was more than90%.In the free DOX group,DCHI group and RSSDCHI group,the relative cell activity decreased with the increase of drug concentration.The relative cell activity of RSSDCHI group was slightly lower than that of DCHI group,and the relative cell activity of RSSDCHI group was similar to that of free DOX of the same concentration（P>0.05）.In the laser irradiation group,with the same Ce6 concentration,the relative cell activity of the DCHI+laser group was significantly lower than that of the Ce6+laser group,with a statistical difference（P<0.001）.While the relative cell activity of the RSSDCHI+laser group was significantly lower than that of the DCHI+laser group,with a statistical difference（P<0.001）.2.U87 tumor bearing mice was successfully established.In the 6-12 h and 36-72h after injection of DCHI or RSSDCHI,the Tumor-to-normal tissue（T/NT）ratio of tumor bearing mice in RSSDCHI group was significantly higher than that in DCHI group（P<0.05）.The fluorescence intensity ratios of tumor/heart,tumor/liver,tumor/spleen,tumor/lung,and tumor/kidney of the RSSDCHI group were significantly higher than those of the DCHI group（P<0.05）.3.RSSDCHI+laser group had the best effect on tumor treatment.The relative tumor volume ratio V/V0（V is the observed day tumor volume,V0 is the pre-treatment tumor volume）on the 16th day after treatment of RSSDCHI+laser group was significantly lower than other groups（P<0.05）.Among the other groups,the tumor growth of DCHI+laser group was slower than that of other groups（P<0.05）.The Hematoxylin and Eosin（H&E）staining results of the main organ sections of the tumor-bearing mice in each group 16th day after treatment showed that no significant tissue damage was caused to the main organs of the tumor-bearing mice by various treatment methods.Conclusion1.The HAase/GSH dual-responsive self-monitoring nano-DDS RSSDCHI with HA/RGD dual-targeting function has been developed.RSSDCHI showed obvious cytotoxic effect than chemotherapy alone or PDT alone in vitro.2.Active targeting resulted in the accumulation of RSSDCHI in tumor tissues indicates that RSSDCHI can be used as an effective NIR-II fluorescent prob in vivo to precisely display the tumor.3.Enhanced anti-tumor efficacy of RSSDCHI in vivo was observed.It can be used for drug delivery and tumor diagnosis,drug release monitoring and guided treatment through NIR-II fluorescence imaging. |
