| Objective:In this thesis,we present a multifunctional nanodrug system,which integrates the targeted delivery to specific tumor cells/tissues,p H-stimulus release of anti-tumor drug molecules,as well as synergistic chemo-photothermal therapy.By utilizing novel analytical techniques,we succeed in the evaluation of targeted anti-tumor efficacy of the nanodrug in vitro and vivo.As a result,we propose a rational research strategy,and demonstrate meaningful experimental findings to explore therapeutic nanodrugs for hepatocellular carcinoma(HCC).Methods:The methodologies are described as follows.(1)First,copper sulfide nanoparticles(Cu S NPs)were synthesized by hydrothermal method.Then,dopamine was in-situ polymerized spontaneously in an alkaline condition to produce polydopamine(PDA)encapsulating on the Cu S NPs,thereby forming a dual photo-thermal responsive nanomaterial,Cu S-PDA composite.Further,a p H-response polymer PAA,was chemically coupled to the Cu S-PDA composite via Michael addition reaction,resulting in the nanocarrier Cu S-PDA-PAA(CPP).(2)Second,a classic anti-tumor drug,DOX,was self-assembled electrostatically onto the surface of the nanocarrier,producing the nanodrug,Cu S-PDA-PAA/DOX.(3)Third,anti-GPC3 antibody commonly overexpressed in the hepatocellular carcinoma was linked to the Cu S-PDA-PAA/DOX via amide reaction,ultimately forming the targeted nanodrug(Cu S-PDA-PAA/DOX/GPC3).The prepared nanodrug reaped a variety of intriguing functionalities including targeting drug delivery,p H-stimulus release,and combined chemo-photothermal anti-tumor effect.(4)The Cu S-PDA-PAA nanocarrier was characterized by TEM,XRD,XPS,IR and UV-vis techniques.(5)The relevant performances such as photothermal conversion,stability,biocompatibility,drug-loading efficiency,drug-release behavior,particle size,and zeta potential were optimized.(6)The in vitro targeting uptake ability was investigated by laser confocal scanning microscope(LCSM)and flow cytometry(FCM).The tumor-cells-killing effect in combination with NIR irradiation was examined by MTT method.Moreover,the apoptosis of the tumor cells were revealed by AO/EB staining method.(7)Subcutaneous tumor model in nude mice was built to assess the anti-tumor efficacy of the designed Cu S-PDA-PAA/DOX/GPC3 nanodrug.Results:The core data and significant findings were detailed in the following.(1)TEM test indicated that the Cu S-PDA-PAA nanoparticles were evenly spherical,with the average size of 250 nm.XPS and IR characterizations verified the successful construction of the nanocarrier.UV-vis spectrum suggested that the anti-tumor drug DOX was effectively adsorbed at the surface of the nanocarrier,forming the nanodrug Cu S-PDA-PAA/DOX.The drug-loading efficiency and drug-loading ratio can reach up to 90.0％and 47.6％,respectively when the nanocarrier and drug was mixed in a 1:05 mass ratio.SDS-PAGE analysis confirmed the successful linkage of anti-GPC3 antibody on the nanocarrier,thus endowing the nanodrug with the targeting delivery characteristic.(2)Appealing and stable photothermal conversion performance of the nanocarrier was observed,as confirmed by 4-cycle photothermal on/off operation.In a dispersion solution containing of 50mg/m L nanocarrier,the media temperature can respectively increase by 38.4 ~oC and 56.8 ~oC after 5-min irradiation under 808 nm NIR with the power of 2 W/cm~2and 4 W/cm~2,respectively.The nanocarrier dispersion in p H 7.4 PBS can be stable for at least21 days at 4 ~oC condition.Besides,the nanocarrier showed favorable biocompatibility to normal H9C2 myocardial cells,7702 hepatocytes and Hep G2 tumor cells.The survival rates of the three cell lines examined were greater than 80％,indicative of negligible cytotoxicity.(3)In p H 5.5 weak acidic environment,the 72-hour accumulative release of the nanodrug was as high as83.7％,much lager than 14.6％obtained in p H 7.4 condition,demonstrating a well-characteristic p H-response and drug-release behavior in the tumor microenvironment(TME).(4)The hydrated particle size of the nanocarrier was found to be 294 nm,with the zeta potential of-42.0 m V.In comparison,the particle size increased to 312 nm and the zeta potential rose up to-6.0 m V after loading with the positively charged DOX molecules.Such alteration in surface potential was beneficial to endocytosis by the targeting tumor cells.Significantly,LCSM and FCM results indicated that Cu S-PDA-PAA/DOX/GPC3 nanodrug can specifically target to Hep G2 cells to exert the anti-tumor effect.(5)The survival rates of H9C2 cells and 7702 cells incubated with the nanodrug typically containing of 6mg/m L DOX for 24 h were 54.0％and 62.5％,respectively;by contrast,the survival rate of Hep G2 cells was merely 35.8％.This result clearly demonstrated that the nanodrug can substantially reduce the cytotoxicity to the normal cells while enhancing the targeting tumor-killing ability.Moreover,the survival rate of Hep G2 cells drastically dropped down to10.3％when in alliance with NIR irradiation,well declaring the high-efficiency chemo-photothermal tumor-killing performance.AO/EB staining experiment also corroborated the synergistic anti-tumor ability.(6)The tumor in the modeled mice can be significantly suppressed by the nanodrug.In addition,histology results revealed that the nanodrug can effectively alleviate the toxicity to heart and liver as compared to free DOX.In addition,the nanodrug plus NIR photothermal treatment strengthened the therapeutic efficacy to HCC in vivo.Conclusion:The prepared nanodrug takes several advantages including easy-to-preparation,favorable stability and biocompatibility,and excellent photothermal conversion performance.Remarkably,the highly-efficient payload of anti-tumor drug,targeted delivery and p H-triggered release in the tumor microenvironment enable to achieve the improved remedy of HCC and reduced toxic effects on normal cells/tissues.This work offers scientific supportings,and importantly,opens an avenue for design of novel nanodrugs against HCC. |
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