| Introduction: The American Cancer Society estimates that liver cancer will account for about 41,000 new cancer cases and 29,000 cancer deaths in the United States in 2017,and it is the fifth leading cause of cancer death in men and the eighth leading cause of cancer death in women.Tumor thermal ablation has become an effective treatment for localized hepatocellular carcinoma(HCC).Nevertheless,the technique is constricted from further applications in lesions beyond 3cm by the high rate of local recurrence.The booming nanotechnology has been widely applied in the field of biomedicine and it may provide more ways to improve the situation.Photothermal therapy(PTT),taking advantages of near-infrared(NIR)laser absorbing agents to burn tumors as a novel ablation technique,has gained a great deal of attentions.Various materials,such as Au nanorods,Au nanocages,polypyrrole and graphene have been investigated intensively as PTT agents due to the excellent surface plasmon resonance(SPR)features.Recently,Mo S2 as an ideal PTT agent has been showing the way by its excellent SPR features,high photothermal conversion efficiency,good biocompatibility and low cost.Tan et al.designed and fabricated layered Mo S2 hollow spheres(LMHSs)in a continuous-flow system by chemical aerosol flow method,with remarkable tumor-control efficacy.However,the so far designed LMHSs are about 600 nm in size,with the low efficiency of passing through tumor capillary endothelial cell gaps to realize enhanced permeability and retention(EPR)effect.We in our study fabricated the LMHSs in size of around 300 nm by adding n-propyl alcohol to reduce the surface tension of the droplets,which have been showing much enhanced efficacy of EPR effect by just intravenous injection.To the best of our knowledge,this is the first synthesis of LMHSs that can be used for intravenous administration.In addition,the designed 300 nm LMHSs exhibited strong NIR absorption and high photothermal conversion efficiency.Local recurrence of HCC may be due to the residual cancer cells and the heterogeneity of tumor cells.Therefore,we expect the tumor can be treated with combined ways of PTT as well as chemotherapy to reduce recurrence.Nanomaterials are suitable as carriers for chemotherapeutic agents.Liu et al.had reported that Mo S2 nanosheets loaded doxorubicin(DOX)exhibited excellent tumor control by combining photothermal treatment and chemotherapy.Different from the two-dimentional Mo S2 nanosheets,the hollow structures and layered structures of the LMHSs provide much more space for drug loading.In this study,DOX was loaded into the hollow structure and we got the prepared DOX@Mo S2,which exhibited high biocompatibility.Objective: The characterization,toxicity,and photothermal efficacy of as-made DOX@Mo S2 were studied.As well,both in vitro and in vivo models were applied to verify the anti-cancer efficacy of DOX@Mo S2 in this study.Methods: Synthesis of LMHSs:(NH2)2CS and Na2 Mo O4.2H2 O dissolved in water and npropyl alcohol,which was adjusted acidity with 1 m L HCl with a continuous flow of nitrogen,the droplets obtained from ultrasonic atomizer were taken through the furnace.Loading and release of DOX: Mo S2 and DOX were dissolved in deionized water under sonication.After 15 minutes,the solution was kept under vacuum condition until it was drained.The Dox@Mo S2 nanoparticles were obtained by washing with deionized water and centrifuging at 12000 rpm for 5 min.To evaluate the release rate at different p H values,the NPs were dispersed into the PBS solution with different p H values(5.5 and 7.2).The PBS solution with NPs were placed at 37 °C under constant shaking.The supernatant of the Dox@Mo S2 solution was obtained by centrifugation at different time points and another fresh PBS was added.A UV-visible spectrophotometer at 482 nm was used to measure the amount of DOX release at different time points.Then,we used a standard curve to calculate the total amount.Photothermal effect of DOX@Mo S2: To determine the photothermal effect of the DOX@Mo S2 under NIR irradiation,the samples with various DOX@Mo S2 concentrations were irradiated by an 808 nm laser for 5 min at 2.0 W cm-2.The temperature change was monitored by FLIR.The control group is pure water.Acute toxicity in vivo: To evaluate the toxicity of the as-made DOX@Mo S2 NPs in vivo,female mice were randomly allocated into four groups.These mice were injected with DOX@Mo S2 NPs at various doses of 50 to 200 mg Kg-1 via the tail vein and sacrificed after 20 days with mice treated with PBS as the control group.The blood parameters were collected for blood routine and blood biochemistry examination.Therapeutic experiment in vitro: To investigate the photothermo-chemotherapy effect in vitro,the MTT assay was used to detect the viability of Hep G-2 cells under various treatments.100 ?L Hep G-2 cells were incubated into 96-well plates at a density of 8 × 103 cells per well for 24 h.Then 100 ?L different materials that included Mo S2,free DOX and DOX@Mo S2 at the same DOX concentration(100 ?g m L-1)as same as Mo S2 without DOX were added into each well.We washed cells with PBS and added into fresh culture after incubation for 2h.Then an 808-nm NIR laser was used to irradiate the cells under different power densities(0.4,0.8,1.2 W cm2).Each well was added with 20 ?L MTT solution and the cells were cultured for another 4 h.Then we added 150 ?L dimethyl sulfoxide(DMSO)into each well and shook the 96-well plates for a while.A microplate reader was used to measure the absorbance at 492 nm.Photothermo-chemotherapy in vivo: H22-tumor-bearing mice were randomly allocated into the following four groups: saline,DOX,Mo S2+NIR,DOX@Mo S2 +NIR.The materials were injected into the mice via tail vein.The injection dose of Mo S2 and DOX@Mo S2 was 40 kg-1 and the DOX group is 10 mg Kg-1.The tumor of the mice in the NIR groups was irradiated by an 808 nm NIR laser(1.2W cm-2)for 3min at 12 h postinjection.The tumor and body weight were recorded everyday.The mice were sacrificed and regarded as state of “death”,when the size of the tumor was more than 20 mm in any one direction.In order to carry out histopathological analysis,tumor,liver,heart,lung,and kidney were collected and stained with H&E.Results:The LMHSs possess a spherical morphology according to the SEM image.The TEM image indicates hollow spherical structures and layered structures.The unique structures are extremely suitable for loading anticancer agents.We used a vacuum negative pressure loading method to load doxorubicin(DOX)into the LMHSs.The DOX loading capacity was 32.5%,which was calculated through a standard curve.The high loading capacity may be attributed to the unique structures of the LMHSs which provide the basis for further photothermo-chemotherapy.The samples with different DOX@Mo S2 concentrations were irradiated by an 808 nm laser to test the photothermal effect.The temperature increment showed obvious concentration dependence.The highest temperature was 59.7? at a concentration of 1 mg m L-1 after irradiation for 5 min.As we expected,the data showed that the nanomaterials possessed excellent photothermal effect which is suitable for tumor ablation.To investigate the release behavior in vitro,the NPs of DOX@Mo S2 were dispersed into the PBS solution with different p H values(5.0 and 7.4)at 37 oC.The DOX releasing rate reached 36.9% when the p H value was 5.0 after 24h,while 13.7% at p H of 7.4,which indicated that the DOX could be released more in tumor tissue due to its acidic microenvironment.In order to evaluate the toxicity and safe injection-dose of the as-made materials in vivo,the blood routine,including hemoglobin(HGB),platelet(PLT),mean capacity hemoglobin(MCH),mean corpuscular volume(MCV),mean corpuscular hemoglobin concentration(MCHC),red blood cells(RBC)and white blood cells(WBC),and the biochemistry,including aspertate aminotransferase(AST),alanine aminotransferase(ALT),urea(UREA),and creatinine(CREA)were evaluated.In addition,body weights of each group were carefully recorded.To our delight,even the 200 mg Kg-1 group did not show obvious abnormalities in the body weight and an upward trend can be found in each group,which indicated the very low toxicity of the NPs.We found no significant abnormalities in biochemistry and routine parameters except that ALT,AST,CREA,and WBC of 200 mg Kg-1 group were slightly higher than the control group but still maintained in normal range.Encouraged by the above findings,we further evaluate the photothermo-chemotherapy effect of the as-made DOX@Mo S2 in vitro.Cell viability of DOX@Mo S2+NIR group reduced to about 75%,50%,33%,and 27% at the power density of 0,0.4,0.8,and 1.2 W cm-2 respectively.Though the cells treated with Mo S2 showed the same trend,it obviously weaker than that of the DOX@Mo S2 +NIR group,which may be due to that some of the cancer cells are not sensitive to thermal ablation(Figure 4c).As we expected,laser irradiation exerted no significant influence on cell killing caused by free DOX: the cell viability was about 63%,44%,46%,and 42% at the power density of 0,0.4,0.8,and 1.2 W cm-2 and the findings were consistent with other scientists' conclusions.Inspired by the ideal therapy effect in vitro,we further explored the photothermo-chemotherapy effect of the as-made DOX@Mo S2 in vivo.The tumors treated with DOX@Mo S2 NPs under NIR irradiation were almost cured with no recurrence over 15 days.As we know,local recurrence is the main reason of thermal ablation failure in HCC,so the LMHSs provide a new approach for the treatment of HCC.The mean volume of tumor was 1325,736,and 248 mm3 in the control,DOX,and Mo S2+NIR groups respectively.Though the tumors in DOX or Mo S2+NIR group were significantly smaller than those in the control group,the treatment effect can not compare with that of the DOX@Mo S2+NIR group,which may be attributed to tumor cells heterogeneity-different cells from the same tumor have different sensitivities to a single treatment-and it may also explain the ideal treatment effect in the DOX@MoS2+NIR group.Conclusion: In summary,DOX@Mo S2 as photothermo-chemotherapy nanoplatforms has been constructed successfully through a simple method and it exhibits excellent treatment effect with no recurrence.The DOX@Mo S2 nanoplatforms have homogeneous morphology,ideal size,high drug-loading capacity,good biocompatibility,excellent photothermal effect and ideal therapeutic effect.It is the first time to synthetize LMHSs that can be used for intravenous administration.The unique structures of the NPs provide more space for drug loading.Under NIR laser irradiation,most of the tumor cells may be killed by the high temperature,and the residual tumor cells or those not sensitive to thermal ablation will be killed by the DOX that released at the tumor site.We found no recurrence or death in the DOX@Mo S2 group even on the 40 th day.The as-made DOX@Mo S2 NPs as photothermo-chemotherapy platforms not only possess high biocompatibility but exhibit remarkable synergistic anti-cancer effects both in vitro and in vivo.In addition,it can effectively inhibit tumor recurrence which is main reason of thermal ablation failure in HCC.These findings indicate that the as-made DOX@Mo S2 NPs are high potential for future clinical application. |
PDF Full Text Request