Multifunctional Dendritic Large Pore Mesoporous Silica Nanoparticles For Synergistic Effect Of Tumor Radiotherapy Sensitization And Photothermal Therapy

ObjectiveCancer is still one of the main causes of death,and it is significant to explore an appropriate way to kill tumors effectively.In this study,the multifunctional dendritic large pore mesoporous silica nanospheres(DLMSN)were designed as carriers,then copper sulfide(Cu S)nanoparticles were deposited in situ.As oxygen carriers,hemoglobin(Hb)were loaded on DLMSN at the same time.Finally,red blood cell membrane(RBCm)were wrapped to increase the stability and biocompatibility of the nanoparticles,which could avoid being recognized by the body’s immune system,thereby multifunctional nanosystems DLMSN/Cu S/Hb/RBCm were successfully constructed.Because Cu S nanoparticles have strong absorption in the near infrared region,so they can absorb appropriate near-infrared light bands and convert them into thermal energy.Besides,because of containing copper elements which belongs to high quality(Z)element,Cu S nanoparticles can enhance the absorption of radiation which could damage the DNA and other components of cells directly or indirectly.Therefore,the multifunctional nanosystems DLMSN/Cu S/Hb/RBCm which were used to combine with photothermal/radiation therapy(PTT-RT)would be a good way to kill tumors.ContentsThis study is mainly made of two parts:The first part was the synthesis and characterization of the DLMSN/Cu S/Hb/RBCm nanoparticles,including the reaction condition and optimization process of nanoparticles DLMSN/Cu S/Hb/RBCm,the characterizations of morphology,particle size,surface potential,optical properties,elemental composition,and stability of the nanoparticles from each step.Then the photothermal effect and radiosensitization of the DLMSN/Cu S/Hb/RBCm nanoparticles in the aqueous solution were investigated.The second part was to investigate the functions of the DLMSN/Cu S/Hb/RBCm nanoparticles at the cell level,including its ability to be taken up and kill tumor cells,so as to evaluate the cytotoxicity of multifunctional nanosystem DLMSN/Cu S/Hb/RBCm combined with PTT-RT.Methods1.The synthesis and characterization of DLMSN/Cu S/Hb/RBCm nanoparticles and their photothermal effect as well as radiosensitization effect in aqueous solution:A dual-templating method was used to synthesize the DLMSN as a blank carrier,and Cu S nanoparticles were deposited in situ in the surface and the mesopores of the DLMSNfollowed by loading the Hb and warping RBCm to construct the multifunctional DLMSN/Cu S/Hb/RBCm nanoparticles successfully.After that,the transmission electron microscopy(TEM)was used to observe the morphological characteristics of the nanoparticles.Ultraviolet-visible-near infrared(UV-vis-NIR)spectrophotometer was used to detect the optical properties.Dynamic light scattering(DLS)was used to investigate the particle size,polydispersity index(PDI)and surface potential.Energy dispersive X-ray spectroscopy(EDX)was used to analyze the elemental composition of DLMSN/Cu S/Hb/RBCm nanoparticles.Their optical properties and particle size in DDW or 10%fetal bovine(FBS)medium were detected through the UV-vis-NIR and DLS respectively for evaluation of the stability of the nanoparticles.The heating process of nanoparticles in aqueous solution under near infrared(NIR)light were then investigated by recording their heating images using an infrared thermal image,and analyzed statistically the video results with the software IRBIS3prof to explore the photothermal effect of the DLMSN/Cu S/Hb/RBCm nanoparticles under different laser powers and concentrations.The influences of multiple-times laser irradiation for DLMSN/Cu S/Hb/RBCm nanoparticles were investigated to assess its photothermal stability.Finally,the effect of their radiosensitization was evaluated by detecting the ROS level produced after laser irradiation with biological X-ray irradiator(voltage:160.0 k V,current:25.0 m A)using 9,10-anthracenediyl-bis(methylene)dimalonic acid bioreagent(ABDA)as ROS probe.2.The function of DLMSN/Cu S/Hb/RBCm nanoparticles at the cell level:The cytotoxicity of DLMSN/Cu S/Hb/RBCm to normal and tumor cells were investigated using mouse-derived breast tumor 4T1 cells and human-derived umbilical vein endothelial(HUVEC)cells as cell models;A flow cytometer was used to detect the cellular uptake of the DLMSN/Cu S/Hb/RBCm nanoparticles labeled with fluorescence.MTT assay was used to detect the ability of DLMSN/Cu S/Hb/RBCm nanoparticles killing 4T1 cells under different treatment(including PTT alone,RT alone and PTT-RT combined therapy)for evaluating the inhibition function of the multifunctional DLMSN/Cu S/Hb/RBCm nanoparticles on tumor cell growth under PTT-RT combined therapy.Results1.The multifunctional DLMSN/Cu S/Hb/RBCm nanoparticles were successfully synthesized by optimizing the reaction conditions.The results from TEM observation showed that the diameters of different nanoparticles were as follow,DLMSN(102±8 nm),DLMSN/Cu S(104±12 nm),DLMSN/Cu S/Hb(106±10 nm)and DLMSN/Cu S/Hb/RBCm(109±10 nm).The results of UV-vis-NIR characterization indicated that the characteristic absorption of Hb was at about 412nm,and that of Cu S nanoparticle was in the range of 800-1000 nm.The DLMSN/Cu S/Hb/RBCm nanoparticles had both of the characteristic peaks in the range of 412 nm and 800-1000 nm.The particle size and surface potential of each nanoparticle detected by the DLS were as follows,DLMSN(156±7 nm,-28.2±3.1m V),DLMSN/Cu S(168±16 nm,-25.6±3.3 m V),DLMSN/Cu S/Hb(174±5 nm,-15.6±0.7 m V),and DLMSN/Cu S/Hb/RBCm(175±1 nm,-23.9±0.3 m V).EDX spectrum analyzer showed the distribution of elements O,Si,S in the nanparticles.Under different laser powers(0.5,1,1.5 W/cm~2)for 10 min,the temperatures of the nanoparticles(200μg/m L)reached 45,65 and 80℃,respectively.The temperatures of DLMSN/Cu S/Hb/RBCm nanoparticlels solution increaseed with the increase of their concentration under NIR laser irradiation(1.5 W/cm~2,10 min),which indicated that the photothermal effect of the nanoparticles was concentration-dependent.As long as Cu S nanoparticles were contained in the nanoparticles(200μg/m L),the temperature could rise to about 65℃after NIR irradiation,which proved that the heating effect was mainly due to the photothermal conversion function of Cu S nanoparticles.After DLMSN/Cu S/Hb/RBCm nanoparticlels solution were irradiated multiple times under the same laser condition(1.0 W/cm~2,10 min),their photothermal conversion performance didn’t change,indicating their good photothermal stability.After DLMSN/Cu S/Hb/RBCm nanoparticles solutions with different concentrations were irradiated with different doses of X-ray,the level of ROS increased with the increases of concentration and radiation dose,which indicated that the radiation effect of DLMSN/Cu S/Hb/RBCm was concentration-and radiation dose-dependent.2.After DLMSN/Cu S/Hb/RBCm nanoparticles with concentration below 80μg/m L were incubated with 4T1 cells and HUVEC cells for 24 hours,the relative activities of the cells were all above 80%,indicating that the nanoparticles were non-toxic to the cells.The results of cellular uptake experiments showed that DLMSN/Cu S/Hb/RBCm nanoparticles could be taken up by 4T1 cells and increased with the increase of incubation time.Compared with single PTT or RT treatment,the multifunctional DLMSN/Cu S/Hb/RBCm nanoparticles(30μg/m L)combined PTT-RT(NIR laser:1.0 W/cm~2,5 min;X-ray irradiation:4 Gy)had the strongest killing ability to 4T1 cells,which can reduce the relative activity of the cells to about15%.ConclusionsIn this study,DLMSN were used as nanocarriers to deposit Cu S nanoparticles in situ,load Hb and followed by surface modification with RBCm.The results showed that the obtained multifunctional DLMSN/Cu S/Hb/RBCm nanoparticles had good stability and dispersivity.They showed the better cellular uptake ability and good biocompatibility from the results of experiments in vitro.Besides,they could damage cell DNA directly or indirectly to induce cell apoptosis or necrosis by combining heating and ROS induced by NIR laser and X-ray irradition respectively,owing to the good photothermal effect and radiosensitization effect of Cu S nanoparticles DLMSN/Cu S/Hb/RBCm nanoparticles were achieved to inhibit the growth of tumor cells by combining PTT-RT therapy.