Iron-Based Polymer Nanomaterials For Multimodal Combination Therapy Of Breast Cancer

Objective:Breast cancer has become the malignant tumors with the highest incidence rate in the world,and is also one of the leading causes of cancer death in women patients.In clinic,the breast cancer is mainly treated with surgery,chemotherapy,radiotherapy,targeted therapy or immuntherapy.Although the research of new drugs,new technologies and new treatments can improve the five-year survival period of breast cancer,the overall effective rate of treatment is low and the recurrence rate is high,leading to a high mortality rate of breast cancer.Therefore,it is very meaningful to find a new treatment strategy for breast cancer.Due to the special physical and chemical properties,Nanomaterials combining with tumor therapy,which can improve tumor treatment efficacy and enhance tumor prognosis,has become a current research hotspot in the field of tumor treatment.The nanomaterials formed by conjugated polymers not only have the common characteristics of nanomaterials,but also have some unique properties due to the presence of conjugated polymers,such as easy surface modification,controllable particle size,good biocomptibility and low toxicity side effects.Polymer nanomaterials,also named polymer dots(Pdots),have been widely used in the fields of tumor imaging and treatment.In this project,we aim to bulid a multi-functional nano-platform based on polymer nanomaterials.It is expected to be used in the multimodal combined therapy of photothermal,photodanamic,chemodynamic and starvation therapy for improving the treatment effect of breast cancer.Methods:Firstly,an iron loading polymer nanoparticle,named as Pdots@Fe,was synthesized.Based on Pdots@Fe,an iron-loaded polymer nanomaterial modified with glucose oxidase was synthesized and named as Pdots@Fe@GOx.Subsequently,the morphology and particle size of Pdots@Fe and Pdots@Fe@GOx were characterized by transmission electron microscopy and dynamic light scattering.Then,the spectral properties of two nanomaterials were tested using fluorescence and absorption spectrometers.The photothermal,photodynamic and catalytic properties in vitro of polyme nanoparticles were characterized by fluorescent dye.Furthermore,the cellular cytotoxicity and multi-mode therapeutic effect of Pdots@Fe and Pdots@Fe@GOx were evaluated using the CCK8 method.Besides,the uptake and ROS generation capabilities of MCF7 cell for Pdots@Fe and Pdots@Fe@GOx were verified by the fluorescence staining experiments.The main mechenisms of MCF-7 cell death induced by Pdots@Fe@GOx was preliminarily explored by lipid ROS staining,glutathione detection and western blotting.Finally,the MCF-7 breast cancer nude model was constructed and the transport pathway of Pdots@Fe@GOx in vivo was monitored by themal imaging experiments.Then,the therapeutic effect of Pdots@Fe and Pdots@Fe@GOx in vivo were explored through intratumoral injection.The biological safety of Pdots@Fe and Pdots@Fe@GOx were explored through H&E staining.Results:[Synthesis and Characterization of Pdots@Fe]Firstly,we synthesized the polyme nanoparticle with Fe2+ doping using nanoprecipitation method,named as Pdots@Fe.The transmission electron microscope images showed that Pdots@Fe@GOx exhibited a well-defined spherical shape without aggregation.The Zeta potential of Pdots@Fe was measured to be-24 mV,which was significantly higher than that of Pdots(-34 mV),indicating the success doping of Fe2+ in Pdots.The experimental results of absorption and fluorescence spectra showed that Pdots and Pdots@Fe have similar spectral properties,indicating that the doping of Fe2+ have little effect on the spectral properties of Pdots.All these results demonstrated that Fe2+ was successfully doped on Pdots without changing any photophysical properties of Pdots.The temperature change experiment of Pdots@Fe in vitro under near-infrared light irradiation confirmed that Pdots@Fe has a photothermal effect.Then,the good photostability of the Pdots@Fe was defined by the on/off irradiation cycles.The photothermal conversion efficiency of Pdots@Fe was calculaed to be 33.55%.Next,a hydroxyl radical specific fluorescent probe was selected as an indicator to verify the occurrence of Fenton reaction.In addition,the singlet oxygen fluorescent probe also verified the the photodynamic effect,and doping Fe2+ had no impact on the photodynamic therapy efficiency.[The MCF-7 cell therapeutic effects of Pdots@Fe in vitro]Before being used for tumor therapy,we explored the cytocompatibility of Pdots@Fe.Pdots have good biocompatibility and low cytotoxicity within a certain concentration range.Similarly,Pdots@Fe showed neglectable toxicity to the normol cells even at 50μg/mL,indicating that the Pdots@Fe have the excellent cellular biocompatibility in normal cells.In contrast,Pdots@Fe only had toxic effects to MCF-7 cells,and had neglectable cytotoxicity to normal cells.Confocal fluorescence imaging revealed that the Pdots@Fe entered the MCF-7 cells efficiently.After 8 hours,a large amount of Pdots@Fe had been uptake by the tumor cells and located in the cytoplasm.Next,we examined the in vitro cytotoxicity of Pdots and Pdots@Fe.Under NIR laser irradiation,Pdots can cause cell death through photodynamic therapy and photothermal therapy,while Pdots@Fe induced the excellent cell killing efficiency by the combine of photodynamic therapy,photothermal therapy and chemodynamic therapy.These results indicated that the combination of chemodynamic,photothermal and photodanamic therapy based on Pdots@Fe was more effective than single therapeutic mode based on Pdots.Furthermore,the ROS staining experiment confirmed that Pdots@Fe induced MCF-7 cell death through ROS generation.[Anti-tumor effects of Pdots@Fe in vivo]Next,we further explored the combined therapeutic effect of photodanamic,photothermal and chemodynamic therapy in a mouse model.Pdots@Fe could achieve a certain therapeutic effect through chemodynamic therapy without near-infrared laser irradiation.Furthermore,under the near-infrared laser irradiation,the breast cancer inhibitory effect of the Pdots@Fe treatment group was significantly enhanced.The weight statisics and corresponding digital photos of mouse tumors also showed similar results,indicating that Pdots@Fe can achieve certain tumor treatment effects through the combination of photodynamic,photothermal,and chemodynamic treatment strategies,but the breast cancer relapses with the treatment time proongs.[Synthesis and characterization of Pdots@Fe@GOx]In the above study,Pdots@Fe had achieved a certain tumor treatment effect in vivo and in vitro,but,the breast cancer treatment effect in vivo is lower than expected and there is the recurrence of breast cancer,indicating that there are still deficiencies in the treatment of breast cancer by Pdots@Fe.Therefore,based on Pdots@Fe,a new multifunctional polymer nanomaterial,named as Pdots@Fe@GOx,was synthesized to achieve better therapeutic effect and inhibit the recurrence of breast cancer.Moreover,the flow and distribution of Pdots@Fe@GOx in vivo was also observed and the specific mechanism of nanomaterials-induced breast cancer cell death was explored.The transmission electron microscope images showed that Pdots@Fe@GOx exhibited a well-defined spherical shape without aggregation and the diameters of Pdots@Fe@GOx were 100 nm.Similarly,the dynamic light scattering results showed that the hydrodynamic diameters of Pdots@Fe@GOx were about 130 nm,which complied with the trend of TEM results.Furthermore,the Zeta potentials of Pdots@Fe@GOx were increased,indicating that glucose oxidase(GOx)was successfully attached to Pdots@Fe.The absorption and fluorescence characteristics showed that doping Fe2+ and modification of GOx did not alter the optical properties of Pdots.Besides,the results of 3,3’,5,5’-tetramethylbenzidine showed that Pdots@Fe@GOx loading Fe2+ and GOx were successful and could play a good Fenton reaction effect under suitable conditions.Furthermore,the temperature change of Pdots@Fe@GOx with different concentrations and IR thermal images showed that Pdots@Fe@GOx had a good photothermal effect.The photothermal conversion efficiency of Pdots@Fe@GOx was calcutaed to be 28.23%,slightly lower than that of Pdots@Fe.The main resons is that the further loading of GOx has slightly affected for the photothermal performance of Pdots@Fe.The results for six laser on/off cycles demonstrated the Pdots@Fe@GOx,as same as Pdots@Fe,has excellent photostability.Besides,the fluorescence intensity of SOSG indicated that Pdots@Fe@GOx,as same as Pdots@Fe,has good photodynamic performance.[The MCF-7 cell therapeutic effects of Pdots@Fe@GOx in vitro]The cellular cytotoxicity showed that Pdots@Fe@GOx had a more substantial killing effect on cancer cells through intrinsic chemodynamic effect and starvation therapy,compared to Pdots@Fe.The co-localization experiment showed that Pdots@Fe@GOx could enter tumor cells to execute the therapeutic effects at 6 h.The relative viabilities of MCF-7 cells incubated with nanomaterials showed Pdots@Fe@GOx has a better tumor treatment effect than Pdots@Fe without NIR irradiation.Moreover,under NIR irradiation,the cell survival rate of the Pdots@Fe@GOx group was reduced to about 20%,demonstrated the synergistic effect by combining photoaymanic,photothermal,enhanced chemodynamic and starvation therapy.Furthermore,the live/dead staining and Annexin VFITC/propidium iodide flow cytometry showed a similar result.The above results indicated that Pdots@Fe@GOx had a better therapeutic effect.[The mechanism of MCF-7 cell death induced by Pdots@Fe@GOx]Next,we investigated the main mechanisms of Pdots@Fe@GOx mediated chemodynamic treatment leading to tumor cell death.The relative GSH levels in the Pdots@Fe or Pdots@Fe@GOx treated groups decreased gradually with the increase of nanomaterial concentration,indicating that the treatment of iron-loaded nanoparticles significantly reduced the content of GSH.GOx promoted the Fenton reaction and further decreased the GSH level.Futermore,western blotting analysis showed Pdots@Fe@GOx decreased the expression of GPX4.Besides,the lipid peroxidation staining showed the similar results.Finally,the cell viabilities of the tumor cells were investigated after treating the cells with ferroptosis inhibitors or iron chelators,followed by the treatment of Pdots@Fe or Pdots@Fe@GOx.The results showed that the co-culture of ferroptosis inhibitor or iron chelator significantly reduced the cell death rate,proving that ferroptosis was the major mechanism of the cell death caused by the Pdots@Fe@GOx.[Anti-tumor effects of Pdots@Fe@GOx in vivo]The therapeutic potential of Pdots@Fe@GOx was evaluated using the MCF-7 breast cancer mouse model.Firstly,the IR thermal images showed that Pdots was responsible for the PTT treatment and Fe doping and GOx modification had no effect on the photothermal effect.Furthermore,the time-dependent tumor growth curves showed that compared with Pdots@Fe,Pdots@Fe@GOx without NIR irradiation caused better therapeutic effect.Furthermore,under NIR irradiation,Pdots@Fe@GOx showed a better tumor treatment effect than all the above experimental groups and almost completely inhibited tumor recurrence.The results of tumor weight at 14 days also confirmed the above results.Therefore,these results demonstrated that Pdots@Fe@GOx has better therapeutic effect of breast cancer than Pdots@Fe,and can also inhibit tumor recurrence.Moreover,the results of body weight changes of mice during treatment and H&E staining of major organs revealed that Pdots@Fe@GOx had good biocompatibility in vivo and minor side effect.Therefore,the multimodal therapy by Pdots@Fe@GOx was promising to be used for cancer treatment.ConclusionThis subject synthesized an iron loaded polymer nanomaterial(Pdots@Fe)to treat breast cancer through the synergistic effect ofphotothermal,photodynamic and chemodynamic therapy.Then based on Pdots@Fe,GOx was further loaded to synthesize a multifunctional polymer nanomaterial(Pdots@Fe@GOx),which was used for the breast cancer combined treatment of photothermal,photodynamic,starvation and chemodynamic therapy.Compared with Pdots@Fe,this improved material Pdots@Fe@GOx achieved better breast cancer treatment effect.