Nucleur-targeted Nanoparticles Combining Photothermal/photodynamic Therapy And Chemotherapy To Treat Breast Cancer

ObjectiveBreast cancer,as the most common malignant tumor in female patients,is seriously harmful to women health.At present,the commonly clinical treatment methods,such as surgery,radiotherapy,chemotherapy and immunotherapy have drawbacks.Therefore,it is urgent to develop new treatment methods to treat breast cancer.Phototherapy,mainly including photothermal therapy(PTT)and photodynamic therapy(PDT),has been widely investigated for cancer treatment.PTT and PDT have many advantages for breast cancer therapy,e.g.high spatial selectivity,non-invasiveness and low drug resistance.In this study,a heptamethine cyanine dye IR780 was used as a photosensitizer to be coupled with a cell penetrating peptide TAT,and the obtained TAT-IR780 conjugate showed tumor permeability and cell nuclear targeting performance.Then,TAT-IR780 was used as a carrier material to prepare nanoparticles for loading chemotherapeutic drug doxorubicin(DOX),and hence to construct a nano therapeutic system(TID nanoparticles)that can combine PTT/PDT and chemotherapy to treat breast cancer.TID nanoparticles can deliver IR780 and DOX to the deep part of tumor due to their high tumor permeability and nuclear targeting effect,and thus will be able to ablate the breast tumor through exerting PTT/PDT effects of IR780 and suppress the tumor recurrence by using the cytotoxicity of DOX.In addition,we also investigated the photothermal-responsive release of DOX from TID nanoparticles,the synergistic effects and related mechanisms of nuclear-targeting PTT/PDT combined with chemotherapy against breast cancer in this study.ContentsThis study has the following three parts.The first part is preparation and characterization of TID nanoparticles.The main contents include synthesis and chemical confirmation of TAT-IR780,preparation and characterization of TID nanoparticles,in vitro evaluation of drug release performance,of the photothermal properties and the optical stability of TID.The second part is the in vitro antitumor investigations of TID nanoparticles.The main contents include the evaluations of cytotoxicity,cellular uptake and intracellular localization,cell apoptosis,and synergistic effects of PTT/PDT combined with chemotherapy.The third part is the in vivo study of TID nanoparticles.The main contents include the construction of a breast cancer mouse model,the detection of in vivo PTT and PDT efficacies,and the evaluation of in vivo synergistic inhibitory effect of PTT/PDT and chemotherapy on breast cancerMethods1.Preparation and characterization of TID nanoparticles.TAT-IR780 was synthesized through nucleophilic substitution reaction and chemically characterized using Fourier transform infrared spectroscopy,nuclear magnetic resonance spectroscopy and inductively coupled plasma mass spectrometry.The optical property of TAT-IR780 was investigated by the ultraviolet spectroscopic spectroscopy(UV).TAT-IR780 was used as carrier material to prepare nanoparticles for loading DOX by using the nanoprecipitation method,thus obtained TID nanoparticles.The particle size,surface charged property and in vitro stability of TID nanoparticles were detected using the particle size and Zeta potential analyzer.The morphology of TID nanoparticles was observed by transmission electron microscopy(TEM).The optical stability of TID nanoparticles was evaluated by the UV and fluorescence spectroscopy(FL).The in vitro release characteristics of DOX from TID nanoparticles at different pH values with or without laser irradiation were examined using the dynamic dialysis.2.The in vitro antitumor investigations of the TID nanoparticles.The cytotoxicity of TID nanoparticles was assessed in 4T1 cells using the MTT assay and live/dead cell staining method.The cellular internalization and subcellular localization of TID nanoparticles in 4T1 cells was observed by the confocal microscopy,and the cell uptak was quantitatively evaluated by flow cytometry.The inducing effect of TID nanoparticles with laser irradiation on the apoptosis of 4T1 cells was assayed using the flow cytometry.To explore the activation mechanism of cell apoptosis,cytochrome c(Cyt c)in the treated cells was stained by immunofluorescence method and its subcellular localization was observed using the confocal microscopy.The temperature change of TID nanoparticle solution under laser irradiation was monitored by the thermal infrared imager,thus to evaluate the PTT efficacy of TID nanoparticles.To further investigate the nuclear-targeted PTT effect of TID nanoparticles in 4T1 cells,the cellular expression level of HSP60,and the expression level of γ-H2AX and distribution status of 53BP1 in the cell nuclei were detected by the immunofluorescence method.To evaluate the PDT efficacy of TID nanoparticles,DCFH-DA was used as a fluorescence probe to detect the intracellular generation of ROS in the treated 4T1 cells.3.The in vivo studies of TID.The 4T1 cells were injected into the mouse mammary fat pad to construct a mouse model of breast cancer.After intratumoral injection of TID nanoparticles,the temperature change at the tumor site was detected using the thermal infrared imager during laser irradiation,hence to evaluate the PTT efficacy of TID nanoparticles in vivo.At the same time,SOSG was used a fluorescent probe to detect the ROS generation level at the tumor tissue,thus to evaluate the PDT efficacy of TID nanoparticles.After intratumoral injection of TID nanoparticles and local laser irradiation,the tumor volume and the mouse body weight were continuously monitored for 20 d,and furthermore the tumor tissue was examined histophathologically by the H&E,Tunel,Ki67,and γ-H2AX staining after treatment,hence to assess the synergistic effect of TID nanoparticles against breast cancer through combining PTT/PDT and chemotherapy.Results1.A novel photosensitizer TAT-IR780 was successfully synthesized and chemically confirmed.TAT-IR780 had amphophilic property and could self-assemble to form TIR nanoparticles.Then,TAT-IR780 was used as carrier material to prepare TID nanoparticles loading with DOX.TID nanoparticles had a spherical shape and size of about 100 nm with a relatively narrow distribution.Compared to free IR780,TIR and TID nanoparticles exhibited obviously enhanced optical stability.Upon 10-minute laser irradiation(785 nm,1.0 W/cm2),the temperature of the TID nanoparticle solution rapidly rised to 60℃ and maintained for 6 minutes,indicating that TID nanoparticles had a high light-to-heat conversion efficiency.DOX exhibited pH-responsive release property from TID nanoparticles in vitro,and furthermore its release was significantly accelerated after laser irradiation.2.In breat cancer 4T1 cells,TID nanoparticles were efficiently uptaken by 4T1 cells after incubation for 6 h and mainly localized in the near nucleus through the mediation of TAT.After laser irradiation,the expression of HSP60 in 4T1 cells treated with TID nanoparticles was obviously up-regulated,and meanwhile the expression level of γ-H2AX and distribution status of 53BP1 in the nuclei both changed evidently.These results demonstrated that TID nanoparticles had strong PTT efficacy and this nuclear-targeted photothermal effect could induce the direct damage of DNA in the nuclei.At the same time,the intracellular generation of ROS was greatly enhanced,confirming the strong PDT efficacy of TID nanoparticles.In 4T1 cells,TID nanoparticles with laser irradiation notably inhibited the cell growth and induced the cell apoptosis,and showed significant synergistic antitumor effects of PTT/PDT and chemotherapy as compared to TIR nanoparticles and free DOX.Additionally,Cyt c was visibly transferred from the mitochondria to the cytoplasm,indicating the activation of mitochondria-related pathway was a main mechanism of cell apoptosis induced by TID nanoparticles-mediated PDT.3.A mouse model of breast cancer was successfully constructed through the inoculation of 4T1 cells.After intratumoral injection,TID nanoparticles penetrated deep into the tumor,and further combination with local laser irradiation inceased the tumor temperature to and maintained at about 60℃.These results verified the strong PTT efficacy of TID nanoparticles in vivo.At the same time,the generation level of ROS in the tumor tissue was remarkably enhanced,demonstrating that TID nanoparticles also possessed the strong PDT efficacy in vivo.In 4T1 tumor-bearing mice,TID nanoparticles with laser irradiation displayed significant synergistic antitumor effects and efficiently inhibited the tumor growth and recurrence.The results of H&E,Ki67,Tunel,and γ-H2AX staining after treatment showed that the cell damage and apoptosis occured in the tumor tissues.Furthermore,the mouse body weight and the histophathological characteristics of major organs were not evidently changed during the whole treatment period,suggesting that TID nanoparticles-mediated combination treatment had a high biosafety in vivoConclusionsIn this study,a nuclear-targeting therapeutic nanosystem(TID nanoparticles)containing both photosensitizer IR780 and chemotherapeutic durg DOX was designed for combination treatment of breast cancer.TID nanoparticles were prepared from TAT-IR780(a conjugate of IR780 with TAT)and DOX by using a nanoprecipitation method and exhibited very high stability and PTT/PDT efficiency in vitro.In breast cancer 4T1 cells,TID nanoparticles were efficiently uptaken and mainly enriched around the nuclei.By combining with laser irradiation,TID nanoparticles notably killed 4T1 cells and also significantly induced cell apoptosis.In 4T1 tumor-bearing mice,TID nanoparticles remarkably inhibited the tumor growth and recurrence,and also showed a relatively high biosafety.Altogether,TID nanoparticles can exert synergistic therapeutic effects against breast cancer through combining nuclear-targeted PTT/PDT and chemotherapy.