Preparation Of Novel Lipid Vesicles And Its Experimental Study On Triple Negative Breast Cancer

Background:Currently,breast cancer is the most common malignant tumor in the world.Triple negative breast cancer(TNBC)is the most aggressive pathological type of breast cancer,accounting for 15%-20%of breast cancer.Due to the lack of estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER-2),it is not sensitive to endocrine therapy or HER-2 treatment,and lack of standardized treatment plan.At present,neoadjuvant chemotherapy(NAC)is the main treatment for TNBC.However,the overall survival rate of TNBC patients is still low during NAC treatment,and it’s also prone to chemoresistance.It is urgent to find new therapies to broaden the choice of TNBC treatment.Photodynamic therapy(PDT)is a novel non-invasive clinical treatment,which has been approved for clinical application by Food and Drug Administration(FDA)in different countries.Therefore,PDT is expected to become a new treatment option for TNBC patients.PDT can kill tumor cells by producing reactive oxygen species(ROS).However,its disadvantage is that it is easily affected by the oxygen concentration in the tumor tissue,that is,insufficient ROS can be generated in the hypoxic area,which will reduce the efficacy.To solve this bottleneck problem,we believe that the synergistic effect of drugs and PDT can make up for the poor efficacy of PDT in tumor hypoxic area.We assumed that hypoxic-activating drug TH302 and PDT can be applied at the same time,and TH302 can be activated by hypoxia conditions and produce cytotoxic properties to improve the efficacy of hypoxic areas in tumor tissue.Therefore,the first part of this study mainly involves the creation of PDT combined with TH302 and observation of its therapeutic effect on TNBC.We use chitosan oligosaccharide(CO)modified liposomes containing the selected photosensitises HPPH and TH302 and target TNBC.Improve drug delivery efficiency and therapeutic effect.In addition,while PDT produces ROS to kill tumor cells efficiently,it can also improve tumor immunogenicity by inducing immunogenic cell death(ICD).ICD induced by PDT can improve the immunogenicity of TNBC and change TNBC from "cold tumor" to "hot tumor".However,even if the immunogenicity of TNBC is improved,the inhibitory immune cells in the tumor immune microenvironment of TNBC will still hinder the immune system from killing the tumor.What’s more,small extracellular vesicles(sEVs)derived from TNBC will stimulate the activation and expansion of inhibitory immune cells.In view of this treatment bottleneck,we believe that PDT can induce ICD in TNBC and reduce sEVs secreted by TNBC cells and stimulated sEVs produced by PDT,which can improve TNBC tumor immune microenvironment and enhance antitumor effect.We hypothesized that GW4869,a neutral sphinolipase inhibitor,could inhibit the secretion of sEVs from TNBC and enhance the antitumor immune response by synergistic effect with PDT inducing ICD in vivo.Therefore,the second part of this paper mainly involved the creation of PDT combined with GW4869 and observed its efficacy on TNBC.We used primary BALB/c mouse bone marrow mesenchymal stem cells(BMSCs),sEVs derived from BMSCs were used as delivery carriers to improve the delivery efficiency and therapeutic effect of Ce6 and GW4869 in the tumor region.Objective:Part Ⅰ:Prepared novel photosensitive liposome CO-HPPH-TH302/Lipo,to identify the TNBC cells targeting ability of the CO-modified liposome in vitro and in vivo,to confirm the antitumor effect of the novel liposome CO-HPPH-TH302/Lipo on TNBC in vitro and in vivo,and to provide new strategies and measures for the treatment of TNBC.Part Ⅱ:Prepared immunomodulatory photosensitive nanovesicle Ce6-GW4869/sEVs.To verify the TNBC cell targeting ability of Ce6-GW4869/sEVs and improve the TNBC immune microenvironment,and further provide a new strategy and a new approach for TNBC treatment.Methods:Part Ⅰ:The liposome CO-HPPH-TH302/Lipo was prepared by "film hydration method" and"post insertion method".The prepared liposomes were characterized by several ways:(1)the hydrated particle size and Zeta potential of liposomes were detected by Zeta-plus particle size analyzer and potential detector,and the stability of CO-HPPH-TH302/Lipo was also evaluated;(2)the morphology of liposomes was observed by TEM;(3)the UV-visible absorption spectrum of liposomes was detected by UV-visible spectrophotometer and the drug encapsulation rate of both drugs were simultaneously evaluated;(4)DSC was used to detect the phase transition temperature of liposomes.The amount of ROS produced by liposomes was detected by SOSG kit and ESR spectroscopy.ROS detection kit(DCFH-DA probe)detected the ROS production by liposomes in vitro.CCK-8 kit was used to detect the IC50 of PDT and TH302 to provide a basis for subsequent treatment in vitro.In vitro experiments,the targeting ability of liposomes to TNBC was detected by laser confocal microscopy and flow cytometry in vitro;in addition,CCK-8 kit was used to evaluate the proliferation and toxicity effects of CO-HPPH-TH302/Lipo in TNBC cells.In vivo experiment,based on the construction of TNBC orthotopic tumor model,the targeting ability of TNBC was evaluated by optical imaging system.What’s more,the status and body weight of nude mice were observed to evaluate the biosafety of liposomes in vivo.Liposomes were injected into tumor-bearing nude mice by tail veins,and continuous observed the changes of tumor volume.15 days after treatment,removed the tumor tissue and vital organs(heart,liver,spleen,lung,kidney)for HE staining to evaluate the therapeutic effect and toxicity of liposomes.Part Ⅱ:The primary BMSCs were observed and identified by optical microscope,flow cytometric assay and adipogenic/osteogenic differentiation experiment;preparation of immunomodulatory photosensitive nanovesicle Ce6-GW4869/sEVs by electroporation.The particle concentration,average particle size and Zeta potential of Ce6-GW4869/sEVs were detected by nanoparticle tracking analysis(NTA);the protein concentration of Ce6-GW4869/sEVs was detected by BCA kit,the morphology of Ce6-GW4869/sEVs was observed by TEM,the expression of membrane proteins on the surface of Ce6-GW4869/sEVs were detected by Western blotting,the UVvis absorption spectrum of Ce6-GW4869/sEVs was detected by UV-vis spectrophotometer,and the drug encapsulation rate was also evaluated.SOSG kit and ROS kit(DCFH-DA probe)were used to detect the ROS production by Ce6-GW4869/sEVs,CCK-8 assay kit was used to detect the IC50 of Ce6 and safe concentration of GW4869 in vitro,and NTA was used to detect the optimal sEVs inhibitory concentration of GW4869 in vitro,to explore the optimal concentrations of Ce6 and GW4869 in vitro.Then,CCK-8 assay kit was used to evaluate the therapeutic effect of Ce6GW4869/sEVs in vitro.Flow cytometric assay,cellular immunofluorescence assay,ELISA assay and chemiluminescence assay were used to verify the ICD induction ability of Ce6-GW4869/sEVs in vitro.What’s more,the TNBC targeting ability of sEVs was evaluated by fluorescence imaging and frozen section of tumor tissue in vivo;the ICD induction ability of Ce6-GW4869/sEVs was verified by tissue immunofluorescence assay in vivo.The percentage of CD8+T cells in spleen and the level of proinflammatory cytokines IFN-γ and TNF-α in serum were detected by flow cytometry and ELISA,respectively;to evaluate the anti-tumor immune response.The therapeutic effect of Ce6GW4869/sEVs was evaluated by observing the changes in tumor volume after treatment in vivo,HE staining of tumor tissues and the survival time of mice.The toxicity of Ce6-GW4869/sEVs were evaluated by HE staining pathological changes of vital organs(heart,liver,spleen,lung,kidney)and blood biochemical indexes after treatment in vivo.Results:Part Ⅰ:The morphology of novel liposome CO-HPPH-TH302/Lipo was quasi-circular;the hydrated particle size was 128.7±75.0 nm;Zeta potential was 29.97±3.5 mV;the encapsulation rates of HPPH and TH302 were 50%and 82%,and the phase transition temperatures of CO modified liposomes and unmodified CO liposomes were 49.3℃ and 43.5℃,respectively.The optimal therapeutic concentrations of HPPH and TH302 were 25 mg/L and 100 mg/L,which were determined by IC50 in vitro.Targeting experiment showed that CO-HPPH-TH302/Lipo could enter TNBC cells more than non-TNBC cells in vitro.What’s more,In vivo targeting experiment found that compared with the liposomes without CO modification,the aggregation of CO modified liposomes in the tumor area reached the peak at 2 h and lasted to 8 h.In vitro treatment experiment,the survival rates of TNBC cells in PDT group and TH302 group were 30.20±6.87%and 74.48± 1.63%,respectively,and the survival rate of TNBC cells in HPPH-TH302/Lipo group was 15.75 ± 2.99%.The survival rates of TNBC cells and non-TNBC cells in CO-HPPH-TH302/Lipo group were 11.08±0.79%and 13.26±2.00%,respectively.The toxicity evaluation experiments showed that CO-HPPH-TH302/Lipo had no pathological damage to vital organs in vivo.Therapeutic experiment showed that the volume of tumor in the CO-HPPH-TH302/Lipo group was reduced to 0.17±0.04 times of the volume before treatment.Part Ⅱ:The morphology of primary BMSCs was long spindle-shaped and arranged in parallel and vortex shape under optical microscope,the Sca-1 and CD44 were overexpressed on the surface of cells and CD45,CDllb and CD31 were under-expressed on the surface of cells,which could induce adipogenic/osteogenic differentiation.Subsequently,sEVs from BMSCs were obtained and immunomodulatory photosensitive nanovesicles Ce6-GW4869/sEVs were prepared by electroporation.Ce6-GW4869/sEVs were typical tea saucer-like bilayer vesicles under TEM.The particle size distribution of Ce6-GW4869/sEVs was 132.1 ± 115.9 nm,the particle concentration was 3.2×109 particles/mL,and Zeta potential was-22.85±0.41 mV;in addition,the encapsulation rates of Ce6 and GW4869 were 46%and 20%respectively.Then,the optimal therapeutic concentration of Ce6 by IC50 was 15 mg/L and the optimal therapeutic concentration of GW4869 was 5 μM in vitro.It was found that CE6-GW4869/sEVs could lead to CRT protein eversion and release of HMGB1 and ATP in TNBC in vitro,and it could reduce the survival rate of tumor cells to 50.54±9.70%in vitro.In vivo experiments,Ce6-GW4869/sEVs showed good biosafety and can target to tumor regions within 1 h with low toxicity to vital organs in vivo.In the meanwhile,Ce6-GW4869/sEVs could lead to CRT protein eversion and release of HMGB1 in TNBC in vivo,and also demonstrated that Ce6-GW4869/sEVs could inhibit the activation and proliferation of immunosuppressive cells by flow cytometry.The infiltration ratio of Tregs cells and MDSCs in tumor tissues were decreased by 52.53%(P<0.05)and 45.76%(P<0.01)of saline group,respectively.Meanwhile,the infiltration of CD8+T cells in spleen and tumor tissues were increased by 1.58 times(P<0.001)and 2.53 times(P<0.001)of saline group,respectively,which also led to the increase of IFN-y and TNF-α levels by 4.99 times(P<0.001)and 3.32 times(P<0.001)of saline group in serum,respectively.In addition,Ce6-GW4869/sEVs can reduce the tumor volume to 0.34±0.08 times of the original volume in vivo.After treatment,there was no obvious pathological damage in the vital organs of mice by HE stainingConclusion:Part Ⅰ:A novel photosensitive liposome CO-HPPH-TH302/Lipo was prepared,which can passively and actively target TNBC cells by EPR effect and CO targeting CD44.It can inhibit the growth of TNBC cells in vitro and in vivo with good biosafety.Part Ⅱ:On the basis of obtaining primary BALB/c mice BMSCs and BMSCs derived sEVs,a novel immunoregulatory photosensitive nanovesicle Ce6-GW4869/sEVs with good biosafety was prepared,which could induce ICD in vitro and in vivo.Tumor cell growth was inhibited by PDT and ICD induced by PDT combined with GW4869 can reverse tumor immunosuppressive microenvironment and improve anti-tumor immune response.