The Applications Of Cancer Cell Membrane-coated Nanoprobes In Triple-negative Breast Cancer Molecular Imaging Diagnosis And Therapy

Objective: Triple-negative breast cancer(TNBC)is a subtype of breast cancer with a high rate of metastasis and poor prognosis.The diagnosis of TNBC remains challenging in clinic.To achieve the aim of specifically targeted diagnosis and therapy of TNBC,in this dissertation study,we built the cancer cell membrane-coated upconversion nanoparticles(CCm₂₃₁-UCNPs).By using the homologous targeting and immune evasion capabilities of cancer cell membranes,the nanoprobe can specifically target TNBC xenograft models,which could help to diagnose TNBC through a non-invasive molecular imaging method.Methods: Hypotonic lysis and differential centrifugation were used to obtain pure cancer cell membranes of MDA-MB-231 cells.Cancer cell membranes were coated on the upconversion nanoparticles(UCNPs)by physical extrusion to get the CCm₂₃₁-UCNPs.Characterization was measured by the dynamic light scattering(DLS)and transmission electron microscopy(TEM).The protein expression before and after the cancer cell membrane coating was confirmed by the SDS-PAGE and Western blot.CCK-8 assay was used to evaluate the cytotoxicity.The CCm₂₃₁-UCNPs were incubated with cells and the homologous targeting and immune evasion abilities were observed by the confocal laser scanning microscope(CLSM).CCm₂₃₁-UCNPs were injected into MDA-MB-231 and MCF-7 mice xenografts via tail veins for UCL and MRI,respectively.PET imaging was performed by pre-targeting technology and click chemistry reaction between the azido and DBCO.Azido was inserted into cancer cell membranes to build the N₃-DSPE-PEG-CCm₂₃₁-UCNPs and DBCO was labeled with 18 F.N₃-DSPE-PEG-CCm₂₃₁-UCNPs were administrated into mice 24 h before PET imaging.In the biodistribution study,?-counter was used to measure the radioactivity of 18 F and the inductively coupled plasma-atomic emission spectroscopy(ICP-AES)was used to measure the distribution of Gd³⁺,respectively.Biotoxicity of CCm₂₃₁-UCNPs was evaluated on mice for 30 days.Results: The hydrodynamic size of the CCm₂₃₁-UCNPs was 190.47 ± 6.67 nm.The core-shell structure was shown in the TEM.Similar protein profiles before and after the cancer cell membrane coating were shown in the SDS-PAGE and Western blot.No obvious cytotoxicity was observed.The uptake of CCm₂₃₁-UCNPs in the MDA-MB-231 group was extremely higher than that of other cell groups,and the Raw 264.7 cell,a kind of murine macrophages,had nearly no uptake of CCm₂₃₁-UCNPs,which exhibited the homologous targeting and immune evasion abilities of the CCm₂₃₁-UCNPs in vitro.The best time window for the tumor imaging was at 24 h post-injection determined by the UCL imaging results.For the UCL,MRI,and PET imaging,tumor uptake of the CCm₂₃₁-UCNPs group was the highest among all the groups.The MDA-MB-231 xenograft models showed the higher tumor uptake than that of the MCF-7 xenograft models.Lower fluorescence signal of liver was observed in the CCm₂₃₁-UCNPs group than that of the UCNPs group.For the 18 F biodistribution,the MDA-MB-231 xenograft models showed significantly higher tumor uptake(4.05±0.5 %ID/g at 4 h post-injection)than that of the MCF-7 xenograft models(1.20±0.14 %ID/g at 4 h post-injection,P < 0.001).For the Gd³⁺ biodistribution,the liver and spleen uptake of the UCNPs group was 1.82-fold and 1.79-fold,respectively,higher than that of the CCm₂₃₁-UCNPs group.No obvious biotoxicity was observed in mice of the injection of CCm₂₃₁-UCNPs till 30 days.Conclusion: In our study,we successfully built a cancer cell membrane-coated upconversion nanoprobe,CCm₂₃₁-UCNPs.The in vitro and in vivo experiments both confirmed the homologous targeting and immune evasion abilities of the CCm₂₃₁-UCNPs.We used the CCm₂₃₁-UCNPs to perform UCL/MRI/PET multi-modality imaging and differentiated MDA-MB-231 xenograft models through the in vivo tri-modality imaging.We used the molecular imaging technology and combined the biomimetic medicine with nanomedicine to perform the non-invasive,multi-modality,and specific diagnosis to TNBC xenograft models,which provides a new strategy for TNBC diagnosis.Objective: Triple-negative breast cancer(TNBC)patients are often not benefit from the ER and PR targeted endocrinotherapy and the HER2 targeted therapy due to the absence of the ER,PR,and HER2 expression.Chemotherapy is the mostly used treatment of TNBC in the clinic,but the drug resistance extremely limits the therapeutic efficacy.To achieve the aim of specifically targeted treatment of TNBC,in our study,we built the cancer cell membrane-coated oxygen delivery nanoprobe,CCm-HSA-ICG-PFTBA,which can improve the hypoxia at tumor sites and enhance the therapeutic efficacy of the photodynamic therapy(PDT),to relief the tumor growth in TNBC xenograft models.Methods: PFTBA was uniformly mixed with HSA and ICG by sonication to obtain the HSA-ICG-PFTBA.Cancer cell membranes were coated onto HSA-ICG-PFTBA to build the CCm-HSA-ICG-PFTBA.The fluorescence characterization was determined by the UV-vis spectrometry.With the near-infrared(NIR)808 nm laser irradiation,1O2 and ROS were detected in vitro by indicators.In vivo fluorescence imaging was performed to determine the time window of CCm-HSA-ICG-PFTBA at the tumor site.The tumor hypoxia level was monitored by both in vivo ¹⁸F-FMISO PET/CT imaging and ex vivo immunofluorescence staining.PDT was conducted with NIR at 24 h post-injection of the CCm-HSA-ICG-PFTBA into the 4T1 xenograft mice models.The living status of the mice was observed every day,the body weight,the length and width of the tumor were measured.On the 14 th day after the PDT,the mice were euthanized and tumors were weighed,the blood and major organs were obtained for evaluating systematic toxicity.Results: The size of the CCm-HSA-ICG-PFTBA was 131.3 ± 1.08 nm.Similar UV peaks were shown between the CCm-HSA-ICG-PFTBA and ICG.The fluorescence property was stable till 60 h in the dark condition.For in vitro 1O2 detection,the fluorescence intensity of the CCm-HSA-ICG-PFTBA group showed the fastest increase,which was significantly higher than that of the other groups(P < 0.001).For the in vitro ROS measurement,the CCm-HSA-ICG-PFTBA group showed the strongest green fluorescence,which reflected the highest ROS concentration.In vivo fluorescence imaging revealed that the best time window was at 24 h post-injection of the CCm-HSA-ICG-PFTBA.Both in vivo ¹⁸F-FMISO PET imaging and ex vivo immunofluorescence staining results exhibited that the tumor hypoxia was significantly improved at 24 h post-injection of the CCm-HSA-ICG-PFTBA.For in vivo PDT treatment,the tumor volume and weight of the CCm-HSA-ICG-PFTBA with NIR group were both the smallest among all the groups and significantly decreased compared to the untreated group(P < 0.01).No obvious biotoxicity was observed by the injection of CCm-HSA-ICG-PFTBA till 14 days.Conclusion: In our study,we successfully built the cancer cell membrane-coated oxygen delivery nanoprobe CCm-HSA-ICG-PFTBA.With the homologous targeting and immune evasion abilities of the cancer cell membranes,the CCm-HSA-ICG-PFTBA nanoprobe can improve the hypoxia at tumor sites,enhance the PDT efficacy,and relief the tumor growth.The CCm-HSA-ICG-PFTBA was with good biocompatibility.Also,the HSA,ICG,and PFTBA are all FDA-approved materials,which enhance the potential for clinical translation.We combined the biomimetic medicine with nanomedicine to perform the specific targeting treatment to TNBC xenograft models,which provides a new strategy to overcome the drug resistance in the TNBC treatment and a new choice for the combination therapy of TNBC.