Fluorescence/MR Dual-modal Imaging Studies Of Targeted CD44~+ Breast Cancer Stem Cells

Background:Studies have shown that tumor stem cells are the main factors of tumorigenesis,development,invasion,recurrence and drug resistance.If the tumor stem cells can be targeted or selectively killed,it can prevent and treat tumor recurrence and metastasis.In recent years,the successful isolation and identification of breast cancer stem cells(BCSCs)also provide hope for radical cure of breast cancer.However,in order to achieve the above therapeutic strategy,it is necessary to obtain the location,eontent and distribution of BCSCs in the tumor before treatment.Therefore,the sensitive,specific and safe synthesis of BCSCs probes and their in vivo imaging are the prerequisite and key to solve this problem.In this paper,a non-cadmium magnetic quantum dot nano probe targeting BCSCs was synthesized and dual-mode imaging of living fluorescence and MR was achieved.Objective:Synthesis of cadmium-free InP/ZnS@BSA-DTPAGd magnetic quantum dot nanoparticles,targeting monoclonal antibodies against CD44,a fluorescent/MR dual-modal molecular probe(pQDs-CD44mAb)targeting BCSC was prepared,physical and chemical characterization and biological safety assessment were carried out,and study its ability to target fluorescence and MR imaging of CD44+breast cancer stem cells in vitro and in vivo.Methods:1.Preparation and Characterization of InP/ZnS@BSA-DTPAGd ProbesUsing gas-liquid phase method,firstly,InP nucleus was synthesized with indium myristate and zinc phosphide as In precursor and phosphorus source,respectively.followed by coating the surface of the ZnS shell on its surface,the quantum dot of InP/ZnS core/shell structure was obtained by centrifugal precipitation.The BSA-DTPAGd was encapsulated on the surface of the quantum dots by phacoemulsification to form a water-soluble InP/ZnS@BSA-DTPAGd diphasic quantum dots.The main elements of the synthesized InP/ZnS@BSA-DTPAGd water-soluble quantum dots were detected by EDX energy dispersive spectroscopy.The morphology and particle size were determined by transmission electron microscopy(TEM)and particle size analyzer.The fluorescence stability was measured by fluorescence spectrophotometry.The imaging performance and relaxation performance were examined by nuclear magnetic resonance(MRI).2.Biosafety evaluation of InP/ZnS@BSA-DTPAGd probeCCK-8 method was used to detect the survival rate of breast cancer stem cells and DC 2.4 mouse fibroblasts after being incubated with the probe for 24 h,the.influence of the main components(heart,liver,spleen,lung,kidney,intestine)and blood main components in mice were detected by tail vein injection of the probe for 7 days and 30 days.3.In vivo and in vitro imaging studies of InP/ZnS@BSA-DTPAGd-CD44 mAb dual mode probesAfter the synthetic InP/ZnS@BSA-DTPAGd probe surface was covalently modified with CD44 monoclonal antibody,the pQDs-CD44mAb was incubated with the BCSCs of phenotype CD44+,the imaging performance of the cells was detected by laser confocal microscopy and nuclear magnetic resonance(MRI).Intratumoral injection of probes in nude mice bearing a phenotype of CD44+ breast cancer,the imaging ability in vivo was measured by small animal fluorescence and MRI imaging instruments.Results:1.Preparation and Characterization of InP/ZnS@BSA-DTPAGd ProbesThe InP/ZnS@BSA-DTPAGd dual-mode probe was successfully prepared.EDX spectrum analyzer shows that the synthesized magnetic quantum point is composed of In,P,Zn,S,Gd and other elements.The average particle size of the synthesized InP/ZnS@BSA-DTPAGd quantum dots was about 68 nm,the morphology was spherical and the dispersion was uniform and stable.The emission wavelength was about 655 nm by fluorescence spectrophotometer.In vitro MR imaging showed that the intensity of T1-weighted signal increased with the increase of Gd3+concentration.Relaxation efficiency r1 and r2 values were 12.38 mM-1s-1,18.17 mM-1s-1,and the relaxation rate r2/r1 was 1.468.2.Biosafety evaluation of InP/ZnS@BSA-DTPAGd probesThe results of CCK-8 cell viability test showed that the toxicity of the probe decreased with the decrease of probe concentration.The survival rates of BCSC and DC 2.4 cells were still above 85%and 90%,respectively,when the InP/ZnS@BSA-DTPAGd probe reached a relatively high concentration(0.2 ?g/ml).Compared with the control group.ICR mice were injected with InP/ZnS@BSA-DTPAGd probe,and the physiological indexes and the stained sections of the main organs were also within the normal range.3.In vivo and in vitro imaging studies of InP/ZnS@BSA-DTPAGd-CD44 mAb dual-mode probesBCSCs with CD44+were incubated with pQDs-CD44 mAb,non-targeted pQDs and blocked by CD44 antibody for 30 min and then with pQDs-CD44 mAb,laser confocal microscopy showed that pQDs-CD44 mAb showed specific aggregation on the surface of the cell membrane,pQDs show no obvious specific absorption on its surface,there was no obvious fluorescence signal on the cell surface of CD44 antibody blocking group.MR imaging showed that the BCSC incubated by the targeting probe exhibited higher T1 signal intensity compared to non-targeted probes.The in vivo injection of InP/ZnS@BSA-DTPAGd-CD44 mAb dual-mode probe was performed in vitro and the effect of real-time fluorescence imaging and T1-weighted MR imaging was significant.Conclusion:The prepared InP/ZnS@BSA-DTPAGd bimodal probes have excellent optical properties and relaxation properties.Targeted labeled dual mode probe InP/ZnS@BSA-DTPAGd-CD44mAb probe can recognize CD44+breast cancer stem cells in vivo and in vivo and can achieve fluorescence and MR dual mode imaging.