An Experimental Study Of An Integrated Optical-magnetic Resonance Dual-modal Diagnosis And Treatment Probe Targeting EGFR In A Mouse Colorectal Cancer Model

Objective:Colorectal cancer(CRC)is the third most common cancer globally.Recently,the incidence and mortality of CRC is on the rise and it has become a serious threat to human health.The epidermal growth factor receptor(EGFR)activation and signal pathways play an important role in CRC.Nearly 80%of CRC patients have over expression of EGFR.In order to improve the level of diagnosis and treatment of CRC,we design and synthesis a new novel cerasome based optical and magnetic resonance double modal imaging probe which is EGFR-targeted and has photodynamic therapy function.Method:Synthesis of porphyrin-containing liposomal nanohybrid cerasomes decorated with cetuximab,an anti-EGFR antibody,and conjugated with near infrared fluorescence dye IRDye800CW and magnetic resonance imaging(MRI)contrast DOTA-Gd on the nanoparticle surface,to enable in vivo tumor detection and photodynamic therapy(PDT).Then,the particle size and Zeta potential of EGFR-CPIG and IgG-CPIG were monitored by 90Plus/bl-mas DLS analyzer.By transmission electron microscopy(TEM)to detect probe morphology analysis,use Varian 4000 ultraviolet-visible(ultraviolet and visible spectrum,UV-Vis spectrophotometer and thermoluminescence fluorescence spectrophotometer measured the UV/Vis absorption spectra and fluorescence spectrum(photoluminescence,PL).The formation of single-line oxygen(1O2)in EGFR-CPIG and IgG-CPIG was measured by using the disodium salinization detector(ADPA).The measurement of the performance of the probe optical-magnetic resonance imaging was carried out by using the small animal living fluorescence imager and the 7.0T high field strong MR imaging scanner.Secondly,CT26-fluc colorectal cancer cells were used as cell models to observe the effect of the nanometer probe photodynamic therapy on cell proliferation,and analyze the distribution and biosafety evaluation in vivo.Then,in Balb/c male mice subcutaneous inoculation CT26-fLuc cells to establish rat subcutaneous transplantation tumor model of CRC,using small animals living fluorescence imaging,high field strength of 7.0 TMR imaging scanner for small animals living optical,magnetic resonance imaging.Among them,12 mice were randomly divided into two groups:EGFR-CPIG probe group and IgG-CPIG probe group,and the mice were executed after 24 h and the tumor and main organs were taken out to observe the distribution of the body.Finally,72 Tumor-bearing mice were divided into nine treatment groups:(1)PBS,(2)PBS + laser irradiation,(3)IgG-CPIG,(4)EGFR-CPIG,(5)free PD-L1 mAb,(6)IgG-CPIG + laser irradiation,(7)EGFR-CPIG + laser irradiation,(8)IgG-CPIG + laser irradiation + PD-L1 mAb,(9)EGFR-CPIG + laser irradiation + PD-L1 mAb.The tumor growth was monitored by measuring both BLI light intensity and also by measuring tumor volume using digital calipers,mouse weight,tumor volume,BLI were detected every three days.After 21 days of treatment,blood routine analysis was performed,and then the mice were executed and the main organs were removed and stained,and the biosafety of the probe was observed in vivo.Results:The particle sizes of the two probes were about 90 nm,and the morphological rules were evenly distributed.Both probe particles are negatively charged,about-40 mV.The porphyrin package efficiency of both was more than 86%,both of which were about 3%.Both probes are showing IRDye800CW(780 nm)and porphyrin(maximum absorption of about 420 nm)of characteristic peak,which mean porphyrin and fluorescent dyes were successfully encapsulated in the silicon contents.The longitudinal relaxation rates of EGFR-CPIG and IgG-CPIG were 9 mM-1s-1 and 13 mM-1s-1 respectively,indicating a favorable T1 contrast effect for in vivo MRI imaging.The most significant TBR difference was found at 24 h,with approximately 2.1-fold higher signal observed for the EGFR-CPIG group compared to the IgG control group.The results showed that the relative signal enhancement at 24 h was approximately 1.71-fold higher for the EGFR-CPIG compared to the IgG-CPIG control group,the tumors in EGFR-CPIG + laser + PD-L1 mAb treatment group showed complete tumor eradication and no recurrence was found during subsequent 21-day observation,suggesting that EGFR-CPIG-targeted PDT plus PD-L1 mAb immunotherapy showed synergistic therapeutic effects,achieved superior therapeutic efficacy,and prevented tumor recurrences.The two probes were mainly distributed in the liver,with less distribution of heart,spleen,lung and kidney.Biosafety evaluation showed that the two probes did not have obvious toxic side effects and could be used in vivo studies.Conclusion:In conclusion,we successfully developed a NIRF/MRI dual-mode theranostic EGFR-CPIG,which could enable noninvasive and targeted in situ tumor imaging with high spatial information,excellent sensitivity,high T1 relaxivity,good fluorescence properties,and image-guided PDT.To the best of our knowledge,this is the first report of cerasomes targeted to EGFR as agents for dual modality imaging and PDT plus immunotherapy.The results of our study indicated that the EGFR-CPIG system preferentially accumulated at the tumor sites and exhibited high tumor targeting ability owing to the combination of EPR effect and the active tumor targeting ability of EGFR.Our results suggested that the liposomal nanohybrid cerasome nanoparticles possess significant potential for the dual-modality imaging-guided precise PDT of CRC,and that combined EGFR-targeted PDT and immunotherapy could achieve superior therapeutic efficacy without tumor recurrence.