| Human serum albumin(HSA)is the most abundant protein in serum,and it has a long half-life.HSA is one of the most important carriers for transportation of several materials with poor water solubility.Since the 1990s,conjugation with HSA was widely used for extending half-life of drugs.Evans Blue(EB)is a kind of azo dyes which can reversiblely and specifically bind with HSA with high binding affinity.It has been usaully used for detecting the leakage of the blood brain barrier.In the previous studies of our team,we modified the EB and conjugated with chelating agent,NOTA,wihch can be easily labeled with radionuclide,and constructed a novel Positron emission tomography(PET)tracer named NEB.Radionuclide as 18F labeled NEB can be applied for blood pool imaging,tumor imaging,evaluation of vascular permeability and sentinel lymph node imaging.In addition,NEB have advantage in diagnosis of some disease as hepatic hemangioma.In the further studies,we conjugated the molecular drugs with EB derivatives,which significantly extended the circulation time of the drugs.In this study,we further explored the application and development of EB derivatives.This study includes two parts.In the first part.wedeveloped a new targeting radiotherapy method for neuroendocrine tumors(NETS)using a EB derivative,NETs are a kind of malignant tumors that canoriginate in many organs and tissues.In the recent 30 years,as the incidence of NETs has been continuously increasing,more and more attention has been paid to NETs.One of the characteristics of NETs is high expression of somatostatin receptors(SSTR)on the surface of tumor cells.Thus,In the last 20 years,Somatostatin Analogs(SSA),the antagonist of SSTR,is one of the most important targeting and therapeutic drugs for NETs diagnosis and treatment.The most commonly used SSAs is Octreotide.Currently,the most important progress in NETs treatment is using 90Y or 177Lu labeled TATE for therapy,which is called Peptide-receptor radionuclide therapy(PRRT).In 2017,results of phase III clinical trial showed that 177Lu labeled TATE(177Lu-DOTATATE)significantly increased the therapeutic efficacy compared withTATE,Moreover177Lu-DOTATATE has been approved by FDA recently.However.since TATE is a small peptide that can be cleared very fast from kidney.thusthe circulation time for TATE is very short.In order to have a good therapeutic efficacy.increased dosage and repeated administration are required,which may induce transient severe renal toxicity after drug administration.Unfortunately,there is little breakthrough for PRRT treatment in rent years.In this study,we modified TATE with EB derivative and designed a novel compound,EB-TATE,the circulation time of which can be significantly extended based on the high binding affinity of EB and HSA.In addition.DOTA was conjugated with EB derivative,and enabled EB-TATE to be easily labeled with radionuclide such as 90Y and 177Lu for internal exposure therapy,and also.EB-TATE can be easily labeled with some positive electron imaging agents such as 64Cu and 68Ga for PET imaging.We used two types of cell lines,HCT116/SSTR2 and AR42J,with high expression of SSTR2 and one cell line,wide type HCT116,which do not express SSTR2 to fully evaluate the efficacy of EB-TATE.Results showed that chemical modification did not significantly influence the biological activity of TATE.EB-TATE have the similar high binding affinity with both SSTR2 and HSA.Results of PET/CT imaging showed that the circulation time of EB-TATE in the peripheral blood was significantly extended.The accumulation of 86Y-EB-TATE in the tumor tissues of HCT116/SSTR2 nude mice tumor model reached 34.7±7.4%ID/g 48 hours post administration,and the accumulation reached 65.8±6.2%ID/g in AR42J nude mice tumor model,significantly higher than the accumulation of 86Y-TATE.In addition,we evaluated the therapeutic efficacy of 90Y labeled EB-TATE.After administration,the HCT116/SSTR2 and AR42J nude mice tumor models were monitored 180 and 90 days,respectively.During the time of monitoring,tumor size of mice received EB-TATE treatment was significantly inhibited and the survival rate was 100%in the high-dose group.Thusthe therapeutic efficacy was significantly higher than 90Y-TATE.Our study revealed that using EB-TATE is a new effectively targeting therapeutic approach for tumors with high SSTR2 expression.EB-TATE showed high potetial for clinical translation in the furture.Furthermore,EB derivative is a platform that can be conjugated with other peptides or/and small molecule drugs to prolong their circulation time for treatment of other disease.In the second part of this study,we optimized and reconstructed theNEB,and we showed a novel new platform for small molecules delivery which based on EB derivative with significantly enhanced performance.Based on NEB,we assumed to acquire a new probe with longer circulation time,more tumor accumulation and more efficacy in lymphnode imaging.In this study,we designed and synthesized a new molecular probe N(EB)2 with two HSA binding motifs and a NOTA group,which can be easily labeled with radionuclide for PET imaging.For our hypothsis,N(EB)2 is able to bind two HSA at the same time to form a albumin-N(EB)2-albumin dimer.As enhancement of binding affinity and the size effect,N(EB)2 was supposed to show better performance in blood circulation time,lymphnode mapping and tumor imaging.Through molecular docking,transmission electron microscope(TEM),atomic force microscopy(AFM),High resolution liquid chromatography-mass spectrometry(LC-MS),the evidence indicated that N(EB)2 can bind two HSA molecular.Compared with 18F-AlF-NEB,18F-AlF-N(EB)2 showed a significantly longer circulation time.For lymphnode mapping,the N(EB)2 migrated slower in lymphatic vessels,whcih induced a delay from the sentinel lymph node(SLN)to the second lymph node,which is a preferable time-window for differentiating the SLN from the secondary lymph node.In addition,PET imaging and fluorescence imaging enabled N(EB)2 to be a multi-modal probe for lymphnode mapping.Furthermore,results in tumor bearing mice showed more accumulation of N(EB)2 than NEB.The N(EB)2 was proved to be an optimized tracer for lymphatic mapping and tumor imaging.It showed a high potential for clinical use.Furthermore,it may be served as a new attractive platform for long-acting drug delivery with high efficacy and biocompatibility. |
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