Long Circulating NIR-Ⅱ Molecular Fluorophores For Drug Delivery And Image-Guided Therapy

Nanocarriers hold great promise for drug delivery and cancer theranostics because of their prolonged blood circulation and high efficacy.However,the limited data on their in vivo pharmacokinetics and delivery process hamper their clinical applications.Owing to the dynamic real-time imaging characteristics,unparalleled temporal and spatial resolution and strong tissue penetration,second near-infrared window（1-1.7μm,NIR-Ⅱ）fluorescence imaging shows great advantages in tumor imaging and treatment.Herein,based on thienothiadiazole（TTD）organic molecule,a series of NIR-Ⅱ small-molecule fluorophores were synthesized by molecular structural regulation and Gaussian calculation.Furthermore,different modification methods were used to prepare self-assembled fluorescent probes with good water solubility,high stability and long circulation time in vivo.The optical properties,self-assembly ability,pharmacokinetics,biosafety,multiple injection routes,different tumor imaging,drug delivery,image-guide drug therapy and surgical navigation of these TTD-derived probes were explored.The chief study contents are divided into the three parts:Firstly,novel NIR-Ⅱ small-molecule fluorophores were developed.In this study,in view of the molecular structure characteristics of NIR-Ⅱ dyes with donor-acceptor-donor（D-A-D）scaffold such as CH1055,we screened the thiophenothiadiazole（TTD）structure with smaller molecular weight and easier synthetic routes as the electron acceptor of potential NIR-Ⅱ dyes.On this basis,different electron donors were incorporated,and thiophene or alkyl chain substituted thiophene were further introduced as electron bridging units.A series of TTD derived fluorophores HLAn（n=1-4）and L6 were synthesized by several chemical reactions,and their maximum emission wavelength were 967-1000 nm.Such TTD-derived NIR-Ⅱ molecular fluorophores not only showed good optical properties,such as excellent optical stability,high quantum yield and molar absorption coefficient,but also could be easily modified into multifunctional water-soluble biological probes.Secondly,NIR-Ⅱ dyes with long blood circulation time were constructed for accurate drug delivery and imaging guided tumor therapy.In this study,we prepared four self-assembled small-molecule NIR-Ⅱ fluorophores HLAnP（n=1-4）with high fluorescence quantum yields（QYs,0.15%–1.3%）,exceptional nonfouling performance and minimal immunogenicity.HLA4P exhibited ultralong blood half-life（～70 h）,long tumor residence time（>21 days）and high tumor accumulation（T/NT>25）through enhanced permeability and retention（EPR）effect,which could be used for tumor imaging by different administration routes.We prepared NR-II theranostic platform HLA4P-GMB by modifying dye HLA4 with gemcitabine（GMB）and PEG respectively through controllable condensation reaction.This theranostic platform showed good self-assembly and NIR-Ⅱ imaging performance.Because of its excellent imaging performance,HLA4P was further used as a drug carrier（～65%encapsulation efficiency）to improve the therapeutic effect of chemotherapeutics DOX,so as to achieve NIR-Ⅱ image-guided therapy in a long imaging window（>14 days）.These results collectively demonstrate HLA4P as a novel NIR-Ⅱ probe has a high potential for accurate drug delivery and image-guided therapy.Thirdly,self-assembled NIR-Ⅱ organic molecular probes with long circulation persistence were used for routine preoperative tumor evaluation and accurate intraoperative imaging-guided tumor resection.In this study,we developed TTD-derived fluorophores L6-PEG_nk（n=1,2,5）as new-generation NIR-Ⅱ probes with exceptional nonfouling performance and significantly high fluorescence QYs（1.0～1.5%）in water.Among them,L6-PEG_2khas demonstrated super-contrast ratio（T/NT>19）,ultralong blood circulation(t_1/2=59.5 h)and high tumor retention time（>20 days）in vivo.The self-assembling mechanism,binding affinities,various injection methods,immunocompatibility and pharmacokinetics were systematically studied.Most importantly,this super-contrast NIR-Ⅱ fluorophore L6-PEG_2k was successfully used for cancer imaging and NIR-Ⅱ image-guided surgery of subcutaneous or orthotopic U87MG tumor in the animal model via intraperitoneal injection with unprecedented high specificity,unparalleled spatiotemporal resolution,deeper tissue penetration and long surgery time window（48～144 h）.