Second Near-infrared Fluorescent Probe For Imaging-guided Photothermal Immunotherapy Of Glioblastoma

Glioblastoma(GBM)is one of the most aggressive intracranial tumors,which displays high rates of morbidity,disability and recurrence.Nowadays,surgical resection is the most direct method for the treatment of GBM.However,complete resection of the tiny focal tumors is challenged by high infiltration and invasive growth of the GBM tumors,which accounts for post-operative recurrence.The main means of adjuvant postoperative treatment are radiotherapy and chemotherapy which caused great systemic toxic and drug resistance quickly,which leaded to the severe limitation of GBM therapy.Immunotherapy has been widely used in cancer treatment by stimulating strong anti-tumor immune response.However,there were problems such as insufficient infiltration of T lymphocytes and immune escape in GBM tissue,which made the corresponding immunotherapy develop rapidly.The blood-brain barrier(BBB)in the central nervous system prevented drugs from entering GBM.In order to increase the drug dose at the tumor site,researchers modified the nano delivery drug system(NDDS)with different ligands and designed it as active-targeting nanoparticles,while reducing the accumulation in normal tissues.In recent years,second near-infrared(NIR-II)fluorophores have attracted much attention due to weak spontaneous fluorescence and deeper tissue imaging ability in vivo imaging,and remarkable antitumor effect in tumor treatment because of its excellent photothermal ability.Based on the above research background,we synthesized a NIR-Ⅱ fluorophore MRP bearing donor-acceptor-donor(D-A-D)backbone and small molecule inhibitor JPC with reduction response.Then,the prodrug nanoparticles were prepared by self-assembling MRP with a prodrug of JPC,T7 ligand-modified DSPE-PEG5 k and lecithin.T7 peptide could bind to transferrin(Tf)receptor on brain capillary endothelial cells and glioma cells to pass through the BBB for tumor-targeted delivery of JPC and MRP.Finally,we studied the performance of prodrug nanoparticles in physical and chemical characterization,in vivo imaging and photothermal immunotherapy of GBM at the cellular and in vivo levels.The contents of this paper are as follows:Chapter 1.IntroductionIn the chapter,we firstly described the development prospect and application status of NIR-II fluorophore,and then we clarified the limitations of GBM therapy due to its obstruction caused by BBB and diffuse tumor cells growth.At present,according to these limitations,NDDS has been developed rapidly to overcome the BBB for GBM therapy.Then,the immune microenvironment of GBM was briefly introduced.Finally,the purpose,significance and innovation of this paper were analyzed.Chapter 2.Preparation and characterization of prodrug nanoparticlesIn the chapter,we synthesized prodrug nanoparticles TNP@JQ1/MRP by a method derived from nanoprecipitation,in which the nanoparticles had the dual targeting properties attributed to T7 peptide and the ability of stimuli(glutathione(GSH)and PTT)-responsive JQ1 release.First of all,a prodrug named as JPC was synthesized by conjugating JQ1 with P-lyso PC with a disulfide spacer.Then MRP fluorophore was prepared by reacting methylglyoxyl with thiadiazole-fused o-phenylenediamine through Maillard-like reaction,which had high fluorescence quantum yield and photothermal conversion efficiency.It was reported that lauric acid(LA)and stearic acid(SA)(4:1 by weight)could form an organic phase-change material(PCM)with low melting point at 39 ℃.Therefore,T7-DSPE-PEG5K/lecithin/JPC weight ratio of1:1.5:1.5 were used and MRP was encapsulated in the core of the nanoparticles composed of PCM through hydrophobic interaction.DLS and TEM characterization results showed TNP@JQ1/MRP nanoparticles had regular morphology,uniform size and good stability in serum.At the same time,photothermal imaging results showed that prodrug nanoparticles had high photothermal conversion efficiency of 38.8% and significant photothermal stability.The experiment of JQ1-SH release showed that prodrug nanoparticles achieved controllable release of JQ1-SH under 808 nm laser irradiation and GSH reduction.In conclusion,we have successfully developed a dual response prodrug nanoparticles TNP@JQ1 /MRP,which could be used for subsequent cell experiments and experimental studies in vivo.Chapter 3.The antitumor mechanism of prodrug nanoparticles in vitroIn the chapter,we explored the uptake ability,dual targeting,cytotoxicity,phototoxicity,the ability to stimulate dendritic cells(DC)maturation and alleviate immune escape.T7 peptide could interact with Tf receptor,which overexpressed on the surface of G422 and b End.3 cells,and promoted the uptake of prodrug nanoparticles through receptor-mediated endocytosis.The results showed that T7 peptide could enhance G422 and b End.3 cellular uptake of prodrug nanoparticles.Under 808 nm laser irradiation,G422 cells ingesting TNP@JQ1/MRP produced a lot of heat,which made temperature increase in G422 cells and promoted cell death.At the same time,tumor cells would release related antigens to stimulate the DCs maturation.Finally,we found that prodrug nanoparticles reduced IFN-?-induced PD-L1 expression on the surface of tumor cells and improved the behavior of immune escape.In conclusion,prodrug nanoparticles showed active targeting ability at G422 cell level,promoted G422 cell death to release tumor related antigen under 808 nm irradiation,activated tumor immune response and reactivated cytotoxic T lymphocytes(CTLs).Subsequent antitumor experiments can be carried out in vivo.Chapter 4.Fluorescence imaging and combined anti-tumor photothermal immunotherapy in vivoIn the chapter,we focused on the application of prodrug nanoparticles in fluorescence imaging,antitumor therapy and immune response in vivo.At the living level,TNP@JQ1 /MRP showed a satisfactory ability to break through BBB,and accurately identified and accumulated in tumor tissue.In GBM subcutaneous tumor model,the tumors of mice in TNP@JQ1/MRP + laser group were inhibited to the greatest extent.At the same time,the weight of mice and the corresponding main organs slicing results showed that the nanomaterial had biosafety.By analyzing the types and proportion of immune cells in mice,the combined therapy of PTT and JQ1 effectively enhanced the systemic anti-tumor immune response,recruited the infiltration of CTLs in tumor tissues,and overcame immune tolerance.This provided a new method for precise photothermal immunotherapy of GBM.