A Tissue Factor Targeted "O2 Evolving" Nanoparticles For Photodynamic Therapy Against Malignant Lymphoma

Neovascularization is neccessary for oxygen and nutrients requirement of Malignant lymphoma,and its new blood vessel density is significantly higher than that of reactive hyperplasia.Compared with normal blood vessels,tumor angiogenesis is more twisted and disordered,with large gap between endothelial cells and stromal cells,which cause the easy leakage of tissue factor(TF)into blood vessels.Thus tumor vascular endothelial cells highly express TF,even trigger the blood coagulation cascade and cause thrombosis and spontaneous thrombophlebitis(Trousseau’s syndrome).In contrast to the aberrantly high TF expression of tumor vascular endothelium,TF can be released only when the normal tissue is damaged,and the coagulation cascade can be initiated.Therefore,TF can serve as the specific target of malignant lymphoma.Photodynamic therapy(PDT)is a clinical modality that employs a photosensitizer,an excitation light of appropriate wavelength,and oxygen(O2)molecules to generate Reactive Oxygen Species(ROS),resulting in the specific killing of lesions.With specific spatiotemporal selectivity and minimal invasiveness,PDT has become an emerging solution for cancer therapy.The low selectivity of currently available photosensitizers,which causes the treatment-related toxicity and side effects on adjacent normal tissues,is a major limitation for clinical PDT against cancer.Moreover,since PDT process is strongly oxygen-dependent,its therapeutic effect is seriously hindered in hypoxic tumor cells.In our previous study,EGFP-EGF1 modified PEG-PLGA nanoparticles has been successfully established to target TF.We designed a TF-cascade-targeting strategy by combined PDT with PSs(HMME)loaded EGFP-EGF1 modified HMME-loaded PEG-PLGA nanoparticles(ENP-HMME).The resultsshowed that ENP-HMME can initially target to tumor vascular endothelium with high TF expression.Increased ROS release was observed after PDT,and vascular endothelial was damaged,thus inducing more TF and recruiting more ENP-HMME.In vivo imaging showed that before ENP-HMME accumulation in tumor tissue is 2.45 times than control group before irradiation,and up to 3.5 times after irradiation.Our group further assessed the efficacy by PET,the result showed 75%inhibition of tumor activity after PDT.Although ROS damage promoted endothelial cells to release TF,creating TF-target amplification effect,at the same time TF coagulation cascade was activated and cause blood clots which infarct and killing tumor.However,the result did not satisfied as expectation.It is probably that the hypoxia of tissue tumor limit the efficacy of PDT,thus restricting the generation of ROS and induction of TF,which affects the positive feedback effect of ENP accumulation.To overcome this challenge,we herein encapsulated catalase based on ENP-HMME designed in previous study and developed catalase encapsulated ENP-HMME(CENP-HMME).Catalase can decompose hydrogen peroxide into O2,the O2 generated during PDT process efficiently improved the therapeutic efficacy through overcoming the hypoxic problems in tumor tissue.The first part of this paper is the preparation and characterization of CENP-HMME.According to the previous literature,the CENP-HMME were successfully constructed.The particle size is about 160nm,and the PDI is less than 0.22 and can be used in vivo experiments.We also assessed the enzyme activity of catalase encapsulated nanoparticles.Bubbles were observed when catalase encapsulated nanoparticles was added into hydrogen peroxide,a indication of enzymatic activity.The enzyme activity was measured by Goth method and it demonstrated about 10%enzyme activity of free catalase.We also quantified the drug loading capacity(DLC)of HMME,about 0.5~2%,which can meet the needs of follow-up experiments.The second part of this paper is in vitro experiments.We evaluated the targeting effect of CENP-HMME,the ability to generate ROS and TF,and the ability to product 02.TF-expressed human umbilical vein endothelial cell(HUVEC)was used to evaluated targeting effect of EGFP-EGF1 modified nanoparticles.The fluorescence microscopy observation showed that EGFP-EGF1 modified nanoparticles showed higher up take by HUVEC significantly.Further quantitative analysis of flow cytometry showed that the uptake of TF-targeted nanoparticles by HUVEC was about 1.4 times that of non-targeted nanoparticles.We further evaluate the level of ROS generation and TF expression by HUVEC after PDT.The results showed PDT significantly increased ROS generation and TF expression.We also explored the enzyme activity of catalase loaded nanoparticles in a lymphoma cell line,CA46.After incubation with nanoparticles,flow cytometry suggest catalase-loaded nanoparticles of catalase contributed to oxygen generation while nanoparticles withoutcatalase showed no activity.Moreover,we measured the ROS level in CA46,and found that the catalase-loaded nanoparticles significantly lower the ROS level in CA46.The third part is in vivo experiment.We evaluated the targeting ability of CENP-HMME after PDT and simply evaluated the formation of thrombosis and efficacy.The ability to improve the internal oxygen supply is also evaluated.The subcutaneous lymphoma-bearing mice model was established for observing pharmacokinetics distribution of nanoparticles after PDT.In vivo imaging showed that ENP-HMME mediated PDT group has 3.6 times nanoparticles accumulation in tumor than NP mediated PDT group,and CENP-HMME mediated PDT group increased up to 12 times.The tumor tissue section also showed consistent result,moreover the tissue section showed that TF expression of the tumor vascular endothelial enhanced significantly after PDT.We exploited catalase loaded nanoparticles improve tumor oxygenation and overcome hypoxia.Hypoxia inducible factor(HIF)-lα staining assay was carried out to evaluate hypoxic conditions in the tumor after PDT with or without catalase.Results show that the group untreated or treated with nanoparticles without catalase showed a strong expression of HIF-lα.In contrast,for the group treated with catalase encapsulated nanoparticles,the immunohistochemical staining suggested that the expression of HIF-la significantly decreased owing to the generated 02 in tumor that overcame the hypoxia.HE staining showed that PDT-treated group showed microthrombosis and induced necrosis of surrounding tissue.The efficacy was simply evaluate by TUNEL staining,and the results showed that,the CENP-HMME mediated PDT group showed more apoptosis and necrosis than ENP-HMME mediated PDT group.Our research showed that the CENP-HMME mediated PDT has stronger tumor targeting effect,with microthrombosiss formation and stronger anti-tumor efficacy,providing a new platform for tumor treatment.