Photochemical Infarction Of Lymphoma In Mice By Tissue Factor Targeted Nanoparticles

Tissue factor (TF) is best known as the primary initiator of blood coagulation. After vessel injury, the TF/FVII a complex activates the coagulation protease cascade, which leads to fibrin deposition and activation of platelets. An increasing number of studies have found TF was overexpressed on many cancer in solid tumors and haematological malignancy. And newly formed tumor capillary blood vessels distribute many TF because they lack tight junctions between endothelial cells. These TF is not only related closely with tumor angiogenesis and metastasis, but also related closely with thrombophlebitis of cancer patients. And it is called Trousseau’s Syndrome in the clinical. However, the TF distribution in the normal vessels is outside the circulation of the blood and cannot contact with these cells expressed TF, unless these nomal vessels are injured which will then starts the coagulation process. So TF is a specific molecular target for these tumor vascular endothelial cells. Previous studies had been showed the fusion protein EGFP-EGF1was affinity to TF, and EGFP-EGF1conjugated PLGA nanoparticles had the ability of targeting to TF.Photochemical reaction can induce vascular endothelial cells injury, platelet aggregation and thrombus formation. The pathogenesis is:photosensitizer in the vascular endothelial cells will generate a large number of reactive oxygen species when light of a particular wavelength is shone upon them. The reactive oxygen species generated will then react with structural protein and lipid on cell membrane which can start lipid peroxidation to injure the vascular endothelial cells and activate the coagulation protease cascade. In the process of these photochemical reaction, photosensitizer is a carrier of energy and bridge of response that plays the decisive role. of hematoporphyrin monomethyl ethe(HMME) is a new type of photosensitizer which has simple composition and stable properties. Its lasing wavelength is395nm and emission wavelength is613nm. HMME can quickly clean from the normal tissue of human body with low toxicity whose acute and long-term toxicity are both lower than the first generation photosensitizer. As plasma concentration of HMME decreases sharply by phagocytosis and subsequent disruption of the liver kupffer cells, so it is necessary to improve the initial dose to form blockages in other tissues beyond liver. Excessive concentrations can lead to not only liver injure, but also damage of superficial position making protection from light necessary. Therefore, targeted drug delivery can significantly reduce the whole body dosage minimizing the risk of damage to the normal tissue and enhancing the drugs to target sites.HMME will be encapsulated in the EGFP-EGF1fusion protein conjugated PLGA nanoparticles and targeting TF on tumor vessels, and then the tumors will be illuminated at appropriate wavelength. Activated oxygen will damage the endothelium and induce more and more TF expression. So many TF will not only become the target of the nanoparticles, but also activate the blood coagulation a nd starve the tumor cells, which will be a promising choice for many other solid tumors.Part1. The construction and characteristic of hematoporphyrin monomethyl ethe loaded EGFP-EGF1conjugated PLGA nanoparticles ObjectivesTo incorporate the hematoporphyrin monomethyl ether (HMME) into EGFP-EGF1protein-conjugated PEG-PLGA nanoparticles. Then to test characteristic of HMME-ENP. Confirm the targeting characteristic of the HMME-ENP. To evaluate the expression of TF and reactive oxygen species from Brain microvascular endothelial cells (BMECs) induced by HMME associated with cold light irradiation. Methods1) HMME-ENP was developed via an emulsion/solvent evaporation technique. The particle size was tested by the particle size analyzer. The shape of nanoparticle was observed by normal electron microscopy, and the conjugated protein was detected by BCA protein assay. The inside HMME was extracted and the Drug Loading Capability (DLC) was calculated.2) The primary BMECs from rats were extracted through double enzyme digestion method. The TNFa was chosen to be a inducer to establish injured endothelial cells model in vitro. The coumarin-6serving as nanoparticle probe, was entrapped in ENP to investigate its BMECs delivery characteristic. The amounts of reactive oxygen species was determined by Dihydroethidium (DHE) probe.The real-time PCR, flow cytometry and western-blot were detected to evaluate the level of TF expression.Results1) The size of HMME-ENP was about110nm-120nm, the appearance was rounding and uniform. The results of immune-electronic microscopy showed the EGFP-EGF1protein was covalently coupled to the nanoparticles’surface. The Entrapment Efficiency of HMME was4.24ug/mg. The EGF1-EGFP protein efficiency of connection was39.27%.2) The TNFa induced BMECs high expressed TF. The HMME-ENP was uptakebetter by the injured BMECs. The expression of TF and reactive oxygen species from BMECs induced by HMME associated with cold light irradiation were increased.Conclusion1) The nanoparticles size of HMME-ENP was about100nm-120nm, and the protein was combined to nanoparticles’ surface. The characteristic of drug loading capability was suitable to subsequent experiments.2) HMME-ENP can target delivery HMME into the injured BMECs.3) The expression of TF and reactive oxygen species from BMECs induced by HMME associated with cold light irradiation were increased. Part2. The target delivery of hematoporphyrin monomethyl ethe loaded EGFP-EGF1conjugated PLGA nanoparticles in lymphoma-bearing modelObjectivesTo construct the lymphoma-bearing model. To confirm the target delivery of hematoporphyrin monomethyl ethe loaded EGFP-EGF1conjugated PLGA nanoparticles in lymphoma-bearing model. To preliminary evaluation the intervening effect by HMME associated with cold light irradiation to the tumor.Methods1) CA46cells with1*107density were inoculated subcutaneously into right shoulder area in NOD/SCID mice to develop implant-tumor.2) Synthesis of Dir loaded ENPs through the double emulsion method. And their DLC was detected. Dir loaded ENP (Dir-ENP) was injected into lymphoma-bearing mice, and though living imaging and organ imaging to determine the targeting of Dir-ENP in vivo.3) Synthesis of cou-6loaded ENPs through the double emulsion method. Cou-6loaded ENP was injected into lymphoma-bearing mice, and the distribution of HMME-ENP and TF expression were observed by fluorescence after frozen section.4) The thrombus distribution was assessed by H&E staining.Results1)80%NOD/Scid mice formed implant-tumor after3-4weeks of CA46cells inoculation.2) living imaging:a) the density of HMME-ENP in the tumor area was more than HMME-NP; And after cold light irradiation, the density of HMME-ENP in the tumor area was also more than HMME-NP;b) After cold light irradiation, the density of HMME-ENP in the tumor area was increased; c) At6h after cold light irradiation, the density of HMME-ENP in the tumor area was the greatest.3) organ imaging: a) At24h after cold light irradiation, the density of HMME-ENP in hepar was the greatest; c) And other results were consistent with the result above.4) Immunofluorescence assay of the tumor after frozen section indicated that the distribution of HMME-ENP and TF expression were superposed.5) The thrombus and reactive oxygen species in the tumor area of the mice injected HMME-ENP were more than the mice injected HMME-NP.Conclusion1) HMME-ENP can target delivery HMME into the tumor area in lymphoma-bearing mice;2) After cold light irradiation, the density of HMME-ENP, the TF expression, reactive oxygen species and the thrombus in the tumor area were all increased which might present an new strategy for tumor treatment.