Tissue Factor-Targeted "O2-Evolving" Nanoparticles for Photodynamic Therapy in Malignant Lymphoma.

Vascular-targeted PDT (vPDT) has produced promising results in the treatment of many cancers, including drug-resistant ones, but little is known about its efficacy in lymphoma. Unfortunately, the lack of a specific therapeutic target and a hypoxic microenvironment for lymphoma jeopardizes the efficacy of vPDT severely. In this study, we designed a lymphoma tissue factor-targeted "O2-evolving" strategy combining PDT with catalase and HMME-encapsulated, EGFP-EGF1-modified PEG-PLGA nanoparticles (CENPs) to boost PDT efficiency; this combination takes advantage of the low oxygen tension of lymphoma. In our results, CENPs accumulated effectively in the vascular lymphoma in vivo and in vitro, and this accumulation increased further with PDT treatment. Per positron emission tomography imaging, combining CENPs with PDT inhibited lymphoma glucose metabolism significantly. The expression of hypoxia-inducible factor (HIF)-1α in the entrapped catalase groups reduced markedly. These data show that the combined administration of PDT and CENPs can prompt tissue factor-cascade-targeted and self-supply of oxygen and that it has a good therapeutic effect on malignant lymphoma.

EGFP-EGF1-conjugated poly(lactic-co-glycolic acid) nanoparticles, a new diagnostic tool and drug carrier for atherosclerosis.

BACKGROUND: EGFP-EGF1-conjugated poly(lactic-co-glycolic acid) (PLGA) nanoparticle (ENP) has a specific affinity to tissue factor (TF). The aim of this study was to investigate the target delivery of ENP to plaques and its uptake in a mouse model of atherosclerosis in vivo and in vitro. MATERIALS AND METHODS: Coumarin-6- and 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbo cyanine iodide (DiR)-loaded ENPs were synthesized using a double-emulsion method. Mouse vascular smooth muscle cells (VSMCs) were induced with MCP-1 to obtain an increased TF expression. Fluorescence microscopy and flow cytometry assay were performed to examine the uptake of coumarin-6-loaded ENPs in cellular models. An animal model of atherosclerosis was established with an ApoE (-/-) mouse fed with continuous high-fat diets for 14 weeks. DiR-loaded ENPs (DiR-ENPs) were injected via the caudal vein. The distribution of DiR-ENPs was examined through organ imaging and confocal laser scanning microscopy. RESULTS: Results indicated TFs were highly expressed in the cellular model. The uptake of coumarin-6-loaded ENPs was significantly higher than that of common PLGA nanoparticles. Thickening of intima and lipid deposition in the aorta could be observed in atherosclerosis mouse models. Confocal laser scanning microscopy organ imaging showed ENPs accumulated in vessels with atherosclerotic plaques, which coincided with high expressions of TF. CONCLUSION: This study showed that EGFP-EGF1-conjugated PLGA nanoparticles could be effectively delivered to atherosclerotic plaques in vivo and taken up by VSMCs with high TF expressions in vitro. Thus, it could be a promising carrier for targeted therapy of atherosclerosis.

Improved in vivo detection of atherosclerotic plaques with a tissue factor-targeting magnetic nanoprobe.

Rupture of atherosclerotic plaques causes acute cardiovascular and cerebrovascular pathology. Tissue factor (TF) is a key factor that affects the development of atherosclerotic plaques and the formation of thrombus and thus constitutes a potential target for the detection of atherosclerotic plaques. In this study, the conjugation of the fusion protein 'enhanced green fluorescent protein with the first epidermal growth factor domain' (EGFP-EGF1) and superparamagnetic iron oxide nanoparticles (EGFP-EGF1-SPIONs) was explored for molecular imaging of TF-positive atherosclerotic plaques. EGFP-EGF1-SPIONs showed improved accuracy, superior contrast effects, and better cytocompatibility compared with common contrast agents in the detection of atherosclerotic plaques of apolipoprotein E knockout (ApoE-/-) mice using magnetic resonance imaging. In conclusion, EGFP-EGF1-SPION is a promising TF-targeting nanoprobe to precisely and specifically detect atherosclerotic plaques, which may improve molecular imaging diagnosis of cardiovascular and cerebrovascular events for the comprehensive evaluation of atherosclerosis. STATEMENT OF SIGNIFICANCE: Traditional methods can only display the status of atherosclerosis, but not forecast the progress of lesions efficiently. It remains challenging to evaluate the plaques specifically and sensitively. In this study, we constructed a tissue factor-targeted magnetic nanoprobe to specifically detect plaques by magnetic resonance imaging in vivo, which will improve the diagnostic technology for atherosclerotic plaques and offer molecular level guidance to treat atherosclerosis. Furthermore, this strategy has critical clinical significance on prevention, diagnosis and therapeutic evaluation of cardio-cerebral vascular events.

Early diagnosis of cerebral thrombosis by EGFP-EGF1 protein conjugated ferroferric oxide magnetic nanoparticles.

Cerebral thrombosis disease is a worldwide problem, with high rates of morbidity, disability, and mortality. Magnetic resonance imaging diffusion-weighted imaging was used as an important early diagnostic method for cerebral thrombotic diseases; however, its diagnosis time is 2 h after onset. In this study, we designed EGFP-EGF1-NP-Fe3O4 for earlier diagnosis of cerebral thrombosis by taking advantage of EGFP-EGF1 fusion protein, in which EGF1 can bind with tissue factor and enhanced green fluorescent protein has previously been widely used as a fluorescent protein marker. EGFP-EGF1-NP-Fe3O4 or NP-Fe3O4 reaches the highest concentration in the infarction areas in 1 h. To evaluate the targeting ability of EGFP-EGF1-NP-Fe3O4, a fluorochrome dye, Dir, was loaded into the nanoparticle. As shown by the in vivo organ multispectral fluorescence imaging, Dir-loaded EGFP-EGF1-NP-Fe3O4 exhibited higher fluorescence than those of model rats treated with Dir-loaded NP-Fe3O4. Coronal frozen sections and transmission electron microscope further showed that EGFP-EGF1-NP-Fe3O4 was mainly accumulated in the tissue factor exposure region of brain. The data indicated that the EGFP-EGF1-NP-Fe3O4 targeted cerebral thrombosis and might be applied in the early diagnosis of intracranial thrombosis.

A tissue factor-cascade-targeted nanoparticle forsite-directed inducing thrombosis.

Tissue factor is an upstream component of the cascade and a high-expressing factor under phathological conditions. In this study, a tissue factor cascade-targeted strategy for inducing local thrombosis was developed by combining ENP-HMME and photochemistry. In vitro study showed that protein EGFP-EGF1 conjugation to the nanoparticles could significantly contribute to the uptake of nanoparticles by tissue factor over-expressed brain capillary endothelial cells. Three-dimensional imaging and specklegram of brains in vivo showed that tissue factor cascade-targeted strategy successfully induced thrombosis of expected position. As shown by the in vivo multispectral fluorescent imaging, when ENP-HMME was combined with photochemistry, higher accumulation in the infarction hemisphere was observed, which might suggest that the photochemistry inducing tissue factor cascade recruited more ENP-HMME than HMME-loaded nanoparticles (NP-HMME). The data indicated the tissue factor cascade-targeted strategy has potential to induce local thrombosis, and might be applied in the treatment of a variety of hypervascular diseases.

A tissue factor-cascade-targeted strategy to tumor vasculature: a combination of EGFP-EGF1 conjugation nanoparticles with photodynamic therapy.

Tumor requires tumor vasculature to supply oxygen and nutrients so as to support its continued growth, as well as provide a main route for metastatic spread. In this study, a TF-cascade-targeted strategy aiming to disrupt tumor blood vessels was developed by combination of TF-targeted HMME-loaded drug delivery system and PDT. PDT is a promising new modality in the treatment of cancers, which employs the interaction between a tumor-localizing photosensitizer and light of an appropriate wavelength to bring about ROS-induced cell death. In vitro results showed that protein EGFP-EGF1modification could significantly contribute to the uptake of nanoparticles by TF over-expressed BCECs. In vivo multispectral fluorescent imaging, the EGFP-EGF1 conjugated nanoparticles showed significantly higher accumulation in tumor tissues than non-conjugated ones. Tumor tissue slides further presented that EGFP-EGF1 conjugated nanoparticles showed significantly higher accumulation in tumor vasculature than non-conjugated ones. In vitro study demonstrated that PDT increased TF expression of BCECs. In vivo imaging, ex vivo imaging and tumor tissue slides showed that PDT further contribute EGFP-EGF1-NP accumulation in tumor. These promising results indicated that PDT enhanced EGFP-EGF1modified PEG-PLGA nanoparticle accumulation in tumor vaculature. Considering that EGFP-EGF1 conjugation enhanced nanoparticles uptake by TF over-expressed endothelium and PDT increased endothelium TF expression. We conclude that PDT triggered a TF cascade targeted effect. A combination of both EGFP-EGF1 modification and PDT provided a positive feed-back target effect to tumor vessels and might have a great potential for tumor therapy.

Enhanced Antitumor Activity of EGFP-EGF1-Conjugated Nanoparticles by a Multitargeting Strategy.

Tumor stromal cells have been increasingly recognized to interact with tumor parenchyma cells and promote tumor growth. Therefore, we speculated that therapeutics delivery to both parenchyma cells and stromal cells simultaneously might treat a tumor more effectively. Tissue factor (TF) was shown to be extensively located in a tumor and was abundantly sited in both tumor parenchyma cells and stromal cells including neo-vascular cells, tumor-associated fibroblasts, and tumor-associated macrophages, indicating it might function as a favorable target for drug delivery to multiple cell types simultaneously. EGFP-EGF1 is a fusion protein derived from factor VII, the natural ligand of TF. It retains the specific TF binding capability but does not cause coagulation. In the present study, a nanoparticle modified with EGFP-EGF1 (ENP) was constructed as a multitargeting drug delivery system. The protein binding experiment showed EGFP-EGF1 could bind well to A549 tumor cells and other stromal cells including neo-vascular cells, tumor-associated fibroblasts, and tumor-associated macrophages. Compared with unmodified nanoparticles (NP), ENP uptake by A549 cells and those stromal cells was significantly enhanced but inhibited by excessive free EGFP-EGF1. In addition, ENP induced more A549 tumor cell apoptosis than Taxol and NP when paclitaxel (PTX) was loaded. In vivo, ENP accumulated more specially in TF-overexpressed A549 tumors by in vivo imaging, mainly regions unoccupied by factor VII and targeted tumor parenchyma cells as well as different types of stromal cells by immunofluorescence staining. Treatment with PTX-loaded ENP (ENP-PTX) significantly reduced the A549 tumor growth in nude mice while NP-PTX- and Taxol-treated mice had lower response to the therapy. Furthermore, H&E and TUNEL staining revealed that ENP-PTX induced more severe tumor necrosis and more extensive cell apoptosis. Altogether, the present study demonstrated that ENP could target multiple key cell types in tumors through TF, which could be utilized to improve the therapeutic effect of anticancer drugs.

EGFP-EGF1-conjugated nanoparticles for targeting both neovascular and glioma cells in therapy of brain glioma.

As neovascular and glioma cells were closely associated and might be mutually promoted in glioma growth, a dual-targeting strategy targeting to both neovascular and glioma cells would be more promising as compared with those targeting one of them. In this study, we reported a drug delivery system where nanoparticles were decorated with EGFP-EGF1 (ENP), a fusion protein derived from factor VII with special affinity for tissue factor (TF) over-expressed in glioma tissues, to facilitate anti-glioma delivery of paclitaxel (PTX) by targeting both neovascular and glioma cells. In vitro protein binding assay demonstrated that EGFP-EGF1 bound well to C6 cells and perturbed human umbilical vein endothelial cells (HUVEC) with a concentration-dependent manner but not to unperturbed HUVEC. EGFP-EGF1-TF interaction significantly enhanced nanoparticles uptake by perturbed HUVEC and glioma C6 cells as well as nanoparticles penetration in C6 glioma spheroids, and thus improved the cytotoxicity of their payload in both monolayer cells and glioma spheroids models. In vivo imaging of glioma-bearing mice demonstrated the specific accumulation of ENP in glioma tissues. In vivo distribution of nanoparticles intuitively showed ENP mainly sited in both extravascular glioma cells and neovascular cells. Pharmacodynamic results revealed that PTX-loaded ENP (ENP-PTX) significantly prolonged the median survival time of glioma-bearing mice compared with that of any other group. TUNEL assay and H&E staining showed that ENP-PTX treatment induced significantly more cell apoptosis and tumor necrosis compared with other treatments. In conclusion, the results of this contribution demonstrated the great potential of EGFP-EGF1-functionalized nanoparticles for dual-targeting therapy of brain glioma.