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Optimization Of Parameters For Tumor-targeting Of Radio-biologicals

放射生物制剂肿瘤靶向参数的优化

基本信息

批准号：
8952923
负责人：
Chang Hum Paik
金额：
--
依托单位：
CLINICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8952923
关键词：
Active Biological Transport Affect Affinity Antigens Area Binding Biological Blood Vessels Blood flow C-terminal Caliber Carbon Dioxide Cell Culture Techniques Cell Line Cell surface Cells Chemicals Cleaved cell Combined Modality Therapy Confocal Microscopy Coupling Cysteine Detection Disulfides Dithiothreitol Dose Endocytosis Endothelial Cells Endothelium Energy Transfer Extravasation Fluorescence Fluorescence Resonance Energy Transfer Gel Generations Glass Glycoproteins Goals Gold Humidity Image In Vitro Incubated Incubators Indium-111 Integrin Binding Integrin alphaVbeta3 Kinetics Label Left Life Measures Mediating Methods Microscope Modeling Monoclonal Antibodies Mus Neoplasms in Vascular Tissue Neuropilin-1 Nuclear Nucleic Acids Nutrient Oxygen Paclitaxel Pathway interactions Peptides Peripheral Permeability Pharmaceutical Preparations Phosphate Buffer Physiological Processes Process RGD (sequence)Radio Radioactivity Radioisotopes Radiolabeled Reagent Reporting Research Saline Schedule Sedimentation process Sepharose Signal Transduction Site Slide Solid Neoplasm Solutions Staining method Stains Surface Surrogate Markers Technology Testing Therapeutic Therapeutic Index Time Tissues Trypsin Tube Tumor Angiogenesis Tumor Tissue Vascular Permeabilities Widow angiogenesis antiangiogenesis therapy base chromophore cytotoxic fluorescence imaging improved interstitial killings nanoparticle neoplastic cell neovasculature overexpression paraform radiotracer receptor receptor binding receptor expression response tumor uptake

项目摘要

Background: In the previous years, we had investigated the effect of iRGD on tumor uptake of In-111 labeled B3, a murine IgG1k monoclonal antibody directed against the Le-Y antigen in two tumor models, A431 tumor and PC-3 tumor. A431 tumor is a vascular tumor and expresses Le-Y, but not integrin alpha(v)beta(3) and neuropilin-1 on the cell surface. PC-3 tumor expresses a high level of Le-Y and neuropilin-1, but no alpha(v)beta(3) on the cell surface. Therefore, only endothelial cells of angiogenic blood vessels express both alpha(v)beta(3) and neuropilin-1 in these tumor models. The purpose of the studies was to investigate if the size and vascularity of tumors are important factors affecting the level of tumor uptake of co-administered In-111-B3 (3.0 microCi/60 microg) as a result of enhanced vascular permeability via the activation of endocytic transport mechanism mediated by the binding of iRGD (4 micromol/kg) to alpha(v)beta(3), and its subsequent formation of a CendR peptide and its binding to neuropilin-1 receptors on angiogenic blood vessels. Results: For a vascular A431 tumor, the iRGD co-administration increased the Le-Y antigen-mediated tumor accumulation of B3 for a large tumor (1000 cubic mm) by 30% whereas it did not increase the accumulation of B3 in a small tumor (100 cubic mm) compared to a control without iRGD co-administration. However, for a non-vascular PC-3 tumor, the iRGD co-administration did not significantly increase the Le-Y antigen-mediated tumor accumulation of B3 in both a large tumor (>400 cubic mm) and a small tumor (<200 cubic mm). Conclusions: These findings suggest that the iRGD co-administration increased the extravasation of B3 into interstitial space of a larger vascular A431 tumor by 29-31% but did not increase the extravasation of B3 into interstitial space of a smaller or non-vascular PC-3 tumor. -Use of FRET-iRGD to study an alpha(v)beta(3)-specific and neuropilin-1-dependent mechanism of iRGD uptake in tumor cells. Introduction: Fluorescence resonance energy transfer (FRET) is a mechanism describing energy transfer between two chromophores. A donor chromophore may transfer energy to an acceptor chromophore through nonradiative dipole-dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor making FRET extremely sensitive to short distances. In the past year, we used an iRGD conjugated with FRET moieties, Donor (Alexa 488)-CRGDRGPADC-Acceptor (Dabcyl) for testing an alpha(v)beta(3)-specific and neuropilin-1-dependent mechanism of iRGD uptake in tumor cells. This FRET-iRGD, (Alexa 488)-CRGDRGPADC-(Dabcyl) was cyclized by a disulfide bridge formed between two cysteine residues to locate the donor and acceptor in a close distance. Its fluorescence emission is negligible in a phosphate-buffered saline (PBS) solution, but it emits the fluorescence strongly at 520 nm when it is treated in a solution containing trypsin and dithiothreitol (DTT) which proteolytically cleaves the peptide by a conditional C-end Rule (CendR) and reduces the disulfide bridge to produce a CendR motif, Alexa 488-CRGDR. Objectives: In the past year, we undertook our research to investigate if 1) the FRET-iRGD could be used as a sensitive probe for testing an alpha(v)beta(3)-specific and neuropilin-1-dependent mechanism of iRGD uptake in tumor cells and 2) to find an effective time window for the endocytic and exocytic transport pathways activated by the FRET-iRGD binding to alpha(v)beta(3), the formation of the CendR peptide and its binding to neuropilin-1 receptors expressed on U-87 MG cells. For this study, we used in vitro cell cultures including spheroids made of U-87 MG cells. Methods: Three different cell lines, A431 (alpha(v)beta(3)-negative, neuropilin-1-negative), PC-3 (alpha(v)beta(3)-negative, neuropilin-1-positive), and U87MG (alpha(v)beta(3)-positive, neuropilin-1-positive) (30 thousand cells/well), were seeded onto 8 well chamber slides (Nunc Lab Tek) and grown overnight in a medium. The cells were incubated with 5nM of FRET-iRGD for 0, 5, 10 and 20min at 37 degree C. The cells were washed with PBS (1x, pH7.4) after peptide incubation and fixed in 4% (v/v) paraformaldehyde for 10 min at 4 degree C. After PBS (1x, pH7.4) washing, the cells were finally mounted with ProLong Gold antifade reagent with DAPI (Life Technologies). Fluorescence images were collected using an Axioimager M1 microscope (Zeiss). For spheroid generation, U-87 MG cells (2 million cells/well) were seeded into Ultra Low Attachment (ULA) 24-well flat-bottomed plates (Costar). After the plate was incubated in a medium for 4 days at 37 degree C in an incubator (5% CO2, 95% humidity), Cells were maintained a sphere form. For FRET-iRGD, the 4 day-old U-87 MG spheroids were transferred to a new ULA plate with a medium and then incubated cells for 1 h in the presence of 1 micro-M of FRET-iRGD. After 1 h, U-87 MG spheroids (500 micro-m in diameter) were collected in V-bottomed 15 ml Falcon tubes and allowed to sediment. Supernatant was removed by gentle pipette and pellets washed once with phosphate-buffered saline (PBS, pH 7.4, 1x). After repeated sedimentation, supernatant was removed and 4% paraformaldehyde (PFA) was added and left for overnight. The next day, PFA was removed and 250 micro-l of Hoechst 33342 solution was gently added to the fixed U-87 MG spheroids for 40 min to stain the nucleic acid. After spheroids were washed once with PBS, U-87 MG spheroids were embedded in 1% Agarose Gel on glass bottom dish (MatTek Corporation). U-87 MG spheroids were observed with FLUOVIEW FV10i confocal microscopy (Olympus). Results: Among the three different cells tested, only U-87 MG (alpha(v)beta(3)-positive, neuropilin-1-positive) cells showed a positive Alexa-488 signal whereas A431 (alpha(v)beta(3)-negative, neuropilin-1-negative) and PC-3 (alpha(v)beta(3)-negative, neuropilin-1-positive) did not show Alexa-488 signal, indicating that both alpha(v)beta(3) and neuropilin-1 receptor expression is required for the generation and endocytosis of a CendR peptide, Alexa-488-CRGDR. The formation and endocytic transport of Alexa-488-CRGDR into U-87 MG cells occurred as early as 5 min of the cell incubation with the FRET-iRGD, indicating that the processes involving the binding of FRET-iRGD to alpha(v)beta(3) on U-87 MG cells, the proteolytic cleavage of the FRET-iRGD to a C-end Rule (CendR) motif, Alexa-488-CRGDK, and subsequent neuropilin-1 dependent endocytosis of the CendR peptide took place very rapidly. To answer a question on an effective time window for the endocytic and exocytic transport of the CendR peptide, we incubated U-87 MG cell spheroids with FRET-iRGD for 1 h and the presence of the CendR peptide in the spheroids was then determined by the confocal microscopy. The confocal microscopy focused at the center of the spheroid showed that the CendR peptide penetrated to the center of the spheroids within 1 h and that the Alexa-488 intensity in the cells in the peripheral area up to 100 micro-m distance from the surface of the spheroids was much lower than that in the center zone of the spheroids. Conclusion: The formation of the CendR peptide Alexa-488-CRGDR and its endocytosis upon binding to neuropilin-1 on U-87 MG cells occurred within 5 min. However, the majority of the Alexa-488-CRGDR that was internalized in the cells in the peripheral area of the spheroids exocytosed from the cells and penetrated into the core of the U-86 MG spheres. This finding appears to indicate that there is a narrow effective time widow for this active transport pathway so that the administration time of bystander drugs or nanoparticles should be well synchronized within this narrow time window to be effectively transported deeply into tumor cores.