RESUMO
The continuous pursuit of designing an ideal infection imaging agent is a crucial and ongoing endeavor in the field of biomedical research. Duramycin, an antimicrobial peptide exerts its antimicrobial action on bacteria by specific recognition of phosphatidylethanolamine (PE) moiety present on most bacterial membranes, particularly Escherichia coli (E. coli). E. coli membranes contain more than 60% PE. Therefore, duramycin is an attractive candidate for the formulation of probes for in situ visualization of E. coli driven focal infections. The aim of the present study is to develop 99m Tc labeled duramycin as a single-photon emission computed tomography (SPECT)-based agent to image such infections. Duramycin was successfully conjugated with a bifunctional chelator, hydrazinonicotinamide (HYNIC). PE specificity of HYNIC-duramycin was confirmed by a dye release assay on PE-containing model membranes. Radiolabeling of HYNIC-duramycin with 99m Tc was performed with consistently high radiochemical yield (>90%) and radiochemical purity (>90%). [99m Tc]Tc-HYNIC-duramycin retained its specificity for E. coli, in vitro. SPECT and biodistribution studies showed that the tracer could specifically identify E. coli driven infection at 3 h post injection. While 99m Tc-labeled duramycin is employed for monitoring early response to cancer therapy and cardiotoxicity, the current studies have confirmed, for the first time, the potential of utilizing 99m Tc labeled duramycin as an imaging agent for detecting bacteria. Its application in imaging PE-positive bacteria represents a novel and promising advancement.
Assuntos
Bacteriocinas , Escherichia coli , Compostos de Organotecnécio , Compostos de Organotecnécio/química , Distribuição Tecidual , Peptídeos/química , Peptídeos/metabolismoRESUMO
Following the in vivo biodistribution of platelets can contribute to a better understanding of their physiological and pathological roles, and nuclear imaging methods, such as single photon emission tomography (SPECT), provide an excellent method for that. SPECT imaging needs stable labeling of the platelets with a radioisotope. In this study, we report a new method to label platelets with 99mTc, the most frequently used isotope for SPECT in clinical applications. The proposed radiolabeling procedure uses a membrane-binding peptide, duramycin. Our results show that duramycin does not cause significant platelet activation, and radiolabeling can be carried out with a procedure utilizing a simple labeling step followed by a size-exclusion chromatography-based purification step. The in vivo application of the radiolabeled human platelets in mice yielded quantitative biodistribution images of the spleen and liver and no accumulation in the lungs. The performed small-animal SPECT/CT in vivo imaging investigations revealed good in vivo stability of the labeling, which paves the way for further applications of 99mTc-labeled-Duramycin in platelet imaging.
Assuntos
Bacteriocinas , Tomografia Computadorizada de Emissão de Fóton Único , Camundongos , Humanos , Animais , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Peptídeos/metabolismo , Bacteriocinas/metabolismoRESUMO
99mTc-hexamethylpropyleneamine oxime (HMPAO) and 99mTc-duramycin in vivo imaging detects pulmonary oxidative stress and cell death, respectively, in rats exposed to >95% O2 (hyperoxia) as a model of acute respiratory distress syndrome (ARDS). Preexposure to hyperoxia for 48 h followed by 24 h in room air (H-T) is protective against hyperoxia-induced lung injury. This study's objective was to determine the ability of 99mTc-HMPAO and 99mTc-duramycin to track this protection and to elucidate underlying mechanisms. Rats were exposed to normoxia, hyperoxia for 60 h, H-T, or H-T followed by 60 h of hyperoxia (H-T + 60). Imaging was performed 20 min after intravenous injection of either 99mTc-HMPAO or 99mTc-duramycin. 99mTc-HMPAO and 99mTc-duramycin lung uptake was 200% and 167% greater (P < 0.01) in hyperoxia compared with normoxia rats, respectively. On the other hand, uptake of 99mTc-HMPAO in H-T + 60 was 24% greater (P < 0.01) than in H-T rats, but 99mTc-duramycin uptake was not significantly different (P = 0.09). Lung wet-to-dry weight ratio, pleural effusion, endothelial filtration coefficient, and histological indices all showed evidence of protection and paralleled imaging results. Additional results indicate higher mitochondrial complex IV activity in H-T versus normoxia rats, suggesting that mitochondria of H-T lungs may be more tolerant of oxidative stress. A pattern of increasing lung uptake of 99mTc-HMPAO and 99mTc-duramycin correlates with advancing oxidative stress and cell death and worsening injury, whereas stable or decreasing 99mTc-HMPAO and stable 99mTc-duramycin reflects hyperoxia tolerance, suggesting the potential utility of molecular imaging for identifying at-risk hosts that are more or less susceptible to progressing to ARDS.
Assuntos
Lesão Pulmonar Aguda , Hiperóxia , Síndrome do Desconforto Respiratório , Lesão Pulmonar Aguda/diagnóstico por imagem , Animais , Hiperóxia/diagnóstico por imagem , Hiperóxia/metabolismo , Imagem Molecular , Oximas , Ratos , Ratos Sprague-DawleyRESUMO
Phosphatidylethanolamine (PE) is a major phospholipid species with important roles in membrane trafficking and reorganization. Accumulating clinical data indicate that the presence of circulating antibodies against PE is positively correlated with the symptoms of antiphospholipid syndromes (APS), including thrombosis and repeated pregnancy loss. However, PE is generally sequestered inside a normal resting cell, and the mechanism by which circulating anti-PE antibodies access cellular PE remains unknown. The studies presented here were conducted with synthetic PE-binding agents, plasma samples from patients with anti-PE autoimmunity, and purified anti-PE antibodies. The results suggest that the cellular vulnerability to anti-PE antibodies may be mediated by the binding of PE molecules in the membrane of the early endosome. Endosomal PE binding led to functional changes in endothelial cells, including declines in proliferation and increases in the production of reactive oxygen species, as well as the expression of inflammatory molecules. Collectively, our findings provide insight into the etiology of anti-PE autoimmunity and, because endosomes are of central importance in almost all types of cells, could have important implications for a wide range of biological processes.
Assuntos
Anticorpos Antifosfolipídeos/imunologia , Síndrome Antifosfolipídica/imunologia , Endossomos/imunologia , Células Endoteliais da Veia Umbilical Humana/imunologia , Imunoglobulina M/imunologia , Fosfatidiletanolaminas/imunologia , Síndrome Antifosfolipídica/patologia , Endossomos/patologia , Células Endoteliais da Veia Umbilical Humana/patologia , HumanosRESUMO
The objective of this research was to estimate whether a [99mTc]duramycin probe can be used for apoptosis imaging in patients with aortic aneurysm (AA). Vascular smooth muscle cell (SMC) apoptosis has an important influence on AA development. Thus, non-invasive imaging of SMC apoptosis may be able to evaluate AA progress and risk stratification. SMCs were treated with hydrogen peroxide (H2O2; 200 µΜ) or culture medium as a control. Apoptosis was measured using flow cytometry and [99mTc]duramycin to detect the binding efficiency to apoptotic SMCs. C57/BL6 mice were administered angiotensin-II and beta-aminopropionitrile (BAPN) subcutaneously to establish an AA model, or saline for controls. Aortic specimens underwent pathological evaluation and their aortic diameters were measured after 6 weeks. Micro-SPECT/CT scanning of [99mTc]duramycin and 18F-FDG PET detection were performed. SMCs treated with H2O2 showed more apoptosis compared with the control group (67.2 ± 3.8% vs. 16.1 ± 0.6%, P < 0.01). The experimental group showed a high rate of AA formation (70%) compared with no AA formation in the control group. The average aorta diameter was higher and [99mTc]duramycin uptake at the AA site was higher in the experimental group compared with the control group. Compared with the normal aorta in the control group, AA in experiment group had more severe medial degeneration, elastic fiber reduction and fracture, and collagen degeneration. TUNEL staining verified the higher apoptosis rate at the AA site in experiment group compared with the control group (63.9 ± 3.7% in ascending AA, 66.4 ± 4.0% in thoracic AA, vs. 3.5 ± 0.3% in normal aorta, P < 0.01). [99mTc]Duramycin may be an effective probe to evaluate apoptosis in AA.
Assuntos
Aneurisma Aórtico/diagnóstico por imagem , Apoptose , Marcação por Isótopo/métodos , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Angiotensina II , Animais , Aneurisma Aórtico/induzido quimicamente , Bacteriocinas/metabolismo , Técnicas de Diagnóstico por Radioisótopos , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/metabolismo , Radioisótopos , Tecnécio/metabolismo , VasoconstritoresRESUMO
Eukaryotic organisms typically express multiple type IV P-type ATPases (P4-ATPases), which establish plasma membrane asymmetry by flipping specific phospholipids from the exofacial to the cytosolic leaflet. Saccharomyces cerevisiae, for example, expresses five P4-ATPases, including Neo1, Drs2, Dnf1, Dnf2, and Dnf3. Neo1 is thought to be a phospholipid flippase, although there is currently no experimental evidence that Neo1 catalyzes this activity or helps establish membrane asymmetry. Here, we use temperature-conditional alleles (neo1(ts)) to test whether Neo1 deficiency leads to loss of plasma membrane asymmetry. Wild-type (WT) yeast normally restrict most of the phosphatidylserine (PS) and phosphatidylethanolamine (PE) to the inner cytosolic leaflet of the plasma membrane. However, the neo1-1(ts) and neo1-2(ts) mutants display a loss of PS and PE asymmetry at permissive growth temperatures as measured by hypersensitivity to pore-forming toxins that target PS (papuamide A) or PE (duramycin) exposed in the extracellular leaflet. When shifted to a semi-permissive growth temperature, the neo1-1(ts) mutant became extremely hypersensitive to duramycin, although the sensitivity to papuamide A was unchanged, indicating preferential exposure of PE. This loss of asymmetry occurs despite the presence of other flippases that flip PS and/or PE. Even when overexpressed, Drs2 and Dnf1 were unable to correct the loss of asymmetry caused by neo1(ts) However, modest overexpression of Neo1 weakly suppressed loss of membrane asymmetry caused by drs2Δ with a more significant correction of PE asymmetry than PS. These results indicate that Neo1 plays an important role in establishing PS and PE plasma membrane asymmetry in budding yeast.
Assuntos
Adenosina Trifosfatases/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Bacteriocinas/farmacologia , Membrana Celular/genética , Depsipeptídeos/farmacologia , Proteínas de Membrana Transportadoras/genética , Peptídeos/farmacologia , Fosfatidiletanolaminas/genética , Fosfatidilserinas/genética , Proteínas de Transferência de Fosfolipídeos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Phosphatidylethanolamine (PE) is one of the most abundant phospholipids in mammalian plasma membranes. In healthy cells, PE resides predominantly in the inner leaflet of the cell membrane. In dead or dying cells on the other hand, PE is externalized to the outer leaflet of the plasma membrane. The exposure of PE on the cell surface has therefore become an attractive target for the molecular imaging of cell death using single-photon emission computed tomography (SPECT) and positron emission tomography (PET). This has motivated the development of PE-specific probes to measure cell death in vitro and non-invasively in vivo. In this review, we highlight the biological roles of PE on cell membranes, and PE exposure as a biomarker of cell death in disease processes, along with the use of PE-binding molecular probes to target PE for the characterization of cell death on a cellular and tissue level. We specifically emphasize the preclinical applications of radiolabeled duramycin for the non-invasive imaging of cell death in animal models of disease and in tumors after therapy. In addition, we discuss the clinical relevance, limitations and future perspectives of this imaging approach of cell death.
Assuntos
Apoptose/genética , Imagem Molecular/métodos , Neoplasias/diagnóstico por imagem , Fosfatidiletanolaminas/isolamento & purificação , Animais , Bacteriocinas/química , Biomarcadores/metabolismo , Membrana Celular/genética , Modelos Animais de Doenças , Humanos , Camundongos , Neoplasias/genética , Neoplasias/patologia , Peptídeos/química , Fosfatidiletanolaminas/genética , Tomografia por Emissão de Pósitrons , Tomografia Computadorizada de Emissão de Fóton Único/métodosRESUMO
Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial peptides that are characterized by the thioether cross-linked bisamino acids lanthionine (Lan) and methyllanthionine (MeLan). Duramycin contains 19 amino acids, including one Lan and two MeLans, an unusual lysinoalanine (Lal) bridge formed from the ε-amino group of lysine 19 and a serine residue at position 6, and an erythro-3-hydroxy-l-aspartic acid at position 15. These modifications are important for the interactions of duramycin with its biological target, phosphatidylethanolamine (PE). Based on the binding affinity and specificity for PE, duramycin has been investigated as a potential therapeutic, as a molecular probe to investigate the role and localization of PE in biological systems, and to block viral entry into mammalian cells. In this study, we identified the duramycin biosynthetic gene cluster by genome sequencing of Streptomyces cinnamoneus ATCC 12686 and investigated the dur biosynthetic machinery by heterologous expression in Escherichia coli In addition, the analog duramycin C, containing six amino acid changes compared to duramycin, was successfully generated in E. coli The substrate recognition motif of DurX, an α-ketoglutarate/iron(II)-dependent hydroxylase that carries out the hydroxylation of aspartate 15 of the precursor peptide DurA, was also investigated using mutagenesis of the DurA peptide. Both in vivo and in vitro results demonstrated that Gly16 is important for DurX activity. IMPORTANCE: Duramycin is a natural product produced by certain bacteria that binds to phosphatidylethanolamine (PE). Because PE is involved in many cellular processes, duramycin is an antibiotic that kills bacteria, but it has also been used as a molecular probe to detect PE and monitor its localization in mammalian cells and even whole organisms, and it was recently shown to display broad-spectrum inhibition of viral entry into host cells. In addition, the molecule has been evaluated as treatment for cystic fibrosis. We report here the genes that are involved in duramycin biosynthesis, and we produced duramycin by expressing those genes in Escherichia coli We show that duramycin analogs can also be produced. The ability to access duramycin and analogs by production in E. coli opens opportunities to improve duramycin as an antibiotic, PE probe, antiviral, or cystic fibrosis therapeutic.
Assuntos
Bacteriocinas/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Peptídeos/genética , Streptomyces/genética , Proteínas de Bactérias/metabolismo , Bacteriocinas/biossíntese , Escherichia coli/metabolismo , Organismos Geneticamente Modificados/metabolismo , Peptídeos/metabolismo , Streptomyces/metabolismoRESUMO
Our goal was to validate the feasibility of(99m)Tc-duramycin as a potential apoptosis probe for monitoring tumor response to paclitaxel in breast cancer xenografts. The binding of(99m)Tc-duramycin to phosphatidylethanolamine was validated in vitro using paclitaxel-treated human breast carcinoma MDA-MB-231 cells. Female BALB/c mice (n = 5) bearing breast cancer xenografts were randomized into 2 groups and intraperitoneally injected with 40 mg/kg paclitaxel or phosphate-buffered saline.(99m)Tc-duramycin (37-55.5 MBq) was injected at 72 hours posttreatment, and single-photon emission computed tomography/computed tomography was performed at 2 hours postinjection. Apoptotic cells and activated caspase 3 in explanted tumor tissue were measured by flow cytometry. Cellular ultrastructural changes were assessed by light and transmission electron microscopy.(99m)Tc-duramycin with radiochemical purity of >90% exhibited rapid blood clearance and predominantly renal clearance. The tumor-to-muscle ratio in the paclitaxel-treated group (5.29 ± 0.62) was significantly higher than that in the control. Tumor volume was decreased dramatically, whereas tumor uptake of(99m)Tc-duramycin (ex vivo) significantly increased following paclitaxel treatment, which was consistent with apoptotic index, histological findings, and ultrastructural changes. Our data demonstrated the feasibility of(99m)Tc-duramycin for early detection of apoptosis after paclitaxel chemotherapy in breast carcinoma xenografts.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Bacteriocinas/administração & dosagem , Neoplasias da Mama/diagnóstico por imagem , Compostos de Organotecnécio/administração & dosagem , Paclitaxel/administração & dosagem , Compostos Radiofarmacêuticos/administração & dosagem , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Bacteriocinas/farmacocinética , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Estudos de Viabilidade , Feminino , Humanos , Camundongos , Compostos de Organotecnécio/farmacocinética , Paclitaxel/farmacologia , Fosfatidiletanolaminas/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Distribuição Aleatória , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: The goal was to determine the feasibility of mapping the injured-but-not-infarcted myocardium using 99mTc-duramycin in the postischemic heart, with spatial information for its characterization as a pathophysiologically intermediate tissue, which is neither normal nor infarcted. METHODS AND RESULTS: Coronary occlusion was conducted in Sprague Dawley rats with preconditioning and 30-minute ligation. In vivo single-photon emission computed tomography was acquired after 3 hours (n=6) using 99mTc-duramycin, a phosphatidylethanolamine-specific radiopharmaceutical. The 99mTc-duramycin+ areas were compared with infarct and area-at-risk (n=8). Cardiomyocytes and endothelial cells were isolated for gene expression profiling. Cardiac function was measured with echocardiography (n=6) at 4 weeks. In vivo imaging with 99mTc-duramycin identified the infarct (3.9±2.4% of the left ventricle and an extensive area 23.7±2.2% of the left ventricle) with diffuse signal outside the infarct, which is pathologically between normal and infarcted (apoptosis 1.8±1.6, 8.9±4.2, 13.6±3.8%; VCAM-1 [vascular cell adhesion molecule 1] 3.2±0.8, 9.8±4.1, 15.9±4.2/mm2; tyrosine hydroxylase 14.9±2.8, 8.6±4.4, 5.6±2.2/mm2), with heterogeneous changes including scattered micronecrosis, wavy myofibrils, hydropic change, and glycogen accumulation. The 99mTc-duramycin+ tissue is quantitatively smaller than the area-at-risk (26.7% versus 34.4% of the left ventricle, P=0.008). Compared with infarct, gene expression in the 99mTc-duramycin+-noninfarct tissue indicated a greater prosurvival ratio (BCL2/BAX [B-cell lymphoma 2/BCL2-associated X] 7.8 versus 5.7 [cardiomyocytes], 3.7 versus 3.2 [endothelial]), and an upregulation of ion channels in electrophysiology. There was decreased contractility at 4 weeks (regional fractional shortening -8.6%, P<0.05; circumferential strain -52.9%, P<0.05). CONCLUSIONS: The injured-but-not-infarcted tissue, being an intermediate zone between normal and infarct, is mapped in vivo using phosphatidylethanolamine-based imaging. The intermediate zone contributes significantly to cardiac dysfunction.
Assuntos
Modelos Animais de Doenças , Infarto do Miocárdio , Peptídeos , Compostos Radiofarmacêuticos , Ratos Sprague-Dawley , Tomografia Computadorizada de Emissão de Fóton Único , Animais , Infarto do Miocárdio/patologia , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/diagnóstico por imagem , Masculino , Miocárdio/patologia , Miocárdio/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/metabolismo , Bacteriocinas/metabolismo , Estudos de Viabilidade , Ratos , Perfilação da Expressão Gênica/métodos , Função Ventricular Esquerda , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Compostos de OrganotecnécioRESUMO
BACKGROUND: Imaging of cell death can provide an early indication of treatment response in cancer. [99mTc]Tc-Duramycin is a small-peptide SPECT tracer that recognizes both apoptotic and necrotic cells by binding to phosphatidylethanolamine present in the cell membrane. Preclinically, this tracer has shown to have favorable pharmacokinetics and selective tumor accumulation early after the onset of anticancer therapy. In this first-in-human study, we report the safety, biodistribution and internal radiation dosimetry of [99mTc]Tc-Duramycin in healthy human volunteers. RESULTS: Six healthy volunteers (3 males, 3 females) were injected intravenously with [99mTc]Tc-Duramycin (dose: 6 MBq/kg; 473 ± 36 MBq). [99mTc]Tc-Duramycin was well tolerated in all subjects, with no serious adverse events reported. Following injection, a 30-min dynamic planar imaging of the abdomen was performed, and whole-body (WB) planar scans were acquired at 1, 2, 3, 6 and 23 h post-injection (PI), with SPECT acquisitions after each WB scan and one low-dose CT after the first SPECT. In vivo 99mTc activities were determined from semi-quantitative analysis of the images, and time-activity curves were generated. Residence times were calculated from the dynamic and WB planar scans. The mean effective dose was 7.61 ± 0.75 µSv/MBq, with the kidneys receiving the highest absorbed dose (planar analysis: 43.82 ± 4.07 µGy/MBq, SPECT analysis: 19.72 ± 3.42 µGy/MBq), followed by liver and spleen. The median effective dose was 3.61 mSv (range, 2.85-4.14). The tracer cleared slowly from the blood (effective half-life of 2.0 ± 0.4 h) due to high plasma protein binding with < 5% free tracer 3 h PI. Excretion was almost exclusively renal. CONCLUSION: [99mTc]Tc-Duramycin demonstrated acceptable dosimetry (< 5 mSv) and a favorable safety profile. Due to slow blood clearance, optimal target-to-background ratios are expected 5 h PI. These data support the further assessment of [99mTc]Tc-Duramycin for clinical treatment response evaluation. TRIAL REGISTRATION: NCT05177640, Registered April 30, 2021, https://clinicaltrials.gov/study/NCT05177640 .
RESUMO
Cell death is involved in numerous pathological conditions such as cardiovascular disorders, ischemic stroke and organ transplant rejection, and plays a critical role in the treatment of cancer. Cell death imaging can serve as a noninvasive means to detect the severity of tissue damage, monitor the progression of diseases, and evaluate the effectiveness of treatments, which help to provide prognostic information and guide the formulation of individualized treatment plans. The high abundance of phosphatidylethanolamine (PE), which is predominantly confined to the inner leaflet of the lipid bilayer membrane in healthy mammalian cells, becomes exposed on the cell surface in the early stages of apoptosis or accessible to the extracellular milieu when the cell suffers from necrosis, thus representing an attractive target for cell death imaging. Duramycin is a tetracyclic polypeptide that contains 19 amino acids and can bind to PE with excellent affinity and specificity. Additionally, this peptide has several favorable structural traits including relatively low molecular weight, stability to enzymatic hydrolysis, and ease of conjugation and labeling. All these highlight the potential of duramycin as a candidate ligand for developing PE-specific molecular probes. By far, a couple of duramycin-based molecular probes such as Tc-99 m-, F-18-, or Ga-68-labeled duramycin have been developed to target exposed PE for in vivo noninvasive imaging of cell death in different animal models. In this review article, we describe the state of the art with respect to in vivo imaging of cell death using duramycin-based molecular probes, as validated by immunohistopathology.
Assuntos
Bacteriocinas , Compostos de Organotecnécio , Animais , Bacteriocinas/química , Morte Celular , Radioisótopos de Gálio , Mamíferos/metabolismo , Sondas Moleculares , Peptídeos/química , Tomografia Computadorizada de Emissão de Fóton Único/métodosRESUMO
OBJECTIVE: The objective of this study is to explore the radiosensitization effects of duramycin against the liver cancer hepatoma cells and relationship to reactive oxygen species (ROS) generation. MATERIALS AND METHODS: MCA-RH 7777 cells were treated with various combinations of duramycin concentrations and radiation doses. After the treatment, cell viabilities were determined by a cell proliferation assay; intracellular ROS levels were detected with the flow cytometric method. RESULTS: MCA-RH 7777 cell viability was found significantly reduced after combining duramycin and radiation exposure (comparing to that of either treatment alone). Increased intracellular ROS levels were observed in cells treated with combinations of duramycin and radiation. CONCLUSION: Duramycin increased the intracellular ROS generation and also increased the radiosensitivity of MCA-RH 7777 cells.
Assuntos
Bacteriocinas/farmacologia , Carcinoma Hepatocelular/terapia , Quimiorradioterapia/métodos , Neoplasias Hepáticas/terapia , Peptídeos/farmacologia , Radiossensibilizantes/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Bacteriocinas/uso terapêutico , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Neoplasias Hepáticas/patologia , Peptídeos/uso terapêutico , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/uso terapêutico , Espécies Reativas de Oxigênio/análise , Espécies Reativas de Oxigênio/metabolismoRESUMO
This study aimed to dynamically monitor myocardial cell death using 99m Tc-Duramycin single-photon emission computed tomography/computed tomography (micro-SPECT/CT) imaging in acute myocardial infarction (AMI) and the anti-apoptosis effect of atorvastatin for cardioprotection. Mice were randomized into three groups: AMI group, AMI with atorvastatin treatment (T-AMI) group, and sham group. Three groups of model mice were randomly selected at day 1 (D1), day 3 (D3), and day 7 (D7) day after surgery with 99m Tc-Duramycin micro-SPECT/CT imaging. The lesion-to-normal myocardial tissue ratio (L/N) average values were 2.62 on D1, 3.89 on D3, and 1.20 on D7 for the uptake of 99m Tc-duramycin in the infarcted region in the AMI group. The sham group presented no positive imaging in myocardium, and the L/N average values were 1.09, 1.14, and 1.10 on D1, D3, and D7, respectively. Meanwhile, 99m Tc-linear-duramycin imaging showed no radioactive uptake in the infarction region. The T-AMI group imaging showed tracer uptake decreased obviously compared to the uptake in the infarcted region in AMI mice. 99m Tc-Duramycin SPECT/CT imaging allowed non-invasive monitoring of myocardial cell death in a mouse model of AMI and an assessment of atorvastatin anti-apoptosis effect for cardioprotection by in vivo molecular imaging.
Assuntos
Apoptose/efeitos dos fármacos , Atorvastatina/farmacologia , Bacteriocinas/química , Cardiotônicos/farmacologia , Infarto do Miocárdio/diagnóstico por imagem , Peptídeos/química , Compostos Radiofarmacêuticos/química , Doença Aguda , Animais , Atorvastatina/uso terapêutico , Bacteriocinas/farmacologia , Cardiotônicos/uso terapêutico , Modelos Animais de Doenças , Camundongos , Infarto do Miocárdio/tratamento farmacológico , Miocárdio/citologia , Miocárdio/metabolismo , Miocárdio/patologia , Peptídeos/farmacologia , Compostos Radiofarmacêuticos/farmacologia , Tomografia Computadorizada com Tomografia Computadorizada de Emissão de Fóton Único , Tecnécio/químicaRESUMO
PURPOSE: Evaluation of [68Ga]NODAGA-duramycin as a positron emission tomography (PET) tracer of cell death for whole-body detection of chemotherapy-induced organ toxicity. PROCEDURES: Tracer specificity of Ga-68 labeled NODAGA-duramycin was determined in vitro using competitive binding experiments. Organ uptake was analyzed in untreated and doxorubicin, busulfan, and cisplatin-treated mice 2 h after intravenous injection of [68Ga]NODAGA-duramycin. In vivo data were validated by immunohistology and blood parameters. RESULTS: In vitro experiments confirmed specific binding of [68Ga]NODAGA-duramycin. Organ toxicities were detected successfully using [68Ga]NODAGA-duramycin PET/X-ray computed tomography (CT) and confirmed by immunohistochemistry and blood parameter analysis. Organ toxicities in livers and kidneys showed similar trends in PET/CT and immunohistology. Busulfan and cisplatin-related organ toxicities in heart, liver, and lungs were detected earlier by PET/CT than by blood parameters and immunohistology. CONCLUSION: [68Ga]NODAGA-duramycin PET/CT was successfully applied to non-invasively detect chemotherapy-induced organ toxicity with high sensitivity in mice. It, therefore, represents a promising alternative to standard toxicological analyses with a high translational potential.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Bacteriocinas , Radioisótopos de Gálio , Rim/efeitos dos fármacos , Rim/diagnóstico por imagem , Fígado/efeitos dos fármacos , Fígado/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Peptídeos , Acetatos/química , Acetatos/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Bacteriocinas/química , Bacteriocinas/farmacocinética , Bussulfano/administração & dosagem , Cisplatino/administração & dosagem , Doxorrubicina/administração & dosagem , Feminino , Radioisótopos de Gálio/química , Radioisótopos de Gálio/farmacocinética , Compostos Heterocíclicos com 1 Anel/química , Compostos Heterocíclicos com 1 Anel/metabolismo , Rim/metabolismo , Rim/patologia , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/patologia , Peptídeos/química , Peptídeos/farmacocinética , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Distribuição TecidualRESUMO
BACKGROUND: Apoptosis in atherosclerotic lesions contributes to plaque vulnerability by lipid core enlargement and fibrous cap attenuation. Apoptosis is associated with exteriorization of phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the cell membrane. Although PS-avid radiolabeled annexin-V has been employed for molecular imaging of high-risk plaques, PE-targeted imaging in atherosclerosis has not been studied. OBJECTIVES: This study sought to evaluate the feasibility of molecular imaging with PE-avid radiolabeled duramycin in experimental atherosclerotic lesions in a rabbit model and compare duramycin targeting with radiolabeled annexin-V. METHODS: Of the 27 rabbits, 21 were fed high-cholesterol, high-fat diet for 16 weeks. Nine of the 21 rabbits received 99mTc-duramycin (test group), 6 received 99mTc-linear duramycin (duramycin without PE-binding capability, negative radiotracer control group), and 6 received 99mTc-annexin-V for radionuclide imaging. The remaining normal chow-fed 6 animals (disease control group) received 99mTc-duramycin. In vivo microSPECT/microCT imaging was performed, and the aortas were explanted for ex vivo imaging and for histological characterization of atherosclerosis. RESULTS: A significantly higher duramycin uptake was observed in the test group compared with that of disease control and negative radiotracer control animals; duramycin uptake was also significantly higher than the annexin-V uptake. Quantitative duramycin uptake, represented as the square root of percent injected dose per cm (âID/cm) of abdominal aorta was >2-fold higher in atherosclerotic lesions in test group (0.08 ± 0.01%) than in comparable regions of disease control animals (0.039 ± 0.0061%, p = 3.70·10-8). Mean annexin uptake (0.060 ± 0.010%) was significantly lower than duramycin (p = 0.001). Duramycin uptake corresponded to the lesion severity and macrophage burden. The radiation burden to the kidneys was substantially lower with duramycin (0.49% ID/g) than annexin (5.48% ID/g; p = 4.00·10-4). CONCLUSIONS: Radiolabeled duramycin localizes in lipid-rich areas with high concentration of apoptotic macrophages in the experimental atherosclerosis model. Duramycin uptake in atherosclerotic lesions was significantly greater than annexin-V uptake and produced significantly lower radiation burden to nontarget organs.
Assuntos
Apoptose/fisiologia , Aterosclerose/metabolismo , Membrana Celular/metabolismo , Imagem Molecular/métodos , Fosfolipídeos/metabolismo , Animais , Aterosclerose/diagnóstico por imagem , Aterosclerose/etiologia , Bacteriocinas/metabolismo , Membrana Celular/patologia , Dieta Hiperlipídica/efeitos adversos , Humanos , Masculino , Peptídeos/metabolismo , Coelhos , Cintilografia/métodosRESUMO
PURPOSE: 99mTc-duramycin imaging enables specific visualization of cell death qualitatively and quantitatively. This study aimed to investigate the potential of 99mTc-duramycin imaging in the early prediction of the curative effect of radiotherapy in combination with or without cetuximab in a nasopharyngeal carcinoma (NPC) model. METHODS: Male BALB/c mice bearing NPC xenografts were randomized into four groups (six mice each group). Group 1 received radiotherapy (RT, 15 Gy/mouse) in combination with cetuximab (CTX, 2 mg/mouse), group 2 received RT (15 Gy/mouse), group 3 was treated using CTX (2 mg/mouse), and group 4, the control group, was treated using a vehicle. 99mTc-duramycin imaging was performed before treatment and 24 h after treatment to evaluate tumor response. Tumor uptake of 99mTc-duramycin was validated ex vivo using γ-counting. Treatment response was further validated by cleaved caspase-3 (CC3) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL). Another four groups were treated parallelly under the same conditions to observe treatment response by tumor volume changes. RESULTS: After 24 h treatment, 99mTc-duramycin uptake in the NPC tumor models were significantly higher in group 1 than in group 2 (P < 0.05), group 3 (P < 0.05), or group 4 (P < 0.05); the uptake also increased notably in comparison with baseline values (P < 0.05). Compared with group 4, group 2 and group 3 both showed significant 99mTc-duramycin uptake in the tumors (P < 0.05). Although the 99mTc-duramycin uptake of group 2 was moderately higher than group 3, there were no significant differences between these two groups (P >0.05). There was a strong positive correlation between tumor 99mTc-duramycin uptake and CC3 (r = 0.893, p < 0.0001) and TUNEL (r = 0.918, P < 0.0001). Tumor volume decreased remarkably in the RT in combination with CTX group on day 5, in the RT alone group on day 7, and was inhibited on day 8 in the CTX alone group, whereas the tumors grew continuously in the control group. CONCLUSIONS: We demonstrated that RT in combination with CTX treatment significantly improved disease control in a NPC xenograft model compared with monotherapy with either. 99mTc-duramycin imaging might be able to reliably identify response to RT in combination with CTX as early as 24 h after therapy initiation in NPC xenograft models. This might help to isolate non-responding patients in a timely manner and avoid unnecessary side effects in the clinic in the future.
Assuntos
Bacteriocinas/administração & dosagem , Cetuximab/farmacologia , Carcinoma Nasofaríngeo/terapia , Neoplasias Nasofaríngeas/terapia , Peptídeos/administração & dosagem , Animais , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/farmacologia , Linhagem Celular Tumoral , Cetuximab/administração & dosagem , Terapia Combinada , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/patologia , Tecnécio/química , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/efeitos da radiação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: Both phosphatidylethanolamine (PE) and phosphatidylserine (PS) can be externalized to the outer cell membrane in apoptosis. Thus the objective was to determine whether PE-targeting 18F-duramycin and PS-targeting 18F-Zn-DPA could be used for imaging apoptosis. METHODS: Duramycin and Zn-DPA were labeled with either 18F-Al or 18F-SFB. U937 cells were incubated with four different concentrations of camptothecin (CPT). For assessing the effect of incubation time on uptake, 37â¯MBq of radiotracer was added to cells incubated for 15, 30, 60, and 120â¯min at 37⯰C. For blocking experiments, 150⯵g duramycin and 40⯵g Zn-DPA were added to cells for 15â¯min prior to the addition of either duramycin or Zn-DPA labeled with 18F. Apoptosis was measured by flow cytometry using an annexin-V/PI kit. Cells were co-stained with Hoechst, Cy5-duramycin, and PSVue480 (FITC-Zn-DPA) to localize fluorescent dye uptake in cells. RESULTS: Apoptosis in cells increased proportionally with CTP as confirmed by both flow cytometry and fluorescent staining. Both FITC-Zn-DPA and FITC-duramycin localized mainly on the cell membrane during early apoptosis and then translocated to the inside during late apoptosis. Uptake of FITC-duramycin, however, was higher than that of FITC-Zn-DPA. Cellular uptake of four different radiotracers was also proportional to the degree of apoptosis, increasing slightly over time and reaching a plateau at about 1â¯h. The blocking experiments demonstrated that uptake in all the control groups was predominantly non-specific, whereas the specific uptake in all the treated groups was at least 50% for both 18F labeled duramycin and Zn-DPA. CONCLUSION: Both PE-targeting 18F-duramycin and PS-targeting 18F-Zn-DPA could be considered as potential radiotracers for imaging cellular apoptosis. Advances in knowledge and implications for patient care: Cellular data support the further development of radiotracers targeting either PE or PS for imaging apoptosis, which can associate with clinical outcome for cancer patients.
Assuntos
Apoptose , Imagem Molecular/métodos , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/metabolismo , Apoptose/efeitos dos fármacos , Bacteriocinas/química , Bacteriocinas/metabolismo , Transporte Biológico , Camptotecina/farmacologia , Linhagem Celular Tumoral , Difenilamina/química , Difenilamina/metabolismo , Radioisótopos de Flúor , Humanos , Peptídeos/química , Peptídeos/metabolismo , RadioquímicaRESUMO
INTRODUCTION: The objective of this study was to investigate the cardioprotective effects of a dodecafluoropentane (DDFP)-based perfluorocarbon emulsion (DDFPe) as an artificial carrier for oxygen delivery to ischemic myocardium, using 99mTc-duramycin SPECT imaging. METHODS: Rat hearts with Ischemia-reperfusion (I/R) was prepared by coronary ligation for 45-min followed by reperfusion. The feasibility of 99mTc-duramycin in detecting myocardial I/R injury and its kinetic profile were first verified in the ischemic hearts with 2-h reperfusion (nâ¯=â¯6). DDFPe (0.6â¯mL/kg) was intravenously administered at 10â¯min after coronary ligation in fifteen rats and saline was given in thirteen rats as controls. 99mTc-duramycin SPECT images were acquired in the DDFPe-treated hearts and saline controls at 2-h (DDFPe-2â¯h, nâ¯=â¯7 and Saline-2â¯h, nâ¯=â¯6) or 24-h (DDFPe-24â¯h, nâ¯=â¯8 and Saline-24â¯h, nâ¯=â¯7) of reperfusion. RESULTS: SPECT images, showing "hot-spot" 99mTc-duramycin uptake in the ischemic myocardium, exhibited significantly lower radioactive retention and smaller hot-spot size in the DDFPe-2â¯h and DDFPe-24â¯h hearts compared to controls. The infarcts in the Saline-24â¯h hearts extended significantly relative to measurements in the Saline-2â¯h. The extension of infarct size did not reach a statistical difference between the DDFPe-2â¯h and DDFPe-24â¯h hearts. Ex vivo measurement of 99mTc-duramycin activity (%ID/g) was lower in the ischemic area of DDFPe-2â¯h and DDFPe-24â¯h than that of the Saline-2â¯h and Saline-24â¯h hearts (Pâ¯<â¯0.05). The area of injured myocardium, delineated by the uptake of 99mTc-duramycin, extended more substantially outside the infarct zone in the controls. CONCLUSIONS: Significant reduction in myocardial I/R injury, as assessed by 99mTc-duramycin cell death imaging and histopathological analysis, was induced by DDFPe treatment after acute myocardial ischemia. 99mTc-duramycin imaging can reveal myocardial cell death in ischemic hearts and may provide a tool for the non-invasive assessment of cardioprotective interventions.
Assuntos
Cardiotônicos/administração & dosagem , Cardiotônicos/farmacologia , Fluorocarbonos/administração & dosagem , Fluorocarbonos/farmacologia , Infarto do Miocárdio/diagnóstico por imagem , Infarto do Miocárdio/metabolismo , Oxigênio/metabolismo , Tomografia Computadorizada de Emissão de Fóton Único , Animais , Bacteriocinas , Humanos , Cinética , Infarto do Miocárdio/patologia , Miocárdio/metabolismo , Compostos de Organotecnécio , Ratos , Ratos Sprague-DawleyRESUMO
Non-invasive imaging of apoptosis in tumors induced by chemotherapy is of great value in the evaluation of therapeutic efficiency. In this study, we report the synthesis, characterization, and utilization of radionuclide technetium-99m (99mTc)-labeled dendrimer-entrapped gold nanoparticles (Au DENPs) for targeted SPECT/CT imaging of chemotherapy-induced tumor apoptosis. Generation five poly(amidoamine) (PAMAM) dendrimers (G5.NH2) were sequentially conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), polyethylene glycol (PEG) modified duramycin, PEG monomethyl ether, and fluorescein isothiocyanate (FI) to form the multifunctional dendrimers, which were then utilized as templates to entrap gold nanoparticles. Followed by acetylation of the remaining dendrimer surface amines and radiolabeling of 99mTc, the SPECT/CT dual mode nanoprobe of tumor apoptosis was constructed. The developed multifunctional Au DENPs before and after 99mTc radiolabeling were well characterized. The results demonstrate that the multifunctional Au DENPs display favorable colloidal stability under different conditions, own good cytocompatibility in the given concentration range, and can be effectively labeled by 99mTc with high radiochemical stability. Furthermore, the multifunctional nanoprobe enables the targeted SPECT/CT imaging of apoptotic cancer cells in vitro and tumor apoptosis after doxorubicin (DOX) treatment in the established subcutaneous tumor model in vivo. The designed duramycin-functionalized Au DENPs might have the potential to be employed as a nanoplatform for the detection of apoptosis and early tumor response to chemotherapy.