RESUMO
OBJECTIVE: The objective of this study is to develop a novel method for monitoring the integrity of motor neurons in vivo by quantifying net retrograde axonal transport. METHODS: The method uses single photon emission computed tomography to quantify retrograde transport to spinal cord of tetanus toxin fragment C (125 I-TTC) following intramuscular injection. We characterized the transport profiles in 3 transgenic mouse models carrying amyotrophic lateral sclerosis (ALS)-associated genes, aging mice, and SOD1G93A transgenic mice following CRISPR/Cas9 gene editing. Lastly, we studied the effect of prior immunization of tetanus toxoid on the transport profile of TTC. RESULTS: This technique defines a quantitative profile of net retrograde axonal transport of TTC in living mice. The profile is distinctly abnormal in transgenic SOD1G93A mice as young as 65 days (presymptomatic) and worsens with disease progression. Moreover, this method detects a distinct therapeutic benefit of gene editing in transgenic SOD1G93A mice well before other clinical parameters (eg, grip strength) show improvement. Symptomatic transgenic PFN1C71G/C71G ALS mice display gross reductions in net retrograde axonal transport, which is also disturbed in asymptomatic mice harboring a human C9ORF72 transgene with an expanded GGGGCC repeat motif. In wild-type mice, net retrograde axonal transport declines with aging. Lastly, prior immunization with tetanus toxoid does not preclude use of this assay. INTERPRETATION: This assay of net retrograde axonal transport has broad potential clinical applications and should be particularly valuable as a physiological biomarker that permits early detection of benefit from potential therapies for motor neuron diseases. ANN NEUROL 2022;91:716-729.
Assuntos
Esclerose Lateral Amiotrófica , Transporte Axonal , Esclerose Lateral Amiotrófica/diagnóstico por imagem , Esclerose Lateral Amiotrófica/genética , Animais , Transporte Axonal/genética , Biomarcadores , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Profilinas , Medula Espinal/diagnóstico por imagem , Medula Espinal/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1 , Toxoide TetânicoRESUMO
For many cancer types, being undetectable from early symptoms or blood tests, or often detected at late stages, medical imaging emerges as the most efficient tool for cancer screening. MRI, ultrasound, X-rays (mammography), and X-ray CT (CT) are currently used in hospitals with variable costs. Diagnostic materials that can detect breast tumors through molecular recognition and amplify the signal at the targeting site in combination with state-of-the-art CT techniques, such as dual-energy CT, could lead to a more precise detection and assist significantly in image-guided intervention. Herein, we have developed a ligand-specific X-ray contrast agent that recognizes α5ß1 integrins overexpressed in MDA-MB-231 breast cancer cells for detection of triple (-) cancer, which proliferates very aggressively. In vitro studies show binding and internalization of our nanoprobes within those cells, towards uncoated nanoparticles (NPs) and saline. In vivo studies show high retention of ~3 nm ligand-PEG-S-AuNPs in breast tumors in mice (up to 21 days) and pronounced CT detection, with statistical significance from saline and iohexol, though only 0.5 mg of metal were utilized. In addition, accumulation of ligand-specific NPs is shown in tumors with minimal presence in other organs, relative to controls. The prolonged, low-metal, NP-enhanced spectral-CT detection of triple (-) breast cancer could lead to breakthrough advances in X-ray cancer diagnostics, nanotechnology, and medicine.
Assuntos
Nanopartículas Metálicas , Neoplasias , Animais , Meios de Contraste/química , Ouro/química , Ligantes , Mamografia/métodos , Nanopartículas Metálicas/química , Camundongos , Tomografia Computadorizada por Raios X/métodosRESUMO
Cell death is involved in many pathological conditions, and there is a need for clinical and preclinical imaging agents that can target and report cell death. One of the best known biomarkers of cell death is exposure of the anionic phospholipid phosphatidylserine (PS) on the surface of dead and dying cells. Synthetic zinc(II)-bis(dipicolylamine) (Zn2BDPA) coordination complexes are known to selectively recognize PS-rich membranes and act as cell death molecular imaging agents. However, there is a need to improve in vivo imaging performance by selectively increasing target affinity and decreasing off-target accumulation. This present study compared the cell death targeting ability of two new deep-red fluorescent probes containing phenoxide-bridged Zn2BDPA complexes. One probe was a bivalent version of the other and associated more strongly with PS-rich liposome membranes. However, the bivalent probe exhibited self-quenching on the membrane surface, so the monovalent version produced brighter micrographs of dead and dying cells in cell culture and also better fluorescence imaging contrast in two living animal models of cell death (rat implanted tumor with necrotic core and mouse thymus atrophy). An (111)In-labeled radiotracer version of the monovalent probe also exhibited selective cell death targeting ability in the mouse thymus atrophy model, with relatively high amounts detected in dead and dying tissue and low off-target accumulation in nonclearance organs. The in vivo biodistribution profile is the most favorable yet reported for a Zn2BDPA complex; thus, the monovalent phenoxide-bridged Zn2BDPA scaffold is a promising candidate for further development as a cell death imaging agent in living subjects.
Assuntos
Morte Celular , Corantes Fluorescentes/química , Imagem Molecular/métodos , Neoplasias/patologia , Compostos Organometálicos/química , Ácidos Picolínicos/química , Timo/patologia , Animais , Atrofia/diagnóstico , Atrofia/patologia , Células CHO , Linhagem Celular Tumoral , Cricetulus , Feminino , Masculino , Camundongos , Microscopia de Fluorescência/métodos , Neoplasias/diagnóstico , Imagem Óptica/métodos , Fosfatidilserinas/análise , Ratos , Ratos Wistar , Timo/citologiaRESUMO
An oxygen sensor-mounted fine-needle biopsy tool was used for in vivo measurement of oxygen levels in tumor xenografts. The system provides a means of measuring the oxygen content in harvested tumor tissue from specific locations. Oxygen in human tumor xenografts in a murine model was observed for over 1 min. Tissues were mapped in relation to oxygen tension (pO2) readings and sampled for conventional cytological examination. Careful modeling of the pO2 readings over 60 seconds yielded a diffusion coefficient for oxygen at the sensor tip, providing additional diagnostic information about the tissue before sampling. Oxygen level measurement may provide a useful adjunct to the use of biomarkers in tumor diagnosis.
RESUMO
PURPOSE: Radiolabeled oligomers complementary to the 16S rRNA in bacteria were investigated as bacterial infection imaging agents. METHODS AND RESULTS: Identical sequences with backbones phosphorodiamidate morpholino (MORF), peptide nucleic acid (PNA), and phosphorothioate DNA (PS-DNA) were (99m)Tc-labeled and evaluated for binding to bacterial RNA. MORF binding to RNA from Escherichia coli strains SM101 and K12 was 4- and 150-fold higher compared to PNA and PS-DNA, respectively. Subsequently MORF oligomer in fluorescence in situ hybridization showed a stronger signal with study MORF compared to control in fixed preparations of two E. coli strains and Klebsiella pneumoniae. Flow cytometry analysis showed study MORF accumulation to be 8- and 80-fold higher compared to the control in live K. pneumoniae and Staphylococcus aureus, respectively. Further, fluorescence microscopy showed increased accumulation of study MORF over control in live E. coli and K. pneumonia. Binding of (99m)Tc-study MORF to RNA from E. coli SM101 and K12 was 30.4 and 117.8pmol, respectively, per 10(10) cells. Mice with K. pneumoniae live or heat-killed (sterile inflammation) in one thigh at 90min for both (99m)Tc-study MORF and control showed higher accumulation in target thighs than in blood and all other organs expect for kidneys and small intestine. Accumulation of (99m)Tc-study MORF was significantly higher (p=0.009) than that of the control in the thigh with sterile inflammation. CONCLUSION: A (99m)Tc-MORF oligomer complimentary to the bacterial 16S rRNA demonstrated binding to bacterial RNA in vitro with specific accumulation into live bacteria. Radiolabeled MORF oligomers antisense to the bacterial rRNA may be useful to image bacterial infection.
Assuntos
Morfolinos/química , Compostos de Organotecnécio/química , RNA Bacteriano/metabolismo , Compostos Radiofarmacêuticos/química , Animais , Infecções Bacterianas/diagnóstico , Escherichia coli/genética , Meia-Vida , Hibridização in Situ Fluorescente , Klebsiella pneumoniae/genética , Camundongos , Microscopia de Fluorescência , Morfolinos/farmacocinética , RNA Ribossômico 16S/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Staphylococcus aureus/genética , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único , Tomografia Computadorizada por Raios XRESUMO
Despite the central role of IFN-γ in vitiligo pathogenesis, systemic IFN-γ neutralization is an impractical treatment option owing to strong immunosuppression. However, most patients with vitiligo present with <20% affected body surface area, which provides an opportunity for localized treatments that avoid systemic side effects. After identifying keratinocytes as key cells that amplify IFN-γ signaling during vitiligo, we hypothesized that tethering an IFN-γâneutralizing antibody to keratinocytes would limit antiâIFN-γ effects on the treated skin for the localized treatment. To that end, we developed a bispecific antibody capable of blocking IFN-γ signaling while binding to desmoglein expressed by keratinocytes. We characterized the effect of the bispecific antibody in vitro, ex vivo, and in a mouse model of vitiligo. Single-photon emission computed tomography/computed tomography biodistribution and serum assays after local footpad injection revealed that the bispecific antibody had improved skin retention, faster elimination from the blood, and less systemic IFN-γ inhibition than the nontethered version. Furthermore, the bispecific antibody conferred localized protection almost exclusively to the treated footpad during vitiligo, which was not possible by local injection of the nontethered antiâIFN-γ antibody. Thus, keratinocyte tethering proved effective while significantly diminishing the off-tissue effects of IFN-γ blockade, offering a safer treatment strategy for localized skin diseases, including vitiligo.
Assuntos
Produtos Biológicos , Vitiligo , Camundongos , Animais , Vitiligo/tratamento farmacológico , Distribuição Tecidual , Queratinócitos/metabolismo , Pele/patologia , Produtos Biológicos/uso terapêuticoRESUMO
Recent advances in small-animal molecular imaging instrumentation combined with well characterized antibody-labeling chemistry have enabled detailed in vivo measurements of antibody distribution in mouse models. This article reviews the strengths and limitations of in vivo antibody imaging methods with a focus on positron emission tomography and single-photon emission computed tomography and a brief discussion of the role of optical imaging in this application. A description of the basic principles behind the imaging techniques is provided along with a discussion of radiolabeling methods relevant to antibodies. Practical considerations of study design and execution are presented through a discussion of sensitivity and resolution tradeoffs for these techniques as defined by modality, signaling probe (isotope or fluorophore) selection, labeling method, and radiation dosimetry. Images and analysis results from a case study are presented with a discussion of output data content and relevant informatics gained with this approach to studying antibody pharmacokinetics.
Assuntos
Anticorpos/metabolismo , Diagnóstico por Imagem/métodos , Animais , Fluorescência , Processamento de Imagem Assistida por Computador , Luminescência , Camundongos , Farmacocinética , Física , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/química , Tomografia Computadorizada de Emissão de Fóton Único/métodosRESUMO
While (188)Re has been used successfully in mice for tumor radiotherapy by MORF/cMORF pretargeting, previous radiolabeling of the amine-derivatized cMORF with (90)Y, a longer physical half-life nuclide, was not very successful. After developing a method involving a prepurification heating step during conjugation that increases labeling efficiency and label stability, the biodistribution of (90)Y-DOTA-Bn-SCN-cMORF ((90)Y-DOTA-cMORF) was measured in normal mice and in MORF-CC49 pretargeted mice that bear LS174T tumors. Absorbed radiation doses were then estimated and compared to those estimated for (188)Re. The pharmacokinetics of the (90)Y-DOTA-cMORF in normal mice and in the pretargeted nude mice was similar to that observed previously with (99m)Tc- and (188)Re-MAG(3)-cMORFs. While the (90)Y-DOTA-cMORF cleared rapidly from normal tissues, tumor clearance was very slow and tumor radioactivity accumulation was constant for at least 7 days such that the tumor/blood (T/B) ratio increased linearly from 6 to 25 over this period. Therefore, by extrapolation, normal tissue toxicities following administration of therapeutic doses of (90)Y may be comparable to that observed for (188)Re in which the T/B increased from 5 to 20. In conclusion, radiolabeling of DOTA-cMORF with (90)Y was improved by introducing a prepurification heating step during conjugation. The (90)Y-DOTA-cMORF provided a similar T/B ratio and biodistribution to that of (188)Re-MAG(3)-cMORF and was retained well in the tumor pretargeted with MORF-CC49. Because of the longer physical half-life, the T/NT absorbed radiation dose ratios were improved in most organs and especially in blood.
Assuntos
Compostos Heterocíclicos/química , Compostos Heterocíclicos/farmacocinética , Morfolinos/química , Morfolinos/farmacocinética , Neoplasias/radioterapia , Compostos Organometálicos/química , Compostos Organometálicos/farmacocinética , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Animais , Camundongos , Camundongos NusRESUMO
In principle, a DNA duplex consisting of an antisense fluorophore-conjugated major strand hybridized to a shorter complementary inhibitor-conjugated minor strand should provide fluorescence only in the tumor after intravenous administration if designed to remain intact except in the presence in tumor of its mRNA target. While we have obtained impressive tumor images in mice using this approach, there remains some background fluorescence. In this study, tissue homogenates of selected mouse organs were incubated with a test duplex and the kinetics of duplex dissociation in normal tissues were measured. In this manner we were able to identify the liver as the likely major source responsible for the duplex dissociation providing this fluorescence background. Thereafter liver homogenates were used to screen a series of duplex candidates with variable-length minor strands, and dissociation was measured by gel electrophoresis. The selected fluorophore/inhibitor duplex with improved stability displayed an insignificant (P > 0.05) background fluorescence after administration to SKH-1 normal mice and apparently without affecting target mRNA binding in vitro in cell culture or in vivo in tumor bearing mice.
Assuntos
DNA/análise , Diagnóstico por Imagem/métodos , Fluorescência , Corantes Fluorescentes/química , Neoplasias Cutâneas/metabolismo , Animais , Linhagem Celular Tumoral , DNA/química , Humanos , Masculino , Camundongos , Camundongos NusRESUMO
Noninvasive measurement of human islet cell mass in pancreas or following islet transplantation by nuclear imaging has yet to be achieved. It has been shown using mouse tumor models that pretargeting imaging strategies are sensitive and can greatly increase target to nontarget signal ratios. The objective now is to demonstrate the specific pretargeting of human islet cells in mice. Our pretargeting strategy uses an anti-human islet cell antibody HPi1, conjugated to a phosphorodiamidate morpholino oligomer (MORF) that binds specifically to a (99m)Tc labeled complementary MORF (cMORF). Sensitivity and specificity of the pretargeting were first validated in culture using a human beta cell line (betalox5) and a negative control human cell line (HEK293). Pretargeting was then used to target and visualize these two cell lines and human islets transplanted subcutaneously in NOD-scid IL2rγ(null) mice. In culture, (99m)Tc accumulation on the betalox5 cells pretargeted by MORF-HPi1 was 100-fold higher than on untreated betalox5 cells or following treatment with native HPi1 and much higher than on the MORF-HPi1 pretargeted control HEK293 cells. Small animal imaging readily localized the transplanted betalox5 cells and human islets, but not the HEK293 cells. Ex vivo counting demonstrated 3-fold higher (99m)Tc accumulation in the transplanted betalox5 cells and human islets than in the control HEK293 cells. The target accumulation was also shown to increase linearly with increased numbers of the implanted betalox5 cells. These results demonstrate specific binding of radioactivity and successful imaging of human betalox5 cells and human islets transplanted in mice. Thus MORF/cMORF pretargeting may be useful to measure noninvasively human islet cell mass within the pancreas or following islet transplantation.
Assuntos
Anticorpos/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/transplante , Morfolinas/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , MorfolinosRESUMO
Our objective was to compare the performance of a micro-single photon emission computed tomography (micro-SPECT) with that of a micro-positron emission tomography (microPET) in a Her2+ tumored mice using an anti-Her2 nanoparticle radiolabeled with (99m)Tc and (18)F. Camera performance was first compared using phantoms; then a tumored mouse administered the (99m)Tc-nanoparticle was imaged on a Bioscan NanoSPECT/CT, while another tumored mouse received the identical nanoparticle, labeled now with (18)F, and was imaged on a Philips Mosaic HP PET camera. The nanoparticle was radiolabeled with (99m)Tc via MAG(3) chelation and with (18)F via SFB as an intermediate. Phantom imaging showed that the resolution of the SPECT camera was clearly superior, but even with 4 heads and multipinhole collimators, detection sensitivity was 15-fold lower. Radiolabeling of the nanoparticle by chelation with (99m)Tc was considerably easier and safer than manual covalent attachment of (18)F. Both cameras provided accurate quantitation of radioactivity over a broad range. In conclusion, when deciding between (99m)Tc vs (18)F, an advantage rests with the chelation of (99m)Tc over covalent attachment of (18)F, achieved manually or otherwise, but with these small animal cameras, this choice also results in trading lower sensitivity for higher resolution.
Assuntos
Morfolinas , Neoplasias Experimentais/diagnóstico , Tomografia por Emissão de Pósitrons/métodos , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Animais , Radioisótopos de Flúor , Camundongos , Morfolinas/administração & dosagem , Morfolinas/química , Morfolinos , Nanopartículas/química , Transplante de Neoplasias , Compostos de Organotecnécio/química , Imagens de Fantasmas , Tomografia por Emissão de Pósitrons/instrumentação , Estreptavidina/química , Tomografia Computadorizada de Emissão de Fóton Único/instrumentaçãoRESUMO
Combining two or more different imaging modalities in the same agent can be of considerable value in molecular imaging. We describe the use of streptavidin nanoparticle-based complexes as multimodality imaging agents to achieve tumor detection in a mouse model by both fluorescence and nuclear imaging. Up to four biotinylated functionalities can be readily attached to these streptavidin nanoparticles without apparent influence on their properties and with reasonable pharmacokinetics and therefore may be ideally suited for multimodality imaging. By binding a biotinylated anti-Her2 Herceptin antibody to provide tumor targeting, a biotinylated DOTA chelator labeled with (111)ln and a biotinylated Cy5.5 fluorophore to a streptavidin nanoparticle, we demonstrated multimodality imaging in SUM190 (Her2+) tumor bearing mice on both an IVIS fluorescence camera and a NanoSPECT/CT small animal nuclear camera. The imaging results show high tumor accumulation and strong tumor-to-normal tissue contrast by both fluorescence and nuclear imaging. The subsequent biodistribution study confirmed the specific tumor accumulation in that tumor accumulation of radioactivity at 40 h was 21 ID%/g and therefore much higher than all other tissues including liver, heart, kidney, spleen, and muscle that accumulated 8.7, 2.5, 6.9, 7.2, and 1.9 ID%/g, respectively. In conclusion, the streptavidin nanoparticle under development in this laboratory was used effectively for multimodality imaging of tumor in mice by fluorescence and nuclear detection. Presumably, other imaging modalities could also be considered.
Assuntos
Núcleo Celular/química , Nanopartículas/química , Neoplasias/patologia , Compostos Organometálicos , Estreptavidina/química , Animais , Carbocianinas/química , Linhagem Celular Tumoral , Feminino , Fluorescência , Compostos Heterocíclicos com 1 Anel/química , Humanos , Camundongos , Camundongos Nus , Compostos Organometálicos/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
UNLABELLED: When antisense oligomers are intracellular, they migrate to and are retained in the nucleus of tumor cells and therefore may be used to carry Auger electron-emitting radionuclides such as (111)In for effective tumor radiotherapy. METHODS: Our nanoparticle consists of streptavidin that links 3 biotinylated components: the antiHer2 antibody trastuzumab (to improve pharmacokinetics), the tat peptide (to improve cell membrane transport), and the (111)In-labeled antiRIalpha messenger RNA antisense morpholino (MORF) oligomer. RESULTS: As evidence of unimpaired function, tumor cell and nuclear accumulations were orders of magnitude higher after incubation with (99m)Tc-MORF/tat/trastuzumab than after incubation with free (99m)Tc-MORF and significantly higher with the antisense than with the sense MORF. In mice, tumor and normal-tissue accumulations of the (99m)Tc-MORF/tat/trastuzumab nanoparticle were comparable to those of free (99m)Tc-trastuzumab, confirming the improved pharmacokinetics due to the trastuzumab component. Although kidneys, liver, and other normal tissues also accumulated the nanoparticle, immunohistochemical evaluation of tissue sections in mice receiving the Cy3-MORF/tat/trastuzumab nanoparticle showed evidence of nuclear accumulation only in tumor tissue. In a dose escalation study, as measured by the surviving fraction, the nanoparticle significantly increased the kill of SK-BR-3 breast cancer Her2+/RIalpha+ cells, compared with all controls. CONCLUSION: Significant radiation-induced antisense-mediated cytotoxicity of tumor cells in vitro was achieved using an Auger electron-emitting antisense MORF oligomer administered as a member of a 3-component streptavidin-delivery nanoparticle.
Assuntos
DNA Antissenso/administração & dosagem , Portadores de Fármacos/administração & dosagem , Radioisótopos de Índio/administração & dosagem , Nanopartículas/administração & dosagem , Neoplasias/patologia , Neoplasias/radioterapia , Estreptavidina/administração & dosagem , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , DNA Antissenso/química , Feminino , Radioisótopos de Índio/química , Camundongos , Camundongos Nus , Nanopartículas/química , Compostos Radiofarmacêuticos/administração & dosagem , Compostos Radiofarmacêuticos/química , Estreptavidina/química , Resultado do TratamentoRESUMO
Fluorescent conjugated DNA oligonucleotides for antisense targeting of mRNA has the potential of improving tumor/normal tissue ratios over that achievable by nuclear antisense imaging. By conjugating the Cy5.5 emitter to the 3' equivalent end of a 25 mer phosphorothioate (PS) antisense major DNA and hybridizing with a shorter 18 mer phosphodiester (PO) complementary minor DNA (cDNA) with the Black Hole inhibitor BHQ3 on its 5' end (i.e., PS DNA25-Cy5.5/PO cDNA18-BHQ3), we previously achieved antisense optical imaging in mice as a proof of this concept. In a process of optimization, we have now evaluated the stability of a small series of duplexes with variable-length minor strands. From these results, a new study anti-mdr1 antisense duplex was selected with a 10 mer minor strand (i.e., PS DNA25-Cy5.5/PO cDNA10-BHQ3). The new study duplex shows stability in serum environments at 37 degrees C and provides a dramatically enhanced fluorescence in KB-G2 (pgp++) cells when compared with KB-31 (pgp+/-) as evidence of antisense dissociation at its mdr1 mRNA target. The duplex was also administered to KB-G2 tumor bearing mice, and when compared to the duplex used previously, the fluorescence from the tumor thigh was more obvious and the tumor-to-background fluorescence ratio was improved. In conclusion, by a process designed to optimize the duplex for optical antisense tumor targeting, the fluorescence signal was improved both in cells and in tumored mice.
Assuntos
Sondas de DNA/química , Sondas de DNA/metabolismo , DNA Antissenso/química , DNA Antissenso/metabolismo , Neoplasias/metabolismo , Animais , Sequência de Bases , Benzotiazóis/metabolismo , Linhagem Celular Tumoral , Sondas de DNA/genética , DNA Antissenso/genética , Fluorescência , Humanos , Masculino , Camundongos , Hibridização de Ácido Nucleico , Quinolinas/metabolismoRESUMO
PURPOSE: Trastuzumab (Herceptin™) is often internalized following binding to Her2+ tumor cells. The objective of this study was to investigate whether trastuzumab can be used as a specific carrier to deliver antisense oligomers into Her2+ tumor cells both in vitro and in vivo. METHODS: A biotinylated MORF oligomer antisense to RhoC mRNA and its biotinylated sense control were labeled with either lissamine for fluorescence detection or 99mTc for radioactivity detection and were linked to biotinylated trastuzumab via streptavidin. The nanoparticles were studied in SUM190 (RhoC+, Her2+) study and SUM149 (RhoC+, Her2−) control cells in culture and as xenografts in mice. RESULTS: As evidence of unimpaired Her2+ binding of trastuzumab within the nanoparticle, accumulations were clearly higher in SUM190 compared to SUM149 cells and, by whole-body imaging, targeting of SUM190 tumor was similar to that expected for a radiolabeled trastuzumab. As evidence of internalization, fluorescence microscopy images of cells grown in culture and obtained from xenografts showed uniform cytoplasm distribution of the lissamine-MORF. An invasion assay showed decreased RhoC expression in SUM190 cells when incubated with the antisense MORF nanoparticles at only 100 nM. CONCLUSION: Both in cell culture and in animals, the nanoparticle with trastuzumab as specific carrier greatly improved tumor delivery of the antisense oligomer against RhoC mRNA into tumor cells overexpressing Her2 and may be of general utility.
Assuntos
Anticorpos Monoclonais/química , Neoplasias da Mama/metabolismo , Portadores de Fármacos/química , Morfolinas/metabolismo , Nanopartículas/química , Estreptavidina/química , Animais , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais Humanizados , Transporte Biológico , Biotina/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Portadores de Fármacos/metabolismo , Feminino , Humanos , Espaço Intracelular/metabolismo , Camundongos , Morfolinos , Receptor ErbB-2/metabolismo , Estreptavidina/metabolismo , TrastuzumabRESUMO
In treating tumors by pretargeting, the antitumor antibody and the cytotoxic effector (e.g., toxins and radioactivity) are separately administered. Therefore, pretargeting is more complicated with many variables. We are conducting studies to understand the influence of each variable using a novel recognition pair of mutually complementary phosphorodiamidate morpholino oligomers (MORF/cMORF). Earlier we developed a semi-empirical model capable of accurately predicting the behavior of a radiolabeled cMORF effector with variations in dosages and timing. We have now extended the model to predict the effector behavior, in particular, its maximum percent tumor accumulation (MPTA) in mice pretargeted with three different MORF-conjugated antibodies (MN14, B72.3, and CC49). The MN14 and the CC49 target different antigens in the same tumor, whereas the CC49 and the B72.3 target the same antigen but with very different tumor accumulation. By comparing the pretargeting results of these three antibodies with our prediction, we confirmed that the MPTA of the radiolabeled cMORF effector in the LS174T tumor is independent of the antibodies. In conclusion, the MPTA cannot be improved through the use of different pretargeting antibodies, although different antibodies may improve the maximum absolute tumor accumulation, the heterogeneity, and/or the tumor-to-normal tissue ratios of the effector. This conclusion will apply equally well to effectors carrying a fluorescent probe, an anticancer agent, or a radioactive imaging agent.
Assuntos
Anticorpos Monoclonais/farmacocinética , Antineoplásicos/farmacocinética , Morfolinas/farmacocinética , Algoritmos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antineoplásicos/imunologia , Antineoplásicos/imunologia , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/imunologia , Radioisótopos de Índio/metabolismo , Camundongos , Camundongos Nus , Morfolinas/metabolismo , Morfolinos , Neoplasias/imunologia , Neoplasias/metabolismo , Compostos Radiofarmacêuticos/metabolismo , Distribuição TecidualRESUMO
Amplification pretargeting can play an important role in molecular imaging by significantly increasing the accumulation of signal in target tissues. Multiple-step amplification pretargeting offers the potential to greatly improve target localization of effector molecules through the intermediate use of polymers conjugated with multiple copies of complementary oligomers. In this study, PAMAM dendrimer generation 3 (G3) was conjugated with multiple copies of a phosphorodiamidate morpholino (MORF) oligomer. Characterization of the conjugate by native-PAGE and SE-HPLC demonstrated that the conjugation was successful. The average numbers of MORF groups in the G3-MORF conjugate, both attached and accessible to the (99m)Tc labeled complementary MORF (cMORF), were determined. The antitumor antibody CC49 was conjugated with both MORF and cMORF (collectively (c)MORF) at an average of about one group per molecule. Nine of the 32 carboxyl groups of the dendrimer were modified with MORF, of which 90% were accessible in solution to (99m)Tc-cMORF. After purification, the G3-MORF was radiolabeled with tracer (99m)Tc-labeled cMORF (i.e., G3-MORF/(99m)Tc-cMORF) and added to the antibody CC49 previously conjugated with cMORF (i.e., CC49-cMORF/G3-MORF/(99m)Tc-cMORF), the complex demonstrated a single peak on SE-HPLC as evidence of complete hybridization between G3-MORF/(99m)Tc-cMORF and CC49-cMORF. The CC49-(c)MORF were bound to both Protein G and Protein L coated plates, and G3-MORF was added to hybridize with CC49-cMORF before the (99m)Tc-cMORF was added to test amplification pretargeting. In comparison to conventional pretargeting without the G3-MORF, the signal was amplified about 6 and 14 times, respectively, showing that the G3-MORF participated in amplifying the signal. Further amplification studies using the CC49-(c)MORF for LS174T tumor cells in tissue culture also demonstrated clear evidence of signal amplification.
Assuntos
Morfolinas/química , Morfolinas/metabolismo , Poliaminas/química , Animais , Anticorpos Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Dendrímeros , Humanos , Camundongos , Morfolinas/análise , Morfolinos , Compostos de Organotecnécio/química , Poliaminas/análise , Poliaminas/síntese química , Coloração e RotulagemRESUMO
INTRODUCTION: This study investigated radiolabeled bacteriophages for specific detection of infection through gamma imaging. Previously, a (99m)Tc-labeled M13 phage demonstrated specific binding for its host Escherichia coli in vitro and in mice through imaging. METHODS: This study was extended to phages P22, E79, VD-13 and phage 60. Each was radiolabeled with (99m)Tc using the chelator MAG(3), and were evaluated for binding to host and non-host bacteria in vitro and in a mouse infection model. RESULTS: In vitro, each (99m)Tc-phage bound to its host at least 4-fold higher than to non-host bacteria. For example, (99m)Tc-E79 showed 10- to 20-fold greater binding to host Pseudomonas aeruginosa compared to non-host Escherichia coli and Salmonella enterica, and (99m)Tc-phage 60 showed 20-fold greater binding to host Klebsiella pneumoniae over non-hosts. Mice received host or non-host bacteria in one thigh, and 3 h later, the (99m)Tc-phages were administered intravenously. After a further 3 h, the tissues were counted. Liver accumulation was highest for (99m)Tc-E79, averaging 39% compared to an average of 13% for the other (99m)Tc-phages. Animals infected with host bacteria showed infected thigh/normal thigh ratios of 14.2 for (99m)Tc-E79, 2.9 for (99m)Tc-P22, 3.5 for (99m)Tc-VD-13 and 2.1 for (99m)Tc-phage 60. CONCLUSIONS: Although specific host binding was observed in vitro for each of these four (99m)Tc-phages, only (99m)Tc-E79 showed specificity for its host in an in vivo model.
Assuntos
Bactérias/virologia , Infecções Bacterianas/diagnóstico por imagem , Bacteriófagos , Infecções/diagnóstico por imagem , Pertecnetato Tc 99m de Sódio/farmacocinética , Animais , Bacteriófagos/química , Bacteriófagos/patogenicidade , Modelos Animais de Doenças , Escherichia coli/virologia , Interações Hospedeiro-Patógeno , Marcação por Isótopo/métodos , Klebsiella pneumoniae/virologia , Masculino , Camundongos , Pseudomonas aeruginosa/virologia , Cintilografia , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Salmonella enterica/virologia , Pertecnetato Tc 99m de Sódio/química , Especificidade da Espécie , Distribuição TecidualRESUMO
OBJECTIVE: To evaluate the use of technetium Tc 99m-labeled EDTA-biotin monomer ((99m)Tc-EB1) as a scintigraphic imaging agent for soft tissue inflammatory lesions in horses. ANIMALS: 6 healthy adult horses. PROCEDURES: First (phase 1), the agent's safety and blood-tissue clearance and an appropriate imaging protocol were determined in 6 horses. Each horse was injected with (99m)Tc-EB1 (1.1 GBq, IV, once); images were acquired at intervals during the following 24-hour period. Subsequently (phase 2), inflammation was induced via injection of 200 mg (10 mL) of mepivacaine (0.4 mg/kg) into the right neck musculature and perineurally in the proximal palmar metacarpal region of the right forelimb of 2 horses. Six hours after mepivacaine injection, (99m)Tc-EB1 (2.2 GBq, IV, once) was administered; 8 hours after injection, comparative soft tissue images were acquired after administration of technetium (99m)Tc-hydroxymethylene diphosphonate ((99m)Tc-HDP; 7.4 GBq, IV, once). RESULTS: After injections of (99m)Tc-EB1, physical examinations, CBCs, and serum biochemical analyses revealed no abnormalities in any horse. Blood clearance of (99m)Tc-EB1 was rapid (A phase, 2.2 minutes; beta phase, 58 minutes). Soft tissue uptake of (99m)Tc-EB1 was immediate and persisted for as long as 4 hours after injection. At 6 hours after IM and perineural mepivacaine injections, mepivacaine-induced inflammation was detectable by use of (99m)Tc-EB1. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that (99m)Tc-EB1 is safe for use in horses and can identify soft tissue inflammation without concurrent uptake in bone. Compared with (99m)Tc-HDP administration, use of (99m)Tc-EB1 extended the duration of soft tissue scintigraphic image acquisition.
Assuntos
Biotina/análogos & derivados , Ácido Edético , Membro Anterior/diagnóstico por imagem , Doenças dos Cavalos/diagnóstico por imagem , Inflamação/veterinária , Compostos de Organotecnécio , Compostos Radiofarmacêuticos , Animais , Feminino , Membro Anterior/patologia , Doenças dos Cavalos/patologia , Cavalos , Inflamação/diagnóstico por imagem , Inflamação/patologia , Cintilografia/veterináriaRESUMO
OBJECTIVE: Pretargeting with radioactivity has significantly improved tumor to normal tissue radioactivity ratios over conventional antibody imaging in both animal studies and clinical trials. This laboratory is investigating DNA analogues such as phosphorodiamidate morpholinos (MORFs) for pretargeting using technetium-99m ((99m)Tc) for detection. However, the unique properties of fluorescence activation and quenching combined with oligomers with their unique properties of hybridization may be particularly useful when used together for pretargeting with optical detection. The use of linear fluorophore-conjugated oligomer duplexes have been little used in animals, and to our knowledge, have not previously been considered for pretargeting applications. METHODS: A MORF/cDNA pair was selected such that when hybridized, the fluorescence of the Cy5.5-conjugated 25 mer MORF (Cy5.5-MORF25) is inhibited with a BHQ3-conjugated 18 mer complementary DNA (BHQ3-cDNA18). The short BHQ3-cDNA18 was selected to dissociate in the presence of a long cMORF25 in the pretargeted tumor, thus releasing the inhibitor from the Cy5.5 emitter. In this manner, the Cy5.5 fluorescence will be inhibited everywhere but in the target. The dissociation was first examined in vitro by adding the duplex to the cMORF25 both in solution and immobilized on polystyrene microspheres and by surface plasmon resonance (SPR). Thereafter, biotinylated cMORF25 immobilized on streptavidin polystyrene microspheres were administered intramuscularly in one thigh of hairless SKH-1 mice as target while an identical weight of the identical microspheres but without the cMORF25 was administered in the contralateral thigh as control. The animals then received IV the Cy5.5-MORF25/BHQ3-cDNA18 duplex or equal molar dosage of single-chain Cy5.5-MORF25 and were imaged. RESULTS: The SPR studies showed that the immobilized cDNA18 rapidly captured the flowing MORF25 to provide a duplex with a slow dissociation rate constant. Furthermore, when cMORF25 was next allowed to flow over the now immobilized duplex, the cDNA18 was unable to prevent dissociation of the heteroduplex and the formation and release of the cMORF25-MORF25 homoduplex. Images of animals obtained soon after receiving the Cy5.5-MORF25 singlet showed intense whole body fluorescence obscuring the target thigh. However, only 5 minutes after receiving the Cy5.5-MORF25/BHQ3-cDNA18 duplex, the target thigh was clearly visible along with only the kidneys. CONCLUSIONS: This first study of optical pretargeting provides a proof of concept that oligomer pretargeting found to be useful with radioactivity detection is applicable with fluorescent detection as well. In addition, our results demonstrate that by using linear oligomers for optical pretargeting, chain lengths (and base sequences) may be manipulated to provide duplexes with stabilities and fluorescence inhibition optimized for pretargeting and other in vivo applications of optical imaging.